The Bovine Ocular Microbiome: A Multi-Approach Study of Composition and Antimicrobial Activity

The Bovine Ocular Microbiome: A Multi-Approach Study of Composition and Antimicrobial Activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Bovine Ocular Microbiome: A Multi-Approach Study of Composition and Antimicrobial Activity Samat Amat, Devin B. Holman, Sarah M. Luecke, Katherine E. Gzyl, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6253983/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background Despite widespread use of antimicrobials and vaccines, the incidence of infectious bovine keratoconjunctivitis (IBK), or pinkeye, continues to increase in North American beef cow-calf operations. Recent research suggests that there is potential for the commensal ocular microbiome to help mitigate IBK. Therefore, this study characterized the ocular microbiome of cattle with and without IBK using culturing and shotgun metagenomic sequencing and assessed the ability of commensal bacteria to inhibit Moraxella spp. in vitro . Ocular swabs (n = 143) were collected from IBK-affected (n = 102) and healthy cattle (n = 41) before antimicrobial treatment from North Dakota herds. Bacteria were cultured aerobically and anaerobically on five different media and the isolates identified. A subset of swabs (37 IBK; 12 healthy) underwent shotgun metagenomic sequencing. The genomes of 31 isolates, including Moraxella bovoculi , Moraxella bovis , and commensal bacteria, were also sequenced. Fifty-two commensal isolates were screened for inhibition of Moraxella spp. using an agar slab method, with five isolates further tested for inhibition in the presence of culturable ocular microbiome using qPCR. Results The 351 bacterial isolates taxonomically identified represented 61 genera from three phyla. The majority of isolates belonged to Bacillus (25.9%), Streptococcus (11.1%), Staphylococcus (10.1%), and Moraxella (9.4%) genera. Shotgun metagenomic analysis revealed significant differences in the ocular microbiome composition between IBK-affected and healthy cattle (R² = 0.042; P = 0.034). Dominant bacterial species included Cutibacterium acnes , Mannheimia pernigra , Mesomycoplasma bovoculi , Moraxella bovis , and Moraxella bovoculi . Eight bacterial species, including Bifidobacterium globosum and Bacillus licheniformis , were more abundant in healthy cattle, while Arthrobacter luteus was enriched in IBK cases. Thirty-seven high-quality metagenome-assembled genomes were also recovered, with 27% classified as Mesomycoplasma bovoculi . Moraxella spp. genomes exhibited strain-specific antimicrobial resistance and virulence gene diversity. Seventeen commensal isolates inhibited Moraxella , with Weizmannia coagulans , Lentilactobacillus buchneri , and Paenibacillus polymyxa showing strong activity. Selected isolates maintained inhibitory effects in co-culture with the ocular microbiome. Conclusion The ocular surface of beef cattle is inhabited by a diverse microbiome that includes several bacterial strains that have the potential to be used as therapeutics to inhibit IBK pathogens. pinkeye ocular microbiome infectious bovine keratoconjunctivitis metagenomic sequencing whole-genome sequencing antimicrobial resistance cattle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Pinkeye, clinically known as infectious bovine keratoconjunctivitis (IBK), is a highly contagious eye disease that can cause inflammation of the cornea and conjunctiva, negatively affecting animal welfare and reducing profitability in the global beef cattle industry (Dennis and Kneipp, 2021 ; Kneipp, 2021 ; Kuibagarov et al., 2023 ). Bacterial pathogens, together with environmental factors (e.g., season, ultraviolet radiation, dust, and flies) and host factors (e.g., age, genetics, and immune status), influence the pathogenesis of IBK in cattle (Snowder et al., 2005 ; O'Connor et al., 2019 ; Maier et al., 2021a ). Moraxella bovis is considered the primary bacterial agent involved in the development of IBK (HENSON and GRUMBLES, 1960; Nayar and Saunders, 1975 ; Aikman et al., 1985 ). This species can spread rapidly within cattle herds through direct contact or environmental transmission (Brown et al., 1998). Moraxella bovoculi is also an important IBK pathogen (Angelos et al., 2007 ; Gould et al., 2013 ). As such, Moraxella bovis and Moraxella bovoculi have been the main targets for vaccine and antimicrobial-based IBK prevention and control in beef cattle. Currently available vaccines used to prevent IBK outbreaks in cattle herds have shown limited effectiveness in controlled trials (Cullen et al., 2017 ; Maier et al., 2021b ). Therefore, antimicrobials have been the primary means of preventing and controlling the spread of these Moraxella spp. within a cattle herd. However, a recent report showing multidrug resistance in Moraxella bovis and Moraxella bovoculi strains from the bovine ocular surface (Pimenov et al., 2024 ), highlights the necessity of developing antimicrobial alternatives to control IBK (Aslam et al., 2018 ). Emerging evidence from culture-independent, high-throughput sequencing studies in humans (Li et al., 2020 ) suggests that the eye harbors a relatively diverse and dynamic microbial community, and that this ocular microbiota may be targeted to improve resistance against eye infections (Li et al., 2020 ). The human ocular surface has been reported to have a self-sustaining microbiome composed largely of bacteria (98%), with fungi and viruses also present (Wen et al., 2017 ; Ozkan et al., 2019 ; Shivaji et al., 2019 ). This resident microbial community imparts unique functions such as resistance to colonization by pathogens and modulation of intraocular immune and inflammation responses, thereby maintaining ocular health (Willcox, 2013 ; Zegans and Van Gelder, 2014 ; Li et al., 2020 ). Perturbation of the ocular microbiome by factors such as dry eye disease, antimicrobials, infections, and contact lens usage can lead to dysbiosis, resulting in overgrowth of pathogens and intraocular inflammation (Chao et al., 2018 ; Gomes et al., 2020 ; Li et al., 2020 ; Petrillo et al., 2020 ). An altered ocular microbiome has been associated with many ophthalmic diseases in humans (Lu and Liu, 2016 ; Shivaji et al., 2021 ). Thus, restoring the homeostasis of the ocular surface microbiome to promote microbiome-mediated resistance against ophthalmic diseases is an active area of research in human medicine. Although much progress has been made in investigating the compositional and functional features as well as the role of the ocular microbiome in human ocular health, the microbiome of the bovine eye remains less well characterized. This is partly due to the fact that the bovine ocular surface has traditionally been viewed as a habitat primarily for pathogenic microbes associated with IBK. However, a longitudinal study using the 16S rRNA gene sequencing revealed the presence of a complex bacterial microbiota on the ocular surface of pre-weaned beef calves (Bartenslager et al., 2021 ). This study also demonstrated that the ocular microbiota in calves is dynamic, changing in composition, richness, and diversity in response to factors such as age, vaccination, and sampling practices. More recently, Gafen and colleagues identified a reduced relative abundance of members of the Actinobacteriota phylum and a greater relative abundance of Moraxella spp. in IBK-affected eyes compared to normal bovine eyes (Gafen et al., 2023 ). Our lab has also recently identified a relatively rich and site-specific bacterial community in the eyes of healthy newborn calves (Luecke et al., 2023 ). The genus Moraxella was well represented in those samples, and it is currently unclear if early colonization of the eye with Moraxella spp. acts to prime the neonatal immune system against pathogens, or if it predisposes to infection. Fundamental questions also remain regarding how the ocular microbial composition, functional content, and antibiotic-resistance profiles differ between IBK-affected and healthy cattle, and whether commensal bacteria in the healthy ocular microbiome can inhibit Moraxella spp. pathogens. Therefore, the objectives of the present study were to: 1) characterize the ocular microbiome of healthy and IBK-infected beef cattle using culturing and shotgun metagenomic sequencing techniques; 2) investigate the genetic diversity of Moraxella bovis , Moraxella bovoculi , and commensal ocular bacterial isolates through whole-genome sequencing and comparative genomic analysis; and 3) evaluate commensal ocular bacterial isolates for their ability to inhibit the growth of Moraxella bovis and Moraxella bovoculi , with the goal of enhancing resistance to IBK (Fig. 1 ). MATERIALS AND METHODS All experimental procedures were approved by the North Dakota State University (NDSU; Fargo, ND, USA) Institutional Animal Care and Use Committee (protocol ID: 20220029). Ocular swab collection Ocular swabs from the cornea and conjunctiva of cattle exhibiting IBK symptoms (n = 102) as well as control swabs from healthy animals (n = 41) were collected from multiple herds across North Dakota as well as from the NDSU Beef Cattle Research and Teaching Center and the NDSU Veterinary Diagnostic Laboratory (Table 1). Ocular swabs were collected from cattle diagnosed with IBK and healthy herd mates using a Puritan Opti-Swab with Liquid Amies Collection and Transport System (Puritan, Guilford, ME, USA). For swabbing, cattle were placed in a hydraulic squeeze chute and the head was manually restrained. Before swabbing, the eye was wiped with a clean paper towel sprayed with 70% ethanol to remove any debris. The eyelids were then opened, and both the conjunctiva and cornea were gently swabbed. Immediately after sample collection, the swab tips were broken and placed in 1 mL of sterile Amies transport medium and stored on ice for transport to the lab. Upon arrival at the lab, the swab in Amies transport medium was vigorously vortexed, and a 150 µL aliquot of Amies medium was used for culturing. The remaining 850 µL of Amies medium, along with the swab, was used for DNA extraction for shotgun metagenomic sequencing. For this, the tip was cut from the swab and placed into 1 mL of brain heart infusion (BHI) broth containing 20% glycerol. The Amies medium was then centrifuged (4,000 x g for 10 min), and 80% of the supernatant was discarded. The pellet was resuspended with the remaining supernatant, transferred to the BHI-glycerol tube containing the rayon tip of the swab, vortexed for 30 s, and stored at -80°C until DNA extraction. As outlined in Fig. 1, the ocular swab samples collected from the IBK-affected and healthy cattle were subjected to culturing and shotgun metagenomic sequencing. Large-scale bacterial culturing of the ocular microbiome A 150 µL aliquot from the swab samples was mixed with 250 µL of sterile Dulbecco's phosphate-buffered saline and spread onto five different agar plate types (Blood, Columbia Blood [CB], De Man, Rogosa and Sharpe [MRS], Wilkins-Chalgren [WC], and multi-slice agars), to support the growth of a wide range of bacteria. The agar plates were incubated both aerobically and anaerobically for 24-48 h at 37°C. The aerobic plates were supplemented with either 5% or 10% CO 2 (MRS plate). Well-isolated colonies of different morphologies were selected and streaked onto fresh agar and incubated for 24 h. These bacterial isolates were then cryopreserved for DNA extraction and future cultivation by transferring a loopful of each isolate into 100 µL of TE buffer and 1 mL of BHI with 20% glycerol, respectively, and then stored at -80°C. A subset of the preserved bacterial isolates (n = 351) was identified by near full-length 16S rRNA gene sequencing. Isolates for taxonomic identification were randomly selected to include all agar types and culturing conditions. Genomic DNA from these selected isolates was extracted using the Zymo Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions with minor modifications as described previously (Webb et al., 2023). The universal bacterial primers 27F (5′ - AGAGTTTGATCMTGGCTCAG - 3′) (Sunna and Bergquist, 2003) and 1492R (5′ - TACGGYTACCTTGTTACGACTT - 3′) (Reysenbach et al., 1992) were used to amplify the near full-length 16S rRNA gene. Each PCR contained 20 µL of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 12.8 µL of sterile water, 2 µL of each primer (10 µM) (IDT Inc., Coralville, IA, USA), 2 µL of isolate DNA, and 1.2 µL of DMSO (Thermo Fisher Scientific) in a total reaction volume of 40 µL. An Eppendorf Mastercycler thermocycler (Eppendorf, Hamburg, Germany) was then used with the following cycle conditions: initial denaturation at 95°C for 5 min; 35 cycles at 95°C for 45 s, 50°C for 30 s, 72°C for 2 min; and a final extension at 72°C for 5 min. The PCR products were visualized on a 1% (w/v) agarose gel and amplicons were sent to MCLAB (San Francisco, CA, USA) for Sanger sequencing. The resulting 16S rRNA gene sequences were identified using the Basic Local Alignment Search Tool (BLAST) and the non-redundant NCBI nucleotide database. Metagenomic DNA extraction from ocular swabs A subset of 49 ocular swabs was subjected to shotgun metagenomic sequencing. For this, metagenomic DNA was extracted from the ocular swab samples using a modified cetyltrimethylammonium bromide buffer (CTAB) and phenol:chloroform-based method as described previously (Fujimura et al., 2016; Rackaityte et al., 2020; Amat et al., 2021). Briefly, samples were thawed on ice, vigorously vortexed, and the liquid portion was transferred into individual sterile 2 mL screw-cap tubes. Samples were then pelleted via centrifugation at 20,000 × g for 10 min at 22°C. The supernatant was carefully removed, and the associated swab tip was snipped into the screw-cap tube containing the pellet. The beads from a Lysing Matrix E tube (MP Biomedicals, Irvine, CA, USA) were added to the sample tubes and 500 µL of CTAB buffer (10% CTAB, 0.7M NaCl, 240 mM potassium phosphate buffer pH 8.0), prewarmed to 60°C, was transferred to the sample tube and vortexed. Samples were then incubated for 20 min at 65°C with agitation at 800 rpm, followed by further lysis using bead beating at 5.5 m/s for 30 s. Next, 500 µL of room temperature phenol:chloroform:isoamyl (25:24:1) was added and mixed by inversion. The samples were centrifuged at 14,000 × g for 5 min, and the supernatant was transferred to a heavy phase-lock gel tube (Quantabio, Beverly, MA, USA). Chloroform was added to the tube at a 1:1 ratio, inverted to mix, and centrifuged at 14,000 × g for 5 min. The aqueous layer was transferred to a sterile 1.5 mL tube with 1 µL of linear acrylamide solution and vortexed. Two volumes of PEG-NaCl solution were then added, the tube was vortexed again, and then incubated for 2 h at 21°C. After incubation, the samples were centrifuged at 14,000 × g for 30 min, the supernatant was carefully removed, and the resulting DNA pellet washed with 400 µL of 70% ethanol and centrifuged at 14,000 × g for 5 min. This wash step was repeated twice. After the final centrifugation, the ethanol was removed and the pellet air dried for approximately 10 min. The DNA pellet was resuspended in 30 µL of 10 mM Tris-Cl (pH 8.0) and quantified using a NanoDrop One C Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific) followed by further quantification using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific). DNA was then stored at -20°C until shotgun metagenomic library preparation. Shotgun metagenomic sequencing Metagenomic DNA libraries were constructed according to the following workflow. DNA was fragmented with a Covaris (E220) instrument (Covaris, Inc., Woburn, Massachusetts, USA) and the quality was assessed using an Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA). End-repair and dA-tailing were then carried out, followed by magnetic bead purification. Next, the fragments were PCR-amplified using KAPA HiFi HotStart DNA Polymerase, and the products were purified. The libraries were assessed for quality prior to DNA circularization. After circularization, the libraries were further amplified to create DNA nanoballs (DNB). Finally, the libraries were sequenced on a DNA-based nanoball technology DNBSEQ on a DNBSEQ-G50 platform (MGI Tech, Shenzhen, China) with a PE150 flow cell and 2 × 150 bp sequencing length. Shotgun metagenomic data analysis Sequencing adapters were removed from the raw sequences, and low-quality reads were filtered out using a 4-bp sliding window and a quality threshold of 15 in fastp v.0.23.4 (Chen, et al. 2018a). Reads that were shorter than 100 bp were also removed. Bowtie2 v. 2.5.3 (Langmead and Salzberg, 2012) was used to align the reads to the Bos taurus genome (ARS-UCD2.0) and the Escherichia phage phiX174 genome (NC_001422). SAMtools v.1.19.2 (Danecek et al., 2021) and BEDtools v.2.31.1 (Quinlan and Hall, 2010) were then used to obtain the reads that did not map to these genomes. Kraken2 v. 2.1.3 (Wood et al., 2019) and Bracken v. 2.9 (Lu et al., 2017), together with the Genome Taxonomy Database (GTDB) release 220 (Parks et al., 2022), were used to assign taxonomy to the metagenomic reads. The unassembled metagenomic reads were also screened for antimicrobial resistance genes (ARGs) using the resistance gene identifier (RGI) v. 6.0.3 and the Comprehensive Antibiotic Resistance Database (CARD) v.3.2.9 (23) with KMA (Clausen et al., 2018). Reads from each sample were assembled individually and reads from IBK-affected and healthy cattle samples were co-assembled separately using MEGAHIT v. 1.2.9 (Li et al., 2015). The reads from each sample were then aligned to the assembled contigs using Bowtie2 and the contigs were then binned into metagenome-assembled genomes (MAGs) with MetaBAT2 v. 2.15 (Kang et al., 2019). The completeness and contamination of these MAGs were evaluated with CheckM2 v. 1.0.1 (Chklovski, et al. 2023) and MAGs with a completeness of at least 90% and a contamination level of less than 5% were retained. These MAGs were then dereplicated using dRep v. 3.4.5 (Olm et al., 2017) with primary clustering set at 90% average nucleotide identity (ANI) and secondary clustering set at 99% ANI. Taxonomy was then assigned to the dereplicated MAGs using GTDB-tk v. 2.4.0 (Chaumeil, et al. 2022). The MAGs were also screened for ARGs using the RGI and CARD. The relative abundance of the MAGs in the metagenomes was estimated using CoverM v. 0.7.0 (github.com/wwood/CoverM). The functional gene content was also characterized by aligning the metagenomic reads to the Kyoto Encyclopedia of Genes and Genomes (KEGG) release 112.0 (Kanehisa et al., 2025) prokaryotic protein database with DIAMOND v. 2.1.8.162 (Buchfink et al., 2021) and matching the genes to KEGG orthology (KO) groups. DNA extraction from ocular commensal and Moraxella spp. isolates Selected Moraxella bovis (n = 5) and Moraxella bovoculi (n = 13) isolates as well as potential candidate bacterial isolates (n = 13) for inhibition of the growth of Moraxella spp. were subjected to whole-genome sequencing. For this, the isolates were re-grown from BHI-glycerol stocks on blood or MRS agar and passaged twice before a single colony was transferred to 5 mL of BHI or MRS broth and incubated for 24 h at 37°C with 5 or 10% CO 2 . Genomic DNA was extracted from the isolates using the DNeasy Blood and Tissue Kit (Qiagen Inc.). Briefly, 1 mL of 24 h isolate culture was centrifuged at max speed (20,817 × g ) for 5 min at 21°C to pellet the bacterial cells. To increase the mass of the pellet recovered, this step was repeated up to three times. To the cell pellet, 180 µL of enzymatic lysis buffer (20 mM Tris-HCl, pH 8, 2 mM sodium EDTA, 1.2% Triton X-100, lysozyme [100 mg/mL], and mutanolysin [25,000 U/mL]) was added and thoroughly vortexed. Samples were incubated for 1 h at 37 °C with agitation at 800 rpm. This incubation was followed by the addition of 25 µL of proteinase K and 200 µL of buffer AL (without added ethanol), thorough vortexing of samples, and a 30 min incubation at 56°C with agitation at 800 rpm. After the second incubation, samples were mechanically lysed by adding approximately 400 mg of sterile 0.1 mm zircon/silica beads to each tube and bead beating at 6.0 m/s for 40 s using a FastPrep-24 Classic bead beater (MP Biomedicals). Samples were then centrifuged at 13,000 × g for 5 min, and the supernatant was transferred to a new sterile 1.5 mL tube. The remaining steps were followed according to the manufacturer’s instructions. Whole-genome sequencing and analysis Whole-genome sequence libraries for the ocular commensal and Moraxella spp. isolates were prepared and sequenced as we described in our previous publication (Magossi et al., 2025). Whole-genome sequences from bacterial isolates were quality-filtered using SOAPnuke v. 1.5.6 (Chen, et al. 2018b), with adapters and sequences shorter than 150 bp or with a quality threshold of less than 20 removed. The reads were assembled using SPAdes v. 3.15.5 with the “isolate” option and the assembly quality was evaluated using QUAST v. 5.2.0 (Gurevich, et al. 2013). The assemblies were then taxonomically classified using the GTDB-tk v. 2.4.0 and GTDB release 220. The vast majority of the genomes had 500X or greater coverage, which may affect the quality of the assembly. Therefore, the reads were subsampled to 100X for each genome using seqtk v. 1.4-r122 (github.com/lh3/seqtk) and re-assembled with SPAdes. The completeness and contamination of each 100X assembly were then assessed with CheckM2 v. 1.0.1. A phylogenetic tree of the bacterial genomes as well as MAGs from the metagenomes was created using PhyloPhlAn v. 3.1.68 (Asnicar, et al. 2020) and visualized in iTol v. 6.9 (Letunic and Bork 2024). FastANI was used to determine which isolates might belong to the same strain (99.99% shared ANI). The genomes were also screened for ARGs using the RGI and CARD and mapped against the metagenomic reads with CoverM to assess their abundance in the ocular metagenomes. Comparative genomic analysis of Moraxella bovis and Moraxella bovoculi Given their association with IBK, the Moraxella bovis and Moraxella bovoculi MAGs and isolate genomes were further analyzed. Briefly, the Moraxella spp. genomes and MAGs were annotated using Prokka v. 1.14.6 (Seemann, 2014) with default parameters and the proteins flag using the reference genomes in GenBank for Moraxella bovis (GCA_025985205.1) and Moraxella bovoculi (GCA_000988745.3). After annotation, Panaroo v. 1.5.0 (Tonkin-Hill et al., 2020) was used to determine the core genome for each species using the strict stringency mode. The core genes (0.99 threshold) were aligned with MAFFT v. 7.526 (Katoh and Standley, 2013) and a phylogenetic tree created from the core genome alignment with RAxML-ng v.1.2.0 (Kozlov et al., 2019) for each species with bootstrapping with 1,000 replicates and the GTR+GAMMA substitution model. The Moraxella spp. MAGs and genomes were also screened for known virulence genes with DIAMOND with a 90% identity threshold. These virulence genes included the RTX operon genes in Moraxella bovis designated mbxA (AAK84651.1), mbxB (AAP74653.1), mbxC (AAP74652.1), and mbxD (AAP74654.1) as well as the Moraxella bovoculi RTX operon genes mbvA (AKM27892.1), mbvB (ABA42642.1), mbvC (ABA42640.1), and mbvD (ABA42643.1). Additional virulence genes assessed were flpA (filamentous-haemagglutinin-like protein; BAD83731.1), flpB (filamentous-haemagglutinin-like protein; BAD83735.1), fur (ferric uptake regulator; BAC75975.1; KDN24651.1), omp79 (outer membrane protein; BAC92729.1), pilA (type IV pilin; QPW18723.1; STY93141.1), plb (phospholipase B; AAK53448.1), and tolC (outer membrane protein; AAP74655.2; ABA42644.1). Plasmids and MOB genes were identified in the genome assemblies using MOB-suite v. 3.1.9 (Robertson and Nash, 2018) . In vitro antimicrobial activity of ocular commensal bacteria against Moraxella spp. Of the 351 isolates selected for identification, 52 were tested for growth inhibitory effects against Moraxella bovis and Moraxella bovoculi using the agar slab method, as previously described (Amat et al., 2019) (Fig.1B). Briefly, to visualize bacterial antimicrobial activity, a small agar slab (10 mm in diameter) containing the bacterial isolate of interest was placed on the surface of an agar plate with a fresh inoculum of Moraxella bovis (8338-1) or Moraxella bovoculi (42G CB-D strain). For this, 100 µL of a 24-h culture was spread as a lawn onto BHI or MRS agar plates and incubated for 24 h at 37°C with 5 or 10% CO 2 . From these 24-h bacterial lawns, a 10 mm sterilized cork borer was used to cut agar slabs. The agar slabs were placed surface-side down onto a fresh lawn of Moraxella bovis or Moraxella bovoculi on TSA plates supplemented with 5% sheep’s blood . The Moraxella bovis and Moraxella bovoculi plates were prepared by spreading 100 µL of Moraxella bovis or Moraxella bovoculi overnight culture onto the plates and drying for up to 30 min at 37°C with 5% CO 2 . Up to four agar slabs were placed on the Moraxella lawns and a control agar slab that contained no bacteria was included in each screening. After co-incubation for 24 h at 37°C with 5% CO 2 , inhibition of Moraxella bovis or Moraxella bovoculi growth was observed using the zone of inhibition (ZOI) surrounding the agar plug containing the bacteria of interest. The diameter of the ZOI was measured using a digital caliper and reported as the mean diameter. Limosilactobacillus fermentum ATCC9338 was also included in the screening for comparison. Growth-inhibitory effects of selected ocular commensal bacteria against Moraxella bovoculi in the presence of the ocular microbiome Following the agar slab experiment, a selection of candidate bacteria that exhibited relatively strong inhibition of Moraxella growth were further evaluated for their ability to inhibit the growth of Moraxella spp. in the presence of the culturable ocular microbiome. Moraxella bovoculi was first co-incubated with selected commensal isolates in an enriched ocular microbiome culture. The enriched culture of the ocular microbiome was prepared by pooling 100 µL from five healthy ocular swabs cryopreserved in BHI and 20% glycerol and adding 2.5 mL of fresh BHI broth. This mixture was then incubated at 37°C for approximately 20 h with agitation at 200 rpm. Next, 60 µL of this enriched ocular microbiome was combined with 60 µL of Moraxella bovoculi overnight culture, 60 µL of overnight culture of the selected commensal bacterial isolate, and 2.82 mL of fresh BHI broth, for a total volume of 3 mL. Each candidate bacterial strain co-culture was run in quadruplicate. In addition, a cocktail containing all candidate strains was included. Controls included: 1) only the enriched ocular microbiome and BHI broth, 2) only Moraxella bovoculi and BHI broth, and 3) only BHI broth. Prior to incubation, 200 µL of the prepared cultures were saved as baseline (0 h) samples. Cultures were incubated for 24 h at 37°C with agitation at 200 rpm. After incubation, 200 µL from each replicate was used for 24 h samples. DNA from both 0 h and 24 h samples were extracted using the same procedure described above for whole-genome sequencing. Quantitative PCR (qPCR) was used to assess the concentration of Moraxella spp . in each sample to observe whether the selected commensal strains could inhibit Moraxella growth in the presence of the bovine ocular microbiome. The primers ISRdown (5′-GTGAAGTCGTAACAAGGTAGCCGT-3′) and ISRup (5′-ACCGACGCTTATCGCAGGCTATCA-3′) (Loy and Brodersen, 2014) were used to amplify the 16S-23S rRNA intergenic spacer region (ISR). Each qPCR mixture contained 1 µL SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 1 µL of each primer (IDT Inc.), 6.8 µL of molecular biology grade water (Corning, Manassa, VA, USA), and 1.2 µL of DNA template, in a total volume of 20 µL. A CFX96 Touch Real-Time PCR Detection system (Bio-Rad Laboratories Ltd.) with the following conditions were used: an initial denaturation at 98°C for 3 min, followed by 39 cycles at 98°C for 15 s, 60 °C for 1 min, and then a melt curve analysis was performed starting at 65°C for 5 sec, with a 0.5 °C increase per cycle, ending at 95 °C. Standard curves (10 2 to 10 9 gene copies) were produced using the pDrive cloning vector (Qiagen Inc.) containing the PCR product from the 16S-23S rRNA ISR (Angelos et al., 2007). Each standard was run in duplicate and a no-template control (1.2 µL of nuclease-free water) was included. Statistical analysis Permutational multivariate analysis of variance (PERMANOVA) of the Bray-Curtis dissimilarities based on the relative abundance values of archaeal and bacterial species as well as copies per million reads for KOs were calculated in vegan 2.6-4 to assess the effect of IBK on the ocular surface microbiome. The Mann-Whitney test was used to compare the inverse Simpson diversity index values and qPCR results between the IBK-affected and healthy cattle as the data were not normally distributed. Differentially abundant ARGs, microbial species, and KOs in the ocular microbiomes of IBK-affected vs. healthy cattle were identified using MaAsLin2 v. 1.16.0 (Mallick, et al. 2021) in R v. 4.3.2. Only those ARGs and KOs found in at least 25% of the samples, and microbial species with an overall relative abundance of ≥ 0.1%, were included for the differential abundance analyses. RESULTS Summary of the ocular swabs collected A total of 143 ocular swabs were collected: 102 from cattle diagnosed with IBK and 41 from healthy cattle. The majority of the ocular swabs (77%) were collected during the summer, while additional samples were obtained from winter IBK outbreaks (Table 1). The ocular microbiome assessed by large-scale culturing Through extensive anaerobic and anaerobic culturing using 5 different agar plates, 658 isolates were recovered (data not shown). The majority of these isolates (n = 417) were recovered under aerobic conditions. In addition, 58% of the isolates were obtained from culturing of IBK swabs, although this is expected given that a larger number of swabs were collected from IBK-affected cattle. Overall, the highest number of isolates were recovered from WC (anaerobic, n = 154), blood (aerobic, n = 138), and MRS (aerobic, n = 110) and) agar plates. Of the 658 bacterial isolates collected, 351 were selected for taxonomic identification via Sanger sequencing of the near full-length 16S rRNA gene. These isolates represented three bacterial phyla: Bacillota (82.2%), Pseudomonadota (13.5%), and Actinomycetota (5.0%) (Table 2). Among the 61 bacterial genera identified, isolates belonging to Bacillus (26%), Streptococcus (11%), Staphylococcus (11%), Moraxella (9%), and Macrococcus (4%) were most frequently recovered (Table 2). There were also 33 Moraxella isolates were obtained, consisting of both Moraxella bovis and Moraxella bovoculi. Metagenomic sequencing summary The ocular surface microbiome was also characterized using shotgun metagenomic sequencing.The vast majority of the metagenomic reads were derived from the host: 96.4 ± 1.0% (SEM) for samples from healthy cattle and 92.9 ± 1.9% for samples from IBK-affected cattle. After host DNA removal, the average number of reads for the healthy and IBK-affected cattle samples were 2,575,944 ± 791,376 and 4,312,305 ± 1,074,688, respectively. Ocular microbiome of IBK-affected vs. healthy cattle There was a significant (PERMANOVA: P = 0.015), but relatively small difference ( R 2 = 0.05) in the ocular surface microbial community structure between healthy cattle and cattle diagnosed with IBK (Fig. 2A). Microbial species diversity on the ocular surface did not differ between healthy and IBK-affected cattle (Fig. 2B). Overall, the ocular surface microbiomes were dominated by bacterial species such as Cutibacterium acnes , Mannheimia pernigra , Mesomycoplasma bovoculi , Moraxella bovis , and Moraxella bovoculi (Fig. 3). Nine bacterial species were differentially abundant in the ocular microbiomes of healthy vs. IBK-affected cattle (Fig. 4). All but one of these species ( Arthrobacter luteus ), were relatively more abundant in the healthy cattle and included Bacillus licheniformis , Bifidobacterium globosum , Blastomonas ursincola , C. acnes , Lawsonella clevelandensis , Psychrobacter maritimus , Ruminococcus sp900100595, and Ruminococcus sp900316555. The functional content of the ocular surface microbiome was assessed using KOs. In total, 158 KOs (prokaryotic-associated) were identified in at least one ocular sample with ko01100 (sedoheptulose-bisphosphatase), ko01240 (uridine nucleosidase), and ko01230 (mannosyl-oligosaccharide alpha-1,2-mannosidase) most abundant (data not shown). The overall functional composition did not between the IBK-affected and healthy cattle (Fig. S1; PERMANOVA: P > 0.05). Similarly, no KOs were differentially abundant in the ocular microbiomes of IBK-affected vs. healthy cattle ( P > 0.05). Furthermore,none of the ARGs identified in the ocular metagenomes were differentially abundant between the two groups of cattle. Overall, ARGs conferring resistance to aminoglycosides [ aph(3')-Ia , aph (3'')-Ib ], macrolides [ mel , mph (E), msr (E)], sulfonamides ( sul2 ), tetracyclines [ tet (H), tet (L), tet (O), tet (Q), and tet (W)] were most abundant, although there was a lot of variation between samples (Fig. 5). Metagenome-assembled genomes (MAGs) There were also 37 dereplicated high-quality MAGs recovered from the ocular metagenomes, 12 of which appeared to represent novel species (Fig. 6, Supplementary Table S1). The most frequently recovered MAGs (n = 10) were identified as Mesomycoplasma bovoculi . Other identified species included Moraxella bovis , Moraxella bovoculi , Luteimonas excrementigallinarum , M. pernigra , C. acnes , Mycoplasma bovis , Lentibacillus daqui , Desemzia incerta , Lactococcus lactis, and Streptococcus ruminantium . The MAGs were also screened for ARGs with 13 MAGs found to be carrying at least one ARG, including bla BRO-1 in both Moraxella bovis MAGs and in Moraxella bovoculi OUG26 (Supplementary Table S1). Other ARGs of note that were identified included floR (phenicols) in Moraxella bovoculi OUG36 and ant(9)-Ia (aminoglycosides), bla III (beta-lactams), erm (A) (macrolides), f exA (phenicols), msr (I) (macrolides), vanG (vancomycin) in L. daqui OUG33 . Overall, the relatively most abundant MAG in the ocular surface metagenomes was MAG OUG1 which was taxonomically identified only at the order level (Cardiobacteriales). This MAG was also the only one with a significantly different relative abundance between the two groups of cattle ( P < 0.05), although there was considerable inter-individual variability. Isolate-targeted whole-genome sequencing The genomes of 31 bacterial isolates were sequenced, including 5 Moraxella bovis and 13 Moraxella bovoculi genomes (Supplementary Table S1). The remaining 13 genomes were ocular bacterial isolates that showed inhibition against Moraxella , and included Bacillus licheniformis (n =2), W. coagulans (n =2), Weissella paramesenteroides (n =2), B. pumilus , Bacillus safensis , Bacillus subtilis , Levilactobacillus brevis , Lactiplantibacillus plantarum , Streptococcus pluranimalium , and L. buchneri. All 5 Moraxella bovis isolates appeared to represent unique strains based on their ANI (< 99.99%) (Rodriguez-R, et al. 2024) while there were 10 Moraxella bovoculi strains based on this criterion. The Bacillus licheniformis , W. coagulans , and W. paramesenteroides isolates also appeared to be unique strains. These 31 isolate genomes were also screened for the presence of ARGs. Four of the Moraxella bovis and two Moraxella bovoculi isolates carried the beta-lactamase gene bla BRO-1 , which was originally described in Moraxella catarrhalis (Bootsma, et al. 1996). The tetracycline efflux pump gene tet (45) was found in two Moraxella bovoculi isolates (SL21, SL24) belonging to the same strain, as well as the B . subtilis SL29 isolate. Several other ARGs associated with Bacillus spp. were identified including the chloramphenicol acetyltransferase gene cat86 in B . pumilus and B . safensis , the class D beta-lactamase bla BPU-1 in B . pumilus, and fosBx1, a thiol transferase gene conferring resistance to fosfomycin, in B . licheniformis and B . subtilis . When the metagenomic reads were mapped to the isolate genomes, the Moraxella bovis and Moraxella bovoculi genomes were most abundant, while many of the non- Moraxella spp. genomes were not detected, likely due to the sequencing depth (Supplementary Table S2). Similarly, none of the non- Moraxella spp. isolated were assembled and binned into high-quality MAGs from the metagenomes.However, the Moraxella bovis OUG21 MAG shared an ANI of 99.94% with Moraxella bovis isolates SL11 and SL18 and Moraxella bovoculi MAG OUG26 and isolate SL1 had an ANI of 99.97%. Plasmids were reconstructed from several isolates including Moraxella bovis and Moraxella bovoculi ; however, none of the ARGs detected were found to be carried on one of these plasmids. Given their association with IBK, the core genomes of the isolates and MAGs classified as Moraxella bovis and Moraxella bovoculi were identified and used to create phylogenetic trees (Fig. 7). The core genome of the Moraxella bovis isolates (n = 5) and MAGs (n = 2) consisted of 1,887 genes and there were 1,484 genes in the core genome of the Moraxella bovoculi isolates (n = 13) and MAGs (n = 2). These genomes were also screened for the presence of 15 known virulence genes. In Moraxella bovis , fur , omp79 , pilA , and plb were found in all genomes whereas only three isolates (SL7, SL8, and SL12) carried all four RTX-operon genes ( mbxCABD ) as well as tolC (Fig. 7A). One of the large flpA and flpB genes, which are typically plasmid-encoded (Loy et al., 2021), were identified in Moraxella bovis isolates SL11 ( flpA ), SL18 ( flpA ) and SL12 (f lpB ). In agreement with their ANI, isolates SL11 and SL18, along with MAG OUG21, were most closely related based on the phylogenetic tree. The fur and pilA genes were also found in all Moraxella bovoculi isolate genomes and MAGs (Fig. 7B). The Moraxella bovoculi RTX-operon genes ( mbvCABD ) were identified in five isolate genomes (SL1, SL14, SL21, SL23, and SL24) and one MAG (OUG26), along with tolC . Consistent with their ANI values, isolates SL2, SL19, and SL20 appeared to belong to the same strain, as did SL21 and SL24. Inhibition of Moraxella bovis and Moraxella bovoculi growth by ocular surface commensal bacteria as determined by the agar slab method Of the 52 isolates tested for inhibition against Moraxella spp. using the agar slab method, 15 showed a ZOI against Moraxella bovis , ranging from 12.9 mm to 36.7 mm (Fig. 8; Table 3). There were also 13 isolates that inhibited the growth of Moraxella bovoculi with a ZOI that ranged from 13 mm to 22.2 mm (Table 4). Bacillus pumilus (SL25 and 122H MRS-F) and Bacillus velezensis (42G MRS-A Aero) exhibited the strongest inhibition against Moraxella spp., while Lactiplantibacillus pentosus (SL27), Bacillus tequilensis (SL29), and Weizmannia coagulans (SL35 and SL34) showed moderate inhibition. Lentilactobacillus buchneri (SL36) displayed only weak inhibition. Growth-inhibitory effects against Moraxella bovoculi in the presence of the culturable ocular microbiome as determined by qPCR To evaluate the potential of the candidate commensal strains to prevent the proliferation of Moraxella spp. growth within a microbial community, an experiment was designed to challenge the growth of Moraxella bovoculi in the presence of selected bacterial strains and the ocular microbiome. The growth of Moraxella over a 24-h period was measured using qPCR, indicating that the selected bacterial strains inhibited the growth of Moraxella bovoculi within an ocular microbial community (Fig. 9). Among these isolates, B. velezensis (42G MRS-A Aero) was most effective, reducing the concentration of Moraxella bovoculi by 99.97% (Fig. 9). B. pumilus (SL25) reduced Moraxella bovoculi concentration by 99.95%, W. coagulans (SL34) and L. pentosus (SL27) by 99.90%, and L. buchneri (SL36) by 99.86%. Additionally, a cocktail containing each of the aforementioned isolates lowered the concentration of Moraxella by 99.59% after 24 h of co-incubation. Discussion The global cattle industry faces multifaceted challenges, including the growing issue of antimicrobial resistance in bovine bacterial pathogens. Antimicrobial resistance reduces the efficacy of antimicrobials routinely used to control infectious diseases in cattle, posing a significant threat to animal health and production and thereby emphasizing the need for alternative approaches. Advancements in high-throughput sequencing have improved our understanding of the taxonomic and functional features of the commensal microbiome across different cattle body sites. As a result, the microbiome has become a key target for preventing and mitigating infectious diseases, including those caused by antimicrobial-resistant bacteria (Wong and Santiago, 2017 ; Relman and Lipsitch, 2018 ). To leverage the ocular microbiome for improved resistance against IBK in cattle, the first logical step is to characterize and identify its taxonomic and functional signatures in both healthy and IBK-affected cattle using culture and metagenomic sequencing methods. Next, commensal isolates from the eyes of healthy cattle should be screened for their ability to inhibit the growth of IBK-associated Moraxella spp. Synthetic ocular bacterial communities composed of beneficial commensal bacteria could then be developed to modulate the ocular microbiome and enhance resistance to colonization by IBK-associated pathogens. Accordingly, in the present study, we isolated and identified a relatively large number of bacterial isolates (n = 351) from the ocular surface of IBK-affected and healthy beef cattle using various growth media and conditions. As such, this is the first study to characterize the culturable fraction of the bovine ocular microbiome in cattle using both aerobic and anaerobic culturing with multiple growth media. An earlier study by Gafen et al. ( 2023 ) isolated bacteria from conjunctival swabs (n = 387) obtained from 228 IBK-affected and 159 healthy eyes in cattle (dairy and beef) using 5% sheep blood agar and incubation with 10% CO 2 at 37°C for 48 h (Gafen et al., 2023 ). In that study, Moraxella bovis, Moraxella bovoculi, Moraxella osloensis, Trueperella pyogenes, Bacillus spp., and Proteus spp. were isolated at varying prevalences. Overall, the culturing results from Gafen and colleagues ( 2023 ) and our present study suggest that the bovine ocular surface harbors a relatively diverse community of culturable bacteria. This is further supported by culture-independent high-throughput sequencing studies. For example, a recent longitudinal study (139 days) using 16S rRNA gene sequencing of the ocular surface microbiota of 227 pre-weaned beef calves revealed the presence of a relatively rich and diverse bacterial microbiota (Bartenslager et al., 2021 ). Bacterial species within the Moraxella , Mycoplasma, Pasteurella , and Acinetobacter genera were noted to be the most relatively abundant (Bartenslager et al., 2021 ). A similar taxonomic composition of the ocular microbiota has been reported in mature and older dairy and beef cattle. Using 16S rRNA gene (V4 region) sequencing, Gafen and colleagues characterized the conjunctiva of healthy (n = 376) and IBK-affected cattle (n = 228) across different states and farms (Gafen et al., 2023 ). The dominant bacterial genera on the eyes of these mature adult cattle were similar to those reported in the pre-weaned calves. In addition, we previously characterized the ocular microbiota of neonatal beef calves and found that the ocular surface was already colonized at this age by a complex bacterial community dominated by Streptococcus (57.6%) (Luecke et al., 2023 ). Thus, results from culturing and 16S rRNA gene sequencing-based studies provide strong evidence that the bovine ocular surface is colonized by a self-sustaining, diverse, and dynamic bacterial microbiota, with Moraxella and Mycoplasma spp. being relatively abundant. The significant differences in the dominant bacterial genera identified by culturing and 16S rRNA sequencing highlight the necessity of using both culture-dependent (e.g. culturomics) and culture-independent methods for a comprehensive characterization of the ocular microbiome in cattle. Although the sequencing-based studies discussed above, along with our extensive culturing, provided valuable insights into the taxonomic composition, diversity, and dynamics of the bovine ocular microbiota, amplicon-based sequencing lacks taxonomic resolution at the species level and beyond, and does not include functional information. Therefore, in the present study, we used shotgun metagenomic sequencing to characterize the ocular surface microbiome of IBK-affected and healthy cattle. A significant but relatively small difference was found in the microbial species composition of the two groups. This appeared to be driven by differences in the relative abundance of nine bacterial species, eight of which were enriched in the ocular microbiomes of healthy cattle. These enriched bacterial species included B. globosum , commonly isolated from the rumen of cattle (Kelly et al., 2016 ) and historically used as a probiotic in pigs (Apgar et al., 1993 ), as well as B. licheniformis , which has been used to improve feed digestion and milk yield in cattle (Qiao et al., 2010 ). Other bacterial species that were more abundant in healthy cattle included potentially pathogenic species. For example, Lawsonella clevelandensis has been reported to be a rare cause of abdominal, breast, spinal, and liver abscesses (Favila Menezes et al., 2018; Ramesh et al., 2021 ; Nour et al., 2023), and was recently reported to an emerging cause of vascular graft infection and vascular infection in humans (Ramesh et al., 2021 ; Nour et al., 2023). This species has not previously been found in bovine-associated microbial communities. In addition to L. clevelandensis (often isolated from fresh water), C. acnes , a member of human skin microbiome and considered to be both a beneficial commensal and opportunistic pathogen (Mayslich et al., 2021 ) were also enriched in the healthy cattle. Two uncharacterized Ruminococcus spp. ( R . sp900100595 and R . sp900316555) were also more abundant in the eyes of healthy cattle. Species within the Ruminococcus genus are largely host-associated and play a key role in the cattle ruminal microbiome, primarily in the breakdown of forages (La Reau and Suen, 2018 ). The only species significantly greater in abundance in the ocular microbiome of IBK-affected cattle was A. luteus . There is limited information regarding its colonization niche and pathogenicity; however, A. luteus has been reported to produce an endonuclease (AZuI), restriction enzyme that can attach and destroy viral DNA (Roberts et al., 1976 ; Yang et al., 1976 ). The relationship between A. luteus and IBK pathogenesis therefore warrants further research. Surprisingly, the relative abundance of Moraxella bovis or Moraxella bovoculi was not significantly greater in IBK affected cattle as compared to healthy cattle. This suggests that the presence of these species alone is not sufficient to cause IBK or that there is inter-species diversity in terms of pathogenicity. The ocular resistome (all ARGs) did not differ between the IBK-affected and healthy cattle, although there was considerable inter-animal variability. Genes conferring resistance to the tetracyclines, macrolides, sulfonamides and aminoglycosides were most abundant in the metagenomes. Not surprisingly, these classes represent antimicrobials that are frequently used to treat cattle for various infectious or conditions. For example, tetracyclines and macrolides (e.g., tulathromycin) are used to treat IBK in North America. Sulfonamides, although not used for ocular infections, are administered to treat diarrhea in nursing calves and respiratory disease in cattle (Fossen et al., 2023 ). Aminoglycosides are given to cattle for the treatment of various infections and are used topically in the ears and eyes and via intrauterine infusion to treat endometritis and occasionally may be infused into the udder to treat mastitis (Mercer, 2022). Although the ocular swabs in the present study were collected before antimicrobial treatment, our results highlight the persistence of ARGs even in the absence of direct exposure and could partially explain why certain antimicrobials (e.g., tetracycline and tulathromycin) are less effective against IBK. The Moraxella bovis isolate genomes (n = 5) and MAGs (n = 2) recovered from the ocular metagenome appeared to represent unique strains based their ANI and phylogeny, although strains SL11 and SL18 from healthy animals and MAG OUG21 were closely related. Using this same criterion, there were at least 10 strains among the Moraxella bovoculi isolates. This demonstrates the strain diversity of Moraxella bovis and Moraxella bovoculi on the bovine ocular surface. As such, this diversity presents a significant challenge in developing effective vaccines for IBK. The presence of different virulence factors within Moraxella spp. strains may help explain their association with IBK-affected or healthy cattle. Certain virulence genes, such as fur , pilA , plb , and tolC , were shared among all Moraxella bovis genomes. However, the presence of others including the mbxCABD , flpA and flpB genes varied among the five isolates and two MAGs. The flpA and flpB genes encode filamentous-haemagglutinin-like proteins that may facilitate adhesion to host cells and are typically located on a plasmid in Moraxella bovis (Kakuda et al., 2006 ). While all Moraxella bovoculi genomes carried fur and pilA , only six carried the mbvCABD genes. The ubiquity of pilA and tolC in Moraxella bovis and pilA among Moraxella bovoculi genomes is in agreement with previous work (Wynn et al., 2022). Although the hemolysin-encoding RTX operon genes, mbxCABD (in Moraxella bovis ) and mbvCABD (in Moraxella bovoculi ), are associated with pathogenesis (Loy and Brodersen, 2014 ; Loy et al., 2021 ), they were detected in Moraxella bovis isolates from both healthy and IBK-affected cattle. Notably, 27% of the MAGs recovered from the ocular metagenomes were identified as Mesomycoplasma bovoculi , suggesting that this species is predominant in the cattle ocular microbial community. At the same time, the Mesomycoplasma bovoculi genome is relatively small (≈ 700 Kbp) and thus less metagenomic sequencing depth is required to assemble. Although, we did not isolate Mesomycoplasma bovoculi or any related species within the Mesomycoplasma or Mycoplasma genera, this is likely due to the difficulty in culturing these species in vitro (Pitt et al., 2022 ). Mesomycoplasma bovoculi (formerly Mycoplasma bovoculi ) has been reported to predispose cattle to IBK infection (Levisohn et al., 2004 ; Schnee et al., 2015 ). Therefore, characterizing the role of Mesomycoplasma bovoculi in ocular microbiome homeostasis, its interactions with Moraxella bovis and Moraxella bovoculi , and its role in IBK development deserve further research attention. Human studies suggest that the ocular surface microbiome is associated with ocular health (Matysiak et al., 2021 ; Mohamed et al., 2021 ), and that a healthy ocular microbiome may provide resistance against colonization with infectious agents (Teweldemedhin et al., 2017 ; Chiang and Chern, 2022 ). In this study, we identified 15 bacterial isolates that inhibited the growth of Moraxella bovis and 12 that inhibited Moraxella bovoculi growth in vitro . These isolates belonged to 10 different bacterial genera, including Bacillus , Paenibacillus, Lactiplantibacillus , Streptococcus , Weissella , and Weizmannia . Bacillus spp. produced the largest ZOI against Moraxella bovis and Moraxella bovoculi and members of this genus are commonly used as probiotics to inhibit pathogenic bacteria colonization through the production of antimicrobial compounds (e.g., lipopeptides and polyketides) or interference with signaling pathways (Piewngam et al., 2018 ; Fazle Rabbee and Baek, 2020 ). Direct inhibition of pathogen growth by L. pentosus (Behbahani et al., 2024 ), Paenibacillus polymyxa (Wang et al., 2021 ), L. buchneri (Rodríguez et al., 2023 ), L. brevis (Kwun et al., 2024 )d paramesenteroides (Wan et al., 2023 ) has also been reported. Weizmannia coagulans is known for its broad-spectrum antimicrobial activity against foodborne pathogens (Kallur et al., 2023 ; Maresca et al., 2024 ; Wu et al., 2024 ). Thus, the inhibition of Moraxella bovis and Moraxella bovoculi growth by these ocular bacterial species may be due to the production of antimicrobial substances such as lactic acid, bacteriocins, and hydrogen peroxide. Overall, these results suggest that the ocular surface environment is highly competitive with multiple bacteria capable of producing antimicrobial factors that can inhibit Moraxella spp. Bacterial tolerance to antimicrobials can be affected by metabolic cross-feeding (Adamowicz et al., 2018; Aranda-Díaz et al., 2020). Consequently, the antimicrobial concentrations required to inhibit/kill target bacteria differ when they are grown in the presence of other bacteria rather than in monoculture, due to the interactions within the community (e.g., competition for nutrients, biofilm formation, and production of antimicrobial substances by other bacteria) (Aslam et al., 2018 ). Since bacterial antimicrobial tolerance is influenced by the metabolic interdependence and interactions of different species within a microbial community, it is important to select bacterial strains that can inhibit the growth of IBK-associated pathogens in the presence of the bovine ocular microbiome. Here, we selected five isolates ( B. pumilus , B. velezensis , L. pentosus , L. buchneri and W. coagulans ) that displayed relatively strong inhibition of Moraxella bovis and Moraxella bovoculi . These isolates were then further evaluated for their ability to inhibit the growth of Moraxella bovoculi in the presence of the bovine ocular microbiome. The growth of Moraxella bovoculi co-cultured with the ocular microbiome was inhibited by all five strains, as well as a mixture of these strains. This suggests that the selected isolate strains have the potential to inhibit Moraxella spp. on the ocular surface. Conclusion In summary, we characterized the ocular surface microbiome of IBK-affected and healthy cattle using culture-dependent and culture-independent methods. Eight bacterial species were relatively more abundant in the healthy cattle ocular microbiome based on metagenomic sequencing, including B. globosum, B. licheniformis, Ruminococcus sp900316555, and Ruminococcus sp900100595. Using large-scale aerobic and anaerobic culturing, we identified 351 isolates representing 61 different genera. Of the 52 bacterial isolates screened for inhibition of Moraxella bovoculi and Moraxella bovis , 17 inhibited the growth of one of these species. Overall, our results provide deeper insight into the ocular microbiome in cattle. This knowledge is important for the development of antimicrobial alternative approaches for preventing and treating IBK in cattle. Declarations Acknowledgments We thank the veterinarians and producers that assisted in sample collection. The authors are also appreciative of Kelli Maddock at the NDSU Veterinary Diagnostic Laboratory, and Garrett Havelka at the NDSU Beef Unit for assistance with sample collection. Author’s Contributions Conceiving the idea, designing the study, and providing supervision: SA. Sample collection: SA, SML and GS. Sample processing: SML and SA. Data processing, bioinformatics, and statistical analysis: DBH, KEG, AM, GS, SML, SA. Manuscript writing: SA. Manuscript review, editing, and finalizing: SA, DBH. All authors have read and agreed to the published version of the manuscript. Funding The work in this study was funded by the North Dakota State Board of Agricultural Research and Education (Award numbers: 20-21-2022; 22-14-0231; 24-30-0265), and North Dakota Agricultural Experiment Station as part of a start-up package for S.A. Data availability Raw sequence data and genome assemblies are available on NCBI under BioProject accession PRJNA1128155. Other data supporting the findings of this study are presented within the paper and in the supplementary information files. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Aikman, J. G., E. M. Allan, and I. E. Selman. 1985. Experimental production of infectious bovine keratoconjunctivitis. Vet Rec 117(10):234-239. doi: 10.1136/vr.117.10.234 Amat, S., D. B. Holman, K. Schmidt, K. L. McCarthy, S. T. Dorsam, A. K. 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Source Four ND Veterinary Clinics, NDSU Beef Herd, NDSU VDL No. of summer swabs 110 No. of winter swabs 33 Swab type Total number of swabs Aerobic isolates Anaerobic isolates Total isolate number MP CB Blood MRS MP Blood WC CB Healthy 41 30 61 30 43 14 11 74 18 281 IBK 102 41 37 108 67 12 10 80 22 377 Subtotal 143 71 98 138 110 26 21 154 40 658 ND: North Dakota; NDSU: North Dakota State University; VDL: veterinary diagnostic laboratory; IBK: infectious bovine keratoconjunctivitis; CB: Columbia blood agar plate; MRS: De Man, Rogosa Sharpe agar plate; MP: multi-sliced agar plate; Sharpe; WC: Wilkins-Chalgren Table 2. Bacterial isolates (n= 351) isolated and identified from aerobic and anaerobic culturing of ocular swab samples obtained from IBK-affected and healthy cattle. Phylum Genus Number of isolates Percentage of total isolates Total phylum prevalence Actinomycetota Corynebacterium 4 1.10% 5.00% Actinomycetota Trueperella 2 0.60% Actinomycetota Luteococcus 2 0.60% Actinomycetota Arthrobacter 2 0.60% Actinomycetota Cellulosimicrobium 1 0.30% Actinomycetota Streptomyces 1 0.30% Actinomycetota Rothia 1 0.30% Actinomycetota Nocardia 1 0.30% Actinomycetota Cutibacterium 1 0.30% Actinomycetota Curtobacterium 1 0.30% Actinomycetota Micrococcus 1 0.30% Bacillota Bacillus 91 25.90% 82.20% Bacillota Streptococcus 39 11.10% Bacillota Staphylococcus 39 11.10% Bacillota. Macrococcus 14 4.00% Bacillota. Paenibacillus 9 2.60% Bacillota. Caldibacillus 9 2.60% Bacillota. Rummeliibacillus 8 2.30% Bacillota. Aerococcus 8 2.30% Bacillota. Desemzia 7 2.00% Bacillota. Mammaliicoccus 5 1.40% Bacillota. Weizmannia 4 1.10% Bacillota. Niallia 4 1.10% Bacillota. Heyndrickxia 4 1.10% Bacillota. Carnobacterium 4 1.10% Bacillota. Alkalihalobacillus 4 1.10% Bacillota. Robertmurraya 3 0.90% Bacillota. Priestia 3 0.90% Bacillota. Enterococcus 3 0.90% Bacillota Cytobacillus 2 0.60% Bacillota Brevibacillus 2 0.60% Bacillota. Peribacillus 2 0.60% Bacillota. Lysinibacillus 2 0.60% Bacillota. Lentilactobacillus 2 0.60% Bacillota. Helcococcus 2 0.60% Bacillota Weissella 1 0.30% Bacillota Virgibacillus 1 0.30% Bacillota Terribacillus 1 0.30% Bacillota Ruoffia 1 0.30% Bacillota Planococcus 1 0.30% Bacillota Peptoniphilus 1 0.30% Bacillota Paraclostridium 1 0.30% Bacillota Oceanobacillus 1 0.30% Bacillota Levilactobacillus 1 0.30% Bacillota Lactiplantibacillus 1 0.30% Bacillota Lacrimispora 1 0.30% Bacillota Globicatella 1 0.30% Bacillota Fundicoccus 1 0.30% Bacillota Faecalicatena 1 0.30% Bacillota Facklamia 1 0.30% Bacillota Exiguobacterium 1 0.30% Bacillota Clostridium 1 0.30% Pseudomonadota Moraxella 33 9.40% 13.50% Pseudomonadota Suttonella 4 1.10% Pseudomonadota. Mannheimia 3 0.90% Pseudomonadota. Acinetobacter 2 0.60% Pseudomonadota Psychrobacter 1 0.30% Pseudomonadota Pseudoaxnthomonas 1 0.30% Pseudomonadota Pantoea 1 0.30% Pseudomonadota Citrobacter 1 0.30% Pseudomonadota Alysiella 1 0.30% Total 351 100% Table 3. Bacterial isolates that induced zones of inhibition (ZOI) against Moraxella bovis using the agar slab method . Strains Average ZOI*(mm) Bacillus pumilus 122H-MRS-F-Ae 36.7 Bacillus sonorensis MVC02-G 30.2 Bacillus mojavensis 26Z-WC-C-An 29.9 Bacillus velezensis 42G-MRS-A-Ae 29.8 Weissella paramesenteroides SL16 27.7 Lactiplantibacillus pentosus SL27 27.4 Levilactobacillus brevis SL28 26.4 Bacillus tequilensis SL29 26.0 Limosilactobacillus fermentum ATCC9338 22.1 Weizmannia coagulans SL34 21.5 Bacillus australimaris SL31 16.7 Desemzia incerta 17G-WC-G-An 16.0 Streptococcus pluranimalium SL30 15.5 Caldibacillus hisashii 23D-WC-E-An 13.5 Lentilactobacillus buchneri SL36 12.9 *The mean ZOI was obtained from three replicates. Table 4. Bacterial isolates that induced zones of inhibition (ZOI) against Moraxella bovoculi using the agar slab method . Strain Average ZOI* (mm) Bacillus pumilus 122H-MRS-F-Ae 22.2 Bacillus velezensis 2G-MRS-A-Ae 21.9 Bacillus tequilensis SL29 21.1 Lactiplantibacillus pentosus SL27 20.6 Limosilactobacillus fermentum ATCC9338 19.7 Paenibacillus polymyxa 42G-WC-F-An 19.2 Weizmannia coagulans SL34 18.9 Lentilactobacillus buchneri SL36 18.6 Levilactobacillus brevis SL28 16.1 Bacillus australimaris SL31 15.5 Weissella paramesenteroides SL26 15.3 Mammaliicoccus sciuri MVC05-MRS-A-Ae 14.1 Streptococcus pluranimalium SL30 13.0 *The mean ZOI was obtained from three replicates. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6253983","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":430836997,"identity":"2e0f8c0f-ef54-4f17-820b-6afb1589b8a5","order_by":0,"name":"Samat Amat","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYHAD5gMMPGBGAhGKD4AINrYEkrXwGBCnRb79dOLnDwx20fzze749ePPnMAM/e44BXi0GZ3I3SxxgSM6dcYx3u+HctsMMkj1vCGhhyN0A1MKc23CMd5s0b8NhBoMbBGyR73+7+ccBhvrc+cd4nknzAB1mT0gLw43cbUBbDuduOMbDJs3DBrRFgpBfbrzdZnHG4HjuxmNpZpJz29J5JM48KyDgsNzNNyoqqnPnHT78TOLNH2s5/vbkDfgdBrELweQhQvkoGAWjYBSMAkIAAD0aSXL+MT+5AAAAAElFTkSuQmCC","orcid":"","institution":"North Dakota State University","correspondingAuthor":true,"prefix":"","firstName":"Samat","middleName":"","lastName":"Amat","suffix":""},{"id":430836998,"identity":"7a3ebf4f-2aea-48d2-aa8d-2c34bfc5f9fb","order_by":1,"name":"Devin B. Holman","email":"","orcid":"","institution":"Agriculture and Agri-Food Canada","correspondingAuthor":false,"prefix":"","firstName":"Devin","middleName":"B.","lastName":"Holman","suffix":""},{"id":430836999,"identity":"46e00ec4-3e48-44b0-ab5b-4aad98789dd0","order_by":2,"name":"Sarah M. Luecke","email":"","orcid":"","institution":"North Dakota State University","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"M.","lastName":"Luecke","suffix":""},{"id":430837001,"identity":"88e7ab4c-c249-45f4-953c-7d5dea2cd29f","order_by":3,"name":"Katherine E. Gzyl","email":"","orcid":"","institution":"Agriculture and Agri-Food Canada","correspondingAuthor":false,"prefix":"","firstName":"Katherine","middleName":"E.","lastName":"Gzyl","suffix":""},{"id":430837003,"identity":"e2e39bf4-f9bb-4896-9ffc-b70e592873bb","order_by":4,"name":"Muhammad Anas","email":"","orcid":"","institution":"North Dakota State University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Anas","suffix":""},{"id":430837005,"identity":"2321e3d1-bb07-4f93-b9a4-dcf3727dbf06","order_by":5,"name":"Gerald Stokka","email":"","orcid":"","institution":"North Dakota State University","correspondingAuthor":false,"prefix":"","firstName":"Gerald","middleName":"","lastName":"Stokka","suffix":""}],"badges":[],"createdAt":"2025-03-18 14:08:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6253983/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6253983/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79263487,"identity":"a8127017-6424-4a7a-8b94-3391a9f6d96b","added_by":"auto","created_at":"2025-03-26 09:52:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":922096,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic workflow diagram illustrating the experimental design, the processes involved in ocular swab collection and processing, and culture, shotgun metagenomic and isolate-targeted whole-genome sequencing (A), as well as the procedures involved in screening of isolated ocular commensal strains for their \u003cem\u003ein vitro\u003c/em\u003e antimicrobial activity against \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e (B).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6253983/v1/fdf3cc0c9eb667d101a01e9b.png"},{"id":79263486,"identity":"31f6b57c-3e19-4b91-9030-3ef9b3991098","added_by":"auto","created_at":"2025-03-26 09:52:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":124849,"visible":true,"origin":"","legend":"\u003cp\u003eA non-metric multidimensional scaling (NMDS) plot of the Bray Curtis dissimilarities (\u003cstrong\u003eA\u003c/strong\u003e), and inverse Simpson diversity index (\u003cstrong\u003eB\u003c/strong\u003e) for the ocular microbiome of healthy cattle (n = 12) and cattle diagnosed with infectious bovine keratoconjunctivitis (IBK) (n = 37) (\u003cem\u003eP\u003c/em\u003e = 0.54).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6253983/v1/fc3169b18e990e51723247a5.png"},{"id":79263488,"identity":"4d991224-d519-4c23-9b7a-8477bc11d70d","added_by":"auto","created_at":"2025-03-26 09:52:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":221924,"visible":true,"origin":"","legend":"\u003cp\u003eStacked bar chart of the 10 relatively most abundant bacterial species in the ocular surface microbiome of healthy cattle (n = 12) and cattle diagnosed with infectious bovine keratoconjunctivitis (IBK) (n = 37).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6253983/v1/3af96934f7c1abda701da37c.png"},{"id":79265427,"identity":"baeadcbd-d81f-4cbd-b648-5ac6edb45db1","added_by":"auto","created_at":"2025-03-26 10:00:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":273045,"visible":true,"origin":"","legend":"\u003cp\u003ePercent relative abundance of differentially abundant bacterial species in the ocular surface microbiome of cattle that were healthy (n = 12) or diagnosed with infectious bovine keratoconjunctivitis (IBK) (n = 37) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). The point represents the median value and the lines are the 25th and 75th percentiles.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6253983/v1/9b302d9687e8090bb648af9d.png"},{"id":79265428,"identity":"fa807293-c7c7-446b-8196-c710a232b803","added_by":"auto","created_at":"2025-03-26 10:00:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":190586,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of the 25 most abundant antimicrobial resistance genes identified in the ocular surface microbiomes of healthy cattle (n = 12) and cattle diagnosed with infectious bovine keratoconjunctivitis (n = 37). CPM: copies per million reads.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6253983/v1/bdbfc0219acca2ad43c507ed.png"},{"id":79263498,"identity":"03582d95-049e-417f-a67f-956e97ce8f35","added_by":"auto","created_at":"2025-03-26 09:52:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":339063,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree of 31 bacterial isolate genomes and 37 metagenome-assembled genomes (MAGs) from the ocular surface of cattle. Isolates genomes are colored red and MAGs are colored blue in the inner ring.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6253983/v1/eceb04f65aad425365d40198.png"},{"id":79263497,"identity":"8e2c16d6-933b-4ad3-9d7d-1d7ed154baa2","added_by":"auto","created_at":"2025-03-26 09:52:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":198444,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree of A) \u003cem\u003eMoraxella bovis\u003c/em\u003e (n = 7) and B) \u003cem\u003eMoraxella bovoculi \u003c/em\u003e(n = 15)\u003cem\u003e \u003c/em\u003eisolate and metagenome-assembled genomes (MAG). Phylogeny was inferred from the alignment of 1,887 and 1,484 core genes for \u003cem\u003eMoraxella bovis \u003c/em\u003eand\u003cem\u003e Moraxella bovoculi, \u003c/em\u003erespectively. Virulence genes are also indicated as being present or absent in the isolate genomes and MAGs. The horizontal scale bar represents substitutions per nucleotide.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6253983/v1/b750be5e00721206601db1ab.png"},{"id":79263494,"identity":"f139a947-6a9a-49dc-bcc2-a9e41dceed25","added_by":"auto","created_at":"2025-03-26 09:52:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":512602,"visible":true,"origin":"","legend":"\u003cp\u003eThe agar slab method used to visualize antimicrobial activity of commensal bacteria from the bovine ocular surface. In this assay, a small agar slab (10 mm in diameter) containing the bacterial strain of interest was placed on the surface of an agar plate freshly inoculated with \u003cem\u003eMoraxella bovis\u003c/em\u003e or \u003cem\u003eMoraxella bovoculi\u003c/em\u003e. After 24h co-incubation, inhibition of \u003cem\u003eMoraxella bovis \u003c/em\u003eor \u003cem\u003eMoraxella bovoculi\u003c/em\u003e growth was indicated by the formation of a zone of inhibition (ZOI) surrounding the agar slab. \u0026nbsp;Commensal bacterial isolates include \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e (42G-WC-F-An); \u003cem\u003eBacillus pumilus\u003c/em\u003e (122H-MRS-F-Ae); \u003cem\u003eWeissella paramesenteroides\u003c/em\u003e (SL26); \u003cem\u003eLevilactobacillus brevis\u003c/em\u003e (SL28); \u003cem\u003eWeizmannia coagulans\u003c/em\u003e (SL35).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6253983/v1/be05e6eb6a420a452bb5173a.png"},{"id":79265436,"identity":"494576f1-fa5f-4b7a-a347-036819388d88","added_by":"auto","created_at":"2025-03-26 10:00:10","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":238620,"visible":true,"origin":"","legend":"\u003cp\u003eAbundance of\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eMoraxella \u003c/em\u003espp.\u003cem\u003e \u003c/em\u003eas estimated by qPCR before (0 h) and after (24 h) incubation with commensal isolates and an enriched ocular bacterial community.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6253983/v1/5b7ff5f685aa99d46a52e688.png"},{"id":79266710,"identity":"eb73b371-0ac6-4ad0-8622-9854e86a086b","added_by":"auto","created_at":"2025-03-26 10:16:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4737535,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6253983/v1/94b4e702-d9ee-4308-bf54-7a2bae91b8cd.pdf"},{"id":79263485,"identity":"5852acaa-f749-4e3d-9837-1cf9716ea165","added_by":"auto","created_at":"2025-03-26 09:52:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":184664,"visible":true,"origin":"","legend":"","description":"","filename":"supp.docx","url":"https://assets-eu.researchsquare.com/files/rs-6253983/v1/2e960f1fd59ca334abd8ecab.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Bovine Ocular Microbiome: A Multi-Approach Study of Composition and Antimicrobial Activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePinkeye, clinically known as infectious bovine keratoconjunctivitis (IBK), is a highly contagious eye disease that can cause inflammation of the cornea and conjunctiva, negatively affecting animal welfare and reducing profitability in the global beef cattle industry (Dennis and Kneipp, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kneipp, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kuibagarov et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Bacterial pathogens, together with environmental factors (e.g., season, ultraviolet radiation, dust, and flies) and host factors (e.g., age, genetics, and immune status), influence the pathogenesis of IBK in cattle (Snowder et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; O'Connor et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Maier et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). \u003cem\u003eMoraxella bovis\u003c/em\u003e is considered the primary bacterial agent involved in the development of IBK (HENSON and GRUMBLES, 1960; Nayar and Saunders, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Aikman et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). This species can spread rapidly within cattle herds through direct contact or environmental transmission (Brown et al., 1998).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMoraxella bovoculi\u003c/em\u003e is also an important IBK pathogen (Angelos et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Gould et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). As such, \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e have been the main targets for vaccine and antimicrobial-based IBK prevention and control in beef cattle. Currently available vaccines used to prevent IBK outbreaks in cattle herds have shown limited effectiveness in controlled trials (Cullen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Maier et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Therefore, antimicrobials have been the primary means of preventing and controlling the spread of these \u003cem\u003eMoraxella\u003c/em\u003e spp. within a cattle herd. However, a recent report showing multidrug resistance in \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e strains from the bovine ocular surface (Pimenov et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), highlights the necessity of developing antimicrobial alternatives to control IBK (Aslam et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEmerging evidence from culture-independent, high-throughput sequencing studies in humans (Li et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) suggests that the eye harbors a relatively diverse and dynamic microbial community, and that this ocular microbiota may be targeted to improve resistance against eye infections (Li et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The human ocular surface has been reported to have a self-sustaining microbiome composed largely of bacteria (98%), with fungi and viruses also present (Wen et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ozkan et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shivaji et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This resident microbial community imparts unique functions such as resistance to colonization by pathogens and modulation of intraocular immune and inflammation responses, thereby maintaining ocular health (Willcox, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zegans and Van Gelder, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Perturbation of the ocular microbiome by factors such as dry eye disease, antimicrobials, infections, and contact lens usage can lead to dysbiosis, resulting in overgrowth of pathogens and intraocular inflammation (Chao et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Gomes et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Petrillo et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). An altered ocular microbiome has been associated with many ophthalmic diseases in humans (Lu and Liu, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shivaji et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Thus, restoring the homeostasis of the ocular surface microbiome to promote microbiome-mediated resistance against ophthalmic diseases is an active area of research in human medicine.\u003c/p\u003e \u003cp\u003eAlthough much progress has been made in investigating the compositional and functional features as well as the role of the ocular microbiome in human ocular health, the microbiome of the bovine eye remains less well characterized. This is partly due to the fact that the bovine ocular surface has traditionally been viewed as a habitat primarily for pathogenic microbes associated with IBK. However, a longitudinal study using the 16S rRNA gene sequencing revealed the presence of a complex bacterial microbiota on the ocular surface of pre-weaned beef calves (Bartenslager et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This study also demonstrated that the ocular microbiota in calves is dynamic, changing in composition, richness, and diversity in response to factors such as age, vaccination, and sampling practices. More recently, Gafen and colleagues identified a reduced relative abundance of members of the Actinobacteriota phylum and a greater relative abundance of \u003cem\u003eMoraxella\u003c/em\u003e spp. in IBK-affected eyes compared to normal bovine eyes (Gafen et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur lab has also recently identified a relatively rich and site-specific bacterial community in the eyes of healthy newborn calves (Luecke et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The genus \u003cem\u003eMoraxella\u003c/em\u003e was well represented in those samples, and it is currently unclear if early colonization of the eye with \u003cem\u003eMoraxella\u003c/em\u003e spp. acts to prime the neonatal immune system against pathogens, or if it predisposes to infection. Fundamental questions also remain regarding how the ocular microbial composition, functional content, and antibiotic-resistance profiles differ between IBK-affected and healthy cattle, and whether commensal bacteria in the healthy ocular microbiome can inhibit \u003cem\u003eMoraxella\u003c/em\u003e spp. pathogens. Therefore, the objectives of the present study were to: 1) characterize the ocular microbiome of healthy and IBK-infected beef cattle using culturing and shotgun metagenomic sequencing techniques; 2) investigate the genetic diversity of \u003cem\u003eMoraxella bovis\u003c/em\u003e, \u003cem\u003eMoraxella bovoculi\u003c/em\u003e, and commensal ocular bacterial isolates through whole-genome sequencing and comparative genomic analysis; and 3) evaluate commensal ocular bacterial isolates for their ability to inhibit the growth of \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e, with the goal of enhancing resistance to IBK (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eAll experimental procedures were approved by the North Dakota State University (NDSU; Fargo, ND, USA) Institutional Animal Care and Use Committee (protocol ID: 20220029).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOcular swab collection \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOcular swabs from the cornea and conjunctiva of cattle exhibiting IBK symptoms (n = 102) as well as control swabs from healthy animals (n = 41) were collected from multiple herds across North Dakota as well as from the NDSU Beef Cattle Research and Teaching Center and the NDSU Veterinary Diagnostic Laboratory (Table 1). Ocular swabs were collected from cattle diagnosed with IBK and healthy herd mates using a Puritan Opti-Swab with Liquid Amies Collection and Transport System (Puritan, Guilford, ME, USA). For swabbing, cattle were placed in a hydraulic squeeze chute and the head was manually restrained. Before swabbing, the eye was wiped with a clean paper towel sprayed with 70% ethanol to remove any debris. The eyelids were then opened, and both the conjunctiva and cornea were gently swabbed. Immediately after sample collection, the swab tips were broken and placed in 1\u0026thinsp;mL of sterile Amies transport medium and stored on ice for transport to the lab. Upon arrival at the lab, the swab in Amies transport medium was vigorously vortexed, and a 150 \u0026micro;L aliquot of Amies medium was used for culturing. The remaining 850 \u0026micro;L of Amies medium, along with the swab, was used for DNA extraction for shotgun metagenomic sequencing. For this, the tip was cut from the swab and placed into 1 mL of brain heart infusion (BHI) broth containing 20% glycerol. The Amies medium was then centrifuged (4,000 x \u003cem\u003eg\u003c/em\u003e for 10 min), and 80% of the supernatant was discarded. The pellet was resuspended with the remaining supernatant, transferred to the BHI-glycerol tube containing the rayon tip of the swab, vortexed for 30 s, and stored at -80\u0026deg;C until DNA extraction. As outlined in Fig. 1, the ocular swab samples collected from the IBK-affected and healthy cattle were subjected to culturing and shotgun metagenomic sequencing. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLarge-scale bacterial culturing of the ocular microbiome \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 150 \u0026micro;L aliquot from the swab samples was mixed with 250 \u0026micro;L of sterile Dulbecco\u0026apos;s phosphate-buffered saline and spread onto five different agar plate types (Blood, Columbia Blood [CB], De Man, Rogosa and Sharpe [MRS], Wilkins-Chalgren [WC], and multi-slice agars), to support the growth of a wide range of bacteria. The agar plates were incubated both aerobically and anaerobically for 24-48 h at 37\u0026deg;C. The aerobic plates were supplemented with either 5% or 10% CO\u003csub\u003e2\u003c/sub\u003e (MRS plate). Well-isolated colonies of different morphologies were selected and streaked onto fresh agar and incubated for 24 h. These bacterial isolates were then cryopreserved for DNA extraction and future cultivation by transferring a loopful of each isolate into 100 \u0026micro;L of TE buffer and 1 mL of BHI with 20% glycerol, respectively, and then stored at -80\u0026deg;C. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e \u003c/em\u003eA subset of the preserved bacterial isolates (n = 351) was identified by near full-length 16S rRNA gene sequencing. Isolates for taxonomic identification were randomly selected to include all agar types and culturing conditions. Genomic DNA from these selected isolates was extracted using the Zymo Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer\u0026rsquo;s instructions with minor modifications as described previously (Webb et al., 2023). The universal bacterial primers 27F (5\u0026prime; - \u003c/p\u003e\n\u003cp\u003eAGAGTTTGATCMTGGCTCAG - 3\u0026prime;) (Sunna and Bergquist, 2003) and 1492R (5\u0026prime; -\u003c/p\u003e\n\u003cp\u003eTACGGYTACCTTGTTACGACTT - 3\u0026prime;) (Reysenbach et al., 1992) were used to amplify the near full-length 16S rRNA gene. Each PCR contained 20 \u0026micro;L of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 12.8 \u0026micro;L of sterile water, 2 \u0026micro;L of each primer (10 \u0026micro;M) (IDT Inc., Coralville, IA, USA), 2 \u0026micro;L of isolate DNA, and 1.2 \u0026micro;L of DMSO (Thermo Fisher Scientific) in a total reaction volume of 40 \u0026micro;L. An Eppendorf Mastercycler thermocycler (Eppendorf, Hamburg, Germany) was then used with the following cycle conditions: initial denaturation at 95\u0026deg;C for 5 min; 35 cycles at 95\u0026deg;C for 45 s, 50\u0026deg;C for 30 s, 72\u0026deg;C for 2 min; and a final extension at 72\u0026deg;C for 5 min. The PCR products were visualized on a 1% (w/v) agarose gel and amplicons were sent to MCLAB (San Francisco, CA, USA) for Sanger sequencing. The resulting 16S rRNA gene sequences were identified using the Basic Local Alignment Search Tool (BLAST) and the non-redundant NCBI nucleotide database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetagenomic DNA extraction from ocular swabs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA subset of 49 ocular swabs was subjected to shotgun metagenomic sequencing. For this, metagenomic DNA was extracted from the ocular swab samples using a modified cetyltrimethylammonium bromide buffer (CTAB) and phenol:chloroform-based method as described previously (Fujimura et al., 2016; Rackaityte et al., 2020; Amat et al., 2021). Briefly, samples were thawed on ice, vigorously vortexed, and the liquid portion was transferred into individual sterile 2 mL screw-cap tubes. Samples were then pelleted via centrifugation at 20,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at 22\u0026deg;C. The supernatant was carefully removed, and the associated swab tip was snipped into the screw-cap tube containing the pellet. The beads from a Lysing Matrix E tube (MP Biomedicals, Irvine, CA, USA) were added to the sample tubes and 500 \u0026micro;L of CTAB buffer (10% CTAB, 0.7M NaCl, 240 mM potassium phosphate buffer pH 8.0), prewarmed to 60\u0026deg;C, was transferred to the sample tube and vortexed. Samples were then incubated for 20 min at 65\u0026deg;C with agitation at 800 rpm, followed by further lysis using bead beating at 5.5 m/s for 30 s. Next, 500 \u0026micro;L of room temperature phenol:chloroform:isoamyl (25:24:1) was added and mixed by inversion. \u003c/p\u003e\n\u003cp\u003eThe samples were centrifuged at 14,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min, and the supernatant was transferred to a heavy phase-lock gel tube (Quantabio, Beverly, MA, USA). Chloroform was added to the tube at a 1:1 ratio, inverted to mix, and centrifuged at 14,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min. The aqueous layer was transferred to a sterile 1.5 mL tube with 1 \u0026micro;L of linear acrylamide solution and vortexed. Two volumes of PEG-NaCl solution were then added, the tube was vortexed again, and then incubated for 2 h at 21\u0026deg;C. After incubation, the samples were centrifuged at 14,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 30 min, the supernatant was carefully removed, and the resulting DNA pellet washed with 400 \u0026micro;L of 70% ethanol and centrifuged at 14,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min. This wash step was repeated twice. After the final centrifugation, the ethanol was removed and the pellet air dried for approximately 10 min. The DNA pellet was resuspended in 30 \u0026micro;L of 10 mM Tris-Cl (pH 8.0) and quantified using a NanoDrop One\u003csup\u003eC\u003c/sup\u003e Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific) followed by further quantification using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific). DNA was then stored at -20\u0026deg;C until shotgun metagenomic library preparation. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShotgun metagenomic sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetagenomic DNA libraries were constructed according to the following workflow. DNA was fragmented with a Covaris (E220) instrument (Covaris, Inc., Woburn, Massachusetts, USA) and the quality was assessed using an Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA). End-repair and dA-tailing were then carried out, followed by magnetic bead purification. Next, the fragments were PCR-amplified using KAPA HiFi HotStart DNA Polymerase, and the products were purified. The libraries were assessed for quality prior to DNA circularization. After circularization, the libraries were further amplified to create DNA nanoballs (DNB). Finally, the libraries were sequenced on a DNA-based nanoball technology DNBSEQ on a DNBSEQ-G50 platform (MGI Tech, Shenzhen, China) with a PE150 flow cell and 2 \u0026times; 150 bp sequencing length. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShotgun metagenomic data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequencing adapters were removed from the raw sequences, and low-quality reads were filtered out using a 4-bp sliding window and a quality threshold of 15 in fastp v.0.23.4 (Chen, et al. 2018a). Reads that were shorter than 100 bp were also removed. Bowtie2 v. 2.5.3 (Langmead and Salzberg, 2012) was used to align the reads to the \u003cem\u003eBos taurus\u003c/em\u003e genome (ARS-UCD2.0) and the \u003cem\u003eEscherichia\u003c/em\u003e phage phiX174 genome (NC_001422). SAMtools v.1.19.2 (Danecek et al., 2021) and BEDtools v.2.31.1 (Quinlan and Hall, 2010) were then used to obtain the reads that did not map to these genomes. Kraken2 v. 2.1.3 (Wood et al., 2019) and Bracken v. 2.9 (Lu et al., 2017), together with the Genome Taxonomy Database (GTDB) release 220 (Parks et al., 2022), were used to assign taxonomy to the metagenomic reads. The unassembled metagenomic reads were also screened for antimicrobial resistance genes (ARGs) using the resistance gene identifier (RGI) v. 6.0.3 and the Comprehensive Antibiotic Resistance Database (CARD) v.3.2.9 (23) with KMA (Clausen et al., 2018). \u003c/p\u003e\n\u003cp\u003eReads from each sample were assembled individually and reads from IBK-affected and healthy cattle samples were co-assembled separately using MEGAHIT v. 1.2.9 (Li et al., 2015). The reads from each sample were then aligned to the assembled contigs using Bowtie2 and the contigs were then binned into metagenome-assembled genomes (MAGs) with MetaBAT2 v. 2.15 (Kang et al., 2019). The completeness and contamination of these MAGs were evaluated with CheckM2 v. 1.0.1 (Chklovski, et al. 2023) and MAGs with a completeness of at least 90% and a contamination level of less than 5% were retained. These MAGs were then dereplicated using dRep v. 3.4.5 (Olm et al., 2017) with primary clustering set at 90% average nucleotide identity (ANI) and secondary clustering set at 99% ANI. Taxonomy was then assigned to the dereplicated MAGs using GTDB-tk v. 2.4.0 (Chaumeil, et al. 2022). The MAGs were also screened for ARGs using the RGI and CARD. The relative abundance of the MAGs in the metagenomes was estimated using CoverM v. 0.7.0 (github.com/wwood/CoverM). The functional gene content was also characterized by aligning the metagenomic reads to the Kyoto Encyclopedia of Genes and Genomes (KEGG) release 112.0 (Kanehisa et al., 2025) prokaryotic protein database with DIAMOND v. 2.1.8.162 (Buchfink et al., 2021) and matching the genes to KEGG orthology (KO) groups. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction from ocular commensal and \u003cem\u003eMoraxella \u003c/em\u003espp. isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSelected \u003cem\u003eMoraxella bovis\u003c/em\u003e (n = 5) and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e (n = 13) isolates as well as potential candidate bacterial isolates (n = 13) for inhibition of the growth of \u003cem\u003eMoraxella\u003c/em\u003e spp. were subjected to whole-genome sequencing. For this, the isolates were re-grown from BHI-glycerol stocks on blood or MRS agar and passaged twice before a single colony was transferred to 5 mL of BHI or MRS broth and incubated for 24 h at 37\u0026deg;C with 5 or 10% CO\u003csub\u003e2\u003c/sub\u003e.\u003csub\u003e \u003c/sub\u003eGenomic DNA was extracted from the isolates using the DNeasy Blood and Tissue Kit (Qiagen Inc.). Briefly, 1 mL of 24 h isolate culture was centrifuged at max speed (20,817 \u0026times; \u003cem\u003eg\u003c/em\u003e) for 5 min at 21\u0026deg;C to pellet the bacterial cells. To increase the mass of the pellet recovered, this step was repeated up to three times. To the cell pellet, 180 \u0026micro;L of enzymatic lysis buffer (20 mM Tris-HCl, pH 8, 2 mM sodium EDTA, 1.2% Triton X-100, lysozyme [100 mg/mL], and mutanolysin [25,000 U/mL]) was added and thoroughly vortexed. Samples were incubated for 1 h at 37 \u0026deg;C with agitation at 800 rpm. This incubation was followed by the addition of 25 \u0026micro;L of proteinase K and 200 \u0026micro;L of buffer AL (without added ethanol), thorough vortexing of samples, and a 30 min incubation at 56\u0026deg;C with agitation at 800 rpm. After the second incubation, samples were mechanically lysed by adding approximately 400 mg of sterile 0.1 mm zircon/silica beads to each tube and bead beating at 6.0 m/s for 40 s using a FastPrep-24 Classic bead beater (MP Biomedicals). Samples were then centrifuged at 13,000 \u0026times; \u003cem\u003eg \u003c/em\u003efor 5 min, and the supernatant was transferred to a new sterile 1.5 mL tube. The remaining steps were followed according to the manufacturer\u0026rsquo;s instructions. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole-genome sequencing and analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-genome sequence libraries for the ocular commensal and \u003cem\u003eMoraxella\u003c/em\u003e spp. isolates were prepared and sequenced as we described in our previous publication (Magossi et al., 2025). Whole-genome sequences from bacterial isolates were quality-filtered using SOAPnuke v. 1.5.6 (Chen, et al. 2018b), with adapters and sequences shorter than 150 bp or with a quality threshold of less than 20 removed. The reads were assembled using SPAdes v. 3.15.5 with the \u0026ldquo;isolate\u0026rdquo; option and the assembly quality was evaluated using QUAST v. 5.2.0 (Gurevich, et al. 2013). The assemblies were then taxonomically classified using the GTDB-tk v. 2.4.0 and GTDB release 220. The vast majority of the genomes had 500X or greater coverage, which may affect the quality of the assembly. Therefore, the reads were subsampled to 100X for each genome using seqtk v. 1.4-r122 (github.com/lh3/seqtk) and re-assembled with SPAdes. The completeness and contamination of each 100X assembly were then assessed with CheckM2 v. 1.0.1. A phylogenetic tree of the bacterial genomes as well as MAGs from the metagenomes was created using PhyloPhlAn v. 3.1.68 (Asnicar, et al. 2020) and visualized in iTol v. 6.9 (Letunic and Bork 2024). FastANI was used to determine which isolates might belong to the same strain (99.99% shared ANI). The genomes were also screened for ARGs using the RGI and CARD and mapped against the metagenomic reads with CoverM to assess their abundance in the ocular metagenomes. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative genomic analysis of \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven their association with IBK, the \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi \u003c/em\u003eMAGs and isolate genomes were further analyzed. Briefly, the \u003cem\u003eMoraxella\u003c/em\u003e spp. genomes and MAGs were annotated using Prokka v. 1.14.6 (Seemann, 2014) with default parameters and the proteins flag using the reference genomes in GenBank for \u003cem\u003eMoraxella bovis\u003c/em\u003e (GCA_025985205.1) and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e (GCA_000988745.3). After annotation, Panaroo v. 1.5.0 (Tonkin-Hill et al., 2020) was used to determine the core genome for each species using the strict stringency mode. The core genes (0.99 threshold) were aligned with MAFFT v. 7.526 (Katoh and Standley, 2013) and a phylogenetic tree created from the core genome alignment with RAxML-ng v.1.2.0 (Kozlov et al., 2019) for each species with bootstrapping with 1,000 replicates and the GTR+GAMMA substitution model. \u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eMoraxella\u003c/em\u003e spp. MAGs and genomes were also screened for known virulence genes with DIAMOND with a 90% identity threshold. These virulence genes included the RTX operon genes in \u003cem\u003eMoraxella bovis\u003c/em\u003e designated \u003cem\u003embxA\u003c/em\u003e (AAK84651.1), \u003cem\u003embxB\u003c/em\u003e (AAP74653.1), \u003cem\u003embxC\u003c/em\u003e (AAP74652.1), and \u003cem\u003embxD\u003c/em\u003e (AAP74654.1) as well as the \u003cem\u003eMoraxella\u003c/em\u003e \u003cem\u003ebovoculi\u003c/em\u003e RTX operon genes \u003cem\u003embvA\u003c/em\u003e (AKM27892.1), \u003cem\u003embvB\u003c/em\u003e (ABA42642.1), \u003cem\u003embvC\u003c/em\u003e (ABA42640.1), and \u003cem\u003embvD\u003c/em\u003e (ABA42643.1). Additional virulence genes assessed were \u003cem\u003eflpA\u003c/em\u003e (filamentous-haemagglutinin-like protein; BAD83731.1), \u003cem\u003eflpB\u003c/em\u003e (filamentous-haemagglutinin-like protein; BAD83735.1), \u003cem\u003efur\u003c/em\u003e (ferric uptake regulator; BAC75975.1; KDN24651.1), \u003cem\u003eomp79\u003c/em\u003e (outer membrane protein; BAC92729.1), \u003cem\u003epilA\u003c/em\u003e (type IV pilin; QPW18723.1; STY93141.1), \u003cem\u003eplb\u003c/em\u003e (phospholipase B; AAK53448.1), and \u003cem\u003etolC\u003c/em\u003e (outer membrane protein; AAP74655.2; ABA42644.1). Plasmids and MOB genes were identified in the genome assemblies using MOB-suite v. 3.1.9 (Robertson and Nash, 2018) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e antimicrobial activity of ocular commensal bacteria against \u003cem\u003eMoraxella\u003c/em\u003e spp. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOf the 351 isolates selected for identification, 52 were tested for growth inhibitory effects against \u003cem\u003eMoraxella bovis \u003c/em\u003eand \u003cem\u003eMoraxella bovoculi\u003c/em\u003e using the agar slab method, as previously described (Amat et al., 2019) (Fig.1B). Briefly, to visualize bacterial antimicrobial activity, a small agar slab (10 mm in diameter) containing the bacterial isolate of interest was placed on the surface of an agar plate with a fresh inoculum of \u003cem\u003eMoraxella bovis\u003c/em\u003e (8338-1) or \u003cem\u003eMoraxella bovoculi \u003c/em\u003e(42G CB-D strain). For this, 100 \u0026micro;L of a 24-h culture was spread as a lawn onto BHI or MRS agar plates and incubated for 24 h at 37\u0026deg;C with 5 or 10% CO\u003csub\u003e2\u003c/sub\u003e. From these 24-h bacterial lawns, a 10 mm sterilized cork borer was used to cut agar slabs. The agar slabs were placed surface-side down onto a fresh lawn of \u003cem\u003eMoraxella bovis\u003c/em\u003e or \u003cem\u003eMoraxella bovoculi \u003c/em\u003eon TSA plates supplemented with 5% sheep\u0026rsquo;s blood\u003cem\u003e. \u003c/em\u003eThe \u003cem\u003eMoraxella bovis \u003c/em\u003eand \u003cem\u003eMoraxella bovoculi \u003c/em\u003eplates were prepared by spreading 100 \u0026micro;L of \u003cem\u003eMoraxella bovis\u003c/em\u003e or \u003cem\u003eMoraxella bovoculi\u003c/em\u003e overnight culture onto the plates and drying for up to 30 min at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Up to four agar slabs were placed on the \u003cem\u003eMoraxella \u003c/em\u003elawns and a control agar slab that contained no bacteria was included in each screening. After co-incubation for 24 h at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, inhibition of \u003cem\u003eMoraxella bovis \u003c/em\u003eor \u003cem\u003eMoraxella bovoculi\u003c/em\u003e growth was observed using the zone of inhibition (ZOI) surrounding the agar plug containing the bacteria of interest. The diameter of the ZOI was measured using a digital caliper and reported as the mean diameter. \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e ATCC9338 was also included in the screening for comparison.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth-inhibitory effects of selected ocular commensal bacteria against \u003cem\u003eMoraxella bovoculi\u003c/em\u003e in the presence of the ocular microbiome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the agar slab experiment, a selection of candidate bacteria that exhibited relatively strong inhibition of \u003cem\u003eMoraxella\u003c/em\u003e growth were further evaluated for their ability to inhibit the growth of\u003cem\u003e Moraxella \u003c/em\u003espp.\u003cem\u003e \u003c/em\u003ein the presence of the culturable ocular microbiome. \u003cem\u003eMoraxella bovoculi\u003c/em\u003e was first co-incubated with selected commensal isolates in an enriched ocular microbiome culture. The enriched culture of the ocular microbiome was prepared by pooling 100 \u0026micro;L from five healthy ocular swabs cryopreserved in BHI and 20% glycerol and adding 2.5 mL of fresh BHI broth. This mixture was then incubated at 37\u0026deg;C for approximately 20 h with agitation at 200 rpm. \u003c/p\u003e\n\u003cp\u003eNext, 60 \u0026micro;L of this enriched ocular microbiome was combined with 60 \u0026micro;L of \u003cem\u003eMoraxella bovoculi\u003c/em\u003e overnight culture, 60 \u0026micro;L of overnight culture of the selected commensal bacterial isolate, and 2.82 mL of fresh BHI broth, for a total volume of 3 mL. Each candidate bacterial strain co-culture was run in quadruplicate. In addition, a cocktail containing all candidate strains was included. Controls included: 1) only the enriched ocular microbiome and BHI broth, 2) only \u003cem\u003eMoraxella bovoculi \u003c/em\u003eand BHI broth, and 3) only BHI broth. Prior to incubation, 200 \u0026micro;L of the prepared cultures were saved as baseline (0 h) samples. Cultures were incubated for 24 h at 37\u0026deg;C with agitation at 200 rpm. After incubation, 200 \u0026micro;L from each replicate was used for 24 h samples. DNA from both 0 h and 24 h samples were extracted using the same procedure described above for whole-genome sequencing.\u003c/p\u003e\n\u003cp\u003eQuantitative PCR (qPCR) was used to assess the concentration of \u003cem\u003eMoraxella \u003c/em\u003espp\u003cem\u003e. \u003c/em\u003ein each sample to observe whether the selected commensal strains could inhibit \u003cem\u003eMoraxella \u003c/em\u003egrowth in the presence of the bovine ocular microbiome. The primers ISRdown (5\u0026prime;-GTGAAGTCGTAACAAGGTAGCCGT-3\u0026prime;) and ISRup (5\u0026prime;-ACCGACGCTTATCGCAGGCTATCA-3\u0026prime;) (Loy and Brodersen, 2014) were used to amplify the 16S-23S rRNA intergenic spacer region (ISR). Each qPCR mixture contained 1 \u0026micro;L SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 1 \u0026micro;L of each primer (IDT Inc.), 6.8 \u0026micro;L of molecular biology grade water (Corning, Manassa, VA, USA), and 1.2 \u0026micro;L of DNA template, in a total volume of 20 \u0026micro;L. A CFX96 Touch Real-Time PCR Detection system (Bio-Rad Laboratories Ltd.) with the following conditions were used: an initial denaturation at 98\u0026deg;C for 3 min, followed by 39 cycles at 98\u0026deg;C for 15 s, 60 \u0026deg;C for 1 min, and then a melt curve analysis was performed starting at 65\u0026deg;C for 5 sec, with a 0.5 \u0026deg;C increase per cycle, ending at 95 \u0026deg;C. Standard curves (10\u003csup\u003e2 \u003c/sup\u003eto 10\u003csup\u003e9 \u003c/sup\u003egene copies) were produced using the pDrive cloning vector (Qiagen Inc.) containing the PCR product from the 16S-23S rRNA ISR (Angelos et al., 2007). Each standard was run in duplicate and a no-template control (1.2 \u0026micro;L of nuclease-free water) was included.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePermutational multivariate analysis of variance (PERMANOVA) of the Bray-Curtis dissimilarities based on the relative abundance values of archaeal and bacterial species as well as copies per million reads for KOs were calculated in vegan 2.6-4 to assess the effect of IBK on the ocular surface microbiome. The Mann-Whitney test was used to compare the inverse Simpson diversity index values and qPCR results between the IBK-affected and healthy cattle as the data were not normally distributed. Differentially abundant ARGs, microbial species, and KOs in the ocular microbiomes of IBK-affected vs. healthy cattle were identified using MaAsLin2 v. 1.16.0 (Mallick, et al. 2021) in R v. 4.3.2. Only those ARGs and KOs found in at least 25% of the samples, and microbial species with an overall relative abundance of \u0026ge; 0.1%, were included for the differential abundance analyses. \u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eSummary of the ocular swabs collected\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 143 ocular swabs were collected: 102 from cattle diagnosed with IBK and 41 from healthy cattle. The majority of the ocular swabs (77%) were collected during the summer, while additional samples were obtained from winter IBK outbreaks (Table 1). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe ocular microbiome assessed by large-scale culturing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough extensive anaerobic and anaerobic culturing using 5 different agar plates, 658 isolates were recovered (data not shown). The majority of these isolates (n = 417) were recovered under aerobic conditions. In addition, 58% of the isolates were obtained from culturing of IBK swabs, although this is expected given that a larger number of swabs were collected from IBK-affected cattle. Overall, the highest number of isolates were recovered from WC (anaerobic, n = 154), blood (aerobic, n = 138), and MRS (aerobic, n = 110) and) agar plates. Of the 658 bacterial isolates collected, 351 were selected for taxonomic identification via Sanger sequencing of the near full-length 16S rRNA gene. These isolates represented three bacterial phyla: Bacillota (82.2%), Pseudomonadota (13.5%), and Actinomycetota (5.0%) (Table 2). Among the 61 bacterial genera identified, isolates belonging to \u003cem\u003eBacillus \u003c/em\u003e(26%), \u003cem\u003eStreptococcus \u003c/em\u003e(11%), \u003cem\u003eStaphylococcus \u003c/em\u003e(11%), \u003cem\u003eMoraxella \u003c/em\u003e(9%), and\u003cem\u003e Macrococcus \u003c/em\u003e(4%) were most frequently recovered (Table 2). There were also 33 \u003cem\u003eMoraxella \u003c/em\u003eisolates were obtained, consisting of both \u003cem\u003eMoraxella bovis \u003c/em\u003eand \u003cem\u003eMoraxella bovoculi.\u003c/em\u003e \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetagenomic sequencing summary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ocular surface microbiome was also characterized using shotgun metagenomic sequencing.The vast majority of the metagenomic reads were derived from the host: 96.4 \u0026plusmn; 1.0% (SEM) for samples from healthy cattle and 92.9 \u0026plusmn; 1.9% for samples from IBK-affected cattle. After host DNA removal, the average number of reads for the healthy and IBK-affected cattle samples were 2,575,944 \u0026plusmn; 791,376 and 4,312,305 \u0026plusmn; 1,074,688, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOcular microbiome of IBK-affected vs. healthy cattle \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a significant (PERMANOVA: \u003cem\u003eP\u003c/em\u003e = 0.015), but relatively small difference (\u003cem\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/em\u003e = 0.05) in the ocular surface microbial community structure between healthy cattle and cattle diagnosed with IBK (Fig. 2A). Microbial species diversity on the ocular surface did not differ between healthy and IBK-affected cattle (Fig. 2B). Overall, the ocular surface microbiomes were dominated by bacterial species such as \u003cem\u003eCutibacterium acnes\u003c/em\u003e, \u003cem\u003eMannheimia pernigra\u003c/em\u003e, \u003cem\u003eMesomycoplasma bovoculi\u003c/em\u003e, \u003cem\u003eMoraxella bovis\u003c/em\u003e, and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e(Fig. 3). Nine bacterial species were differentially abundant in the ocular microbiomes of healthy vs. IBK-affected cattle (Fig. 4). All but one of these species (\u003cem\u003eArthrobacter luteus\u003c/em\u003e), were relatively more abundant in the healthy cattle and included \u003cem\u003eBacillus licheniformis\u003c/em\u003e, \u003cem\u003eBifidobacterium globosum\u003c/em\u003e, \u003cem\u003eBlastomonas ursincola\u003c/em\u003e, \u003cem\u003eC. acnes\u003c/em\u003e, \u003cem\u003eLawsonella clevelandensis\u003c/em\u003e, \u003cem\u003ePsychrobacter maritimus\u003c/em\u003e, \u003cem\u003eRuminococcus\u003c/em\u003e sp900100595, and \u003cem\u003eRuminococcus\u003c/em\u003e sp900316555.\u003c/p\u003e\n\u003cp\u003eThe functional content of the ocular surface microbiome was assessed using KOs. In total, 158 KOs (prokaryotic-associated) were identified in at least one ocular sample with ko01100 (sedoheptulose-bisphosphatase), ko01240 (uridine nucleosidase), and ko01230 (mannosyl-oligosaccharide alpha-1,2-mannosidase) most abundant (data not shown). The overall functional composition did not between the IBK-affected and healthy cattle (Fig. S1; PERMANOVA: \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). Similarly, no KOs were differentially abundant in the ocular microbiomes of IBK-affected vs. healthy cattle (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). Furthermore,none of the ARGs identified in the ocular metagenomes were differentially abundant between the two groups of cattle. Overall, ARGs conferring resistance to aminoglycosides [\u003cem\u003eaph(3\u0026apos;)-Ia\u003c/em\u003e, \u003cem\u003eaph (3\u0026apos;\u0026apos;)-Ib\u003c/em\u003e], macrolides [\u003cem\u003emel\u003c/em\u003e, \u003cem\u003emph\u003c/em\u003e(E), \u003cem\u003emsr\u003c/em\u003e(E)], sulfonamides (\u003cem\u003esul2\u003c/em\u003e), tetracyclines [\u003cem\u003etet\u003c/em\u003e(H), \u003cem\u003etet\u003c/em\u003e(L),\u003cem\u003e tet\u003c/em\u003e(O),\u003cem\u003e tet\u003c/em\u003e(Q), and \u003cem\u003etet\u003c/em\u003e(W)] were most abundant, although there was a lot of variation between samples (Fig. 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetagenome-assembled genomes (MAGs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere were also 37 dereplicated high-quality MAGs recovered from the ocular metagenomes, 12 of which appeared to represent novel species (Fig. 6, Supplementary Table S1). The most frequently recovered MAGs (n = 10) were identified as \u003cem\u003eMesomycoplasma bovoculi\u003c/em\u003e. Other identified species included \u003cem\u003eMoraxella bovis\u003c/em\u003e, \u003cem\u003eMoraxella bovoculi\u003c/em\u003e, \u003cem\u003eLuteimonas excrementigallinarum\u003c/em\u003e, \u003cem\u003eM. pernigra\u003c/em\u003e, \u003cem\u003eC. acnes\u003c/em\u003e, \u003cem\u003eMycoplasma bovis\u003c/em\u003e, \u003cem\u003eLentibacillus daqui\u003c/em\u003e, \u003cem\u003eDesemzia incerta\u003c/em\u003e, \u003cem\u003eLactococcus lactis,\u003c/em\u003e and \u003cem\u003eStreptococcus ruminantium\u003c/em\u003e. The MAGs were also screened for ARGs with 13 MAGs found to be carrying at least one ARG, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eBRO-1 \u003c/sub\u003ein both \u003cem\u003eMoraxella bovis\u003c/em\u003e MAGs and in \u003cem\u003eMoraxella bovoculi\u003c/em\u003e OUG26 (Supplementary Table S1). Other ARGs of note that were identified included \u003cem\u003efloR\u003c/em\u003e (phenicols) in \u003cem\u003eMoraxella bovoculi\u003c/em\u003e OUG36 and \u003cem\u003eant(9)-Ia \u003c/em\u003e(aminoglycosides), \u003cem\u003ebla\u003c/em\u003eIII (beta-lactams), \u003cem\u003eerm\u003c/em\u003e(A) (macrolides), f\u003cem\u003eexA \u003c/em\u003e(phenicols), \u003cem\u003emsr\u003c/em\u003e(I) (macrolides), \u003cem\u003evanG \u003c/em\u003e(vancomycin)\u003cem\u003e \u003c/em\u003ein \u003cem\u003eL. daqui \u003c/em\u003eOUG33\u003cem\u003e.\u003c/em\u003e Overall, the relatively most abundant MAG in the ocular surface metagenomes was MAG OUG1 which was taxonomically identified only at the order level (Cardiobacteriales). This MAG was also the only one with a significantly different relative abundance between the two groups of cattle (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), although there was considerable inter-individual variability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolate-targeted whole-genome sequencing \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genomes of 31 bacterial isolates were sequenced, including 5 \u003cem\u003eMoraxella bovis\u003c/em\u003e and 13 \u003cem\u003eMoraxella bovoculi\u003c/em\u003e genomes (Supplementary Table S1). The remaining 13 genomes were ocular bacterial isolates that showed inhibition against \u003cem\u003eMoraxella\u003c/em\u003e, and included \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003elicheniformis \u003c/em\u003e(n =2), \u003cem\u003eW. coagulans \u003c/em\u003e(n =2), \u003cem\u003eWeissella paramesenteroides \u003c/em\u003e(n =2), \u003cem\u003eB.\u003c/em\u003e \u003cem\u003epumilus\u003c/em\u003e, \u003cem\u003eBacillus safensis\u003c/em\u003e, \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eLevilactobacillus brevis\u003c/em\u003e, \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e, \u003cem\u003eStreptococcus pluranimalium\u003c/em\u003e, and \u003cem\u003eL. buchneri. \u003c/em\u003eAll 5 \u003cem\u003eMoraxella bovis\u003c/em\u003e isolates appeared to represent unique strains based on their ANI (\u0026lt; 99.99%) (Rodriguez-R, et al. 2024) while there were 10 \u003cem\u003eMoraxella bovoculi\u003c/em\u003e strains based on this criterion. The \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003elicheniformis\u003c/em\u003e, \u003cem\u003eW. coagulans\u003c/em\u003e, and \u003cem\u003eW. paramesenteroides \u003c/em\u003eisolates also appeared to be unique strains. \u003c/p\u003e\n\u003cp\u003eThese 31 isolate genomes were also screened for the presence of ARGs. Four of the \u003cem\u003eMoraxella bovis\u003c/em\u003e and two \u003cem\u003eMoraxella bovoculi\u003c/em\u003e isolates carried the beta-lactamase gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eBRO-1\u003c/sub\u003e, which was originally described in \u003cem\u003eMoraxella catarrhalis \u003c/em\u003e(Bootsma, et al. 1996). The tetracycline efflux pump gene \u003cem\u003etet\u003c/em\u003e(45) was found in two \u003cem\u003eMoraxella bovoculi \u003c/em\u003eisolates (SL21, SL24) belonging to the same strain, as well as the \u003cem\u003eB\u003c/em\u003e. \u003cem\u003esubtilis\u003c/em\u003e SL29 isolate. Several other ARGs associated with \u003cem\u003eBacillus\u003c/em\u003e spp. were identified including the chloramphenicol acetyltransferase gene \u003cem\u003ecat86\u003c/em\u003e in \u003cem\u003eB\u003c/em\u003e. \u003cem\u003epumilus\u003c/em\u003e and \u003cem\u003eB\u003c/em\u003e. \u003cem\u003esafensis\u003c/em\u003e, the class D beta-lactamase \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eBPU-1 \u003c/sub\u003ein\u003csub\u003e \u003c/sub\u003e\u003cem\u003eB\u003c/em\u003e. \u003cem\u003epumilus, \u003c/em\u003eand\u003cem\u003e fosBx1, \u003c/em\u003ea thiol transferase gene conferring resistance to fosfomycin, in \u003cem\u003eB\u003c/em\u003e. \u003cem\u003elicheniformis \u003c/em\u003eand \u003cem\u003eB\u003c/em\u003e. \u003cem\u003esubtilis\u003c/em\u003e. When the metagenomic reads were mapped to the isolate genomes, the \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e genomes were most abundant, while many of the non-\u003cem\u003eMoraxella\u003c/em\u003e spp. genomes were not detected, likely due to the sequencing depth (Supplementary Table S2). Similarly, none of the non-\u003cem\u003eMoraxella\u003c/em\u003e spp. isolated were assembled and binned into high-quality MAGs from the metagenomes.However, the \u003cem\u003eMoraxella bovis\u003c/em\u003e OUG21 MAG shared an ANI of 99.94% with \u003cem\u003eMoraxella bovis\u003c/em\u003e isolates SL11 and SL18 and \u003cem\u003eMoraxella bovoculi \u003c/em\u003eMAG\u003cem\u003e \u003c/em\u003eOUG26 and isolate SL1 had an ANI of 99.97%. Plasmids were reconstructed from several isolates including \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e; however, none of the ARGs detected were found to be carried on one of these plasmids.\u003c/p\u003e\n\u003cp\u003eGiven their association with IBK, the core genomes of the isolates and MAGs classified as \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi \u003c/em\u003ewere identified and used to create phylogenetic trees (Fig. 7). The core genome of the \u003cem\u003eMoraxella bovis\u003c/em\u003e isolates (n = 5) and MAGs (n = 2) consisted of 1,887 genes and there were 1,484 genes in the core genome of the \u003cem\u003eMoraxella bovoculi \u003c/em\u003eisolates (n = 13) and MAGs (n = 2). These genomes were also screened for the presence of 15 known virulence genes. In \u003cem\u003eMoraxella bovis\u003c/em\u003e, \u003cem\u003efur\u003c/em\u003e, \u003cem\u003eomp79\u003c/em\u003e, \u003cem\u003epilA\u003c/em\u003e, and \u003cem\u003eplb\u003c/em\u003e were found in all genomes whereas only three isolates (SL7, SL8, and SL12) carried all four RTX-operon genes (\u003cem\u003embxCABD\u003c/em\u003e) as well as \u003cem\u003etolC \u003c/em\u003e(Fig. 7A). One of the large \u003cem\u003eflpA\u003c/em\u003e and \u003cem\u003eflpB\u003c/em\u003e genes, which are typically plasmid-encoded (Loy et al., 2021), were identified in \u003cem\u003eMoraxella bovis\u003c/em\u003e isolates SL11 (\u003cem\u003eflpA\u003c/em\u003e), SL18 (\u003cem\u003eflpA\u003c/em\u003e) and SL12 (f\u003cem\u003elpB\u003c/em\u003e). In agreement with their ANI, isolates SL11 and SL18, along with MAG OUG21, were most closely related based on the phylogenetic tree. \u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003efur\u003c/em\u003e and \u003cem\u003epilA\u003c/em\u003e genes were also found in all \u003cem\u003eMoraxella bovoculi\u003c/em\u003e isolate genomes and MAGs (Fig. 7B). The \u003cem\u003eMoraxella bovoculi\u003c/em\u003e RTX-operon genes (\u003cem\u003embvCABD\u003c/em\u003e) were identified in five isolate genomes (SL1, SL14, SL21, SL23, and SL24) and one MAG (OUG26), along with \u003cem\u003etolC\u003c/em\u003e. Consistent with their ANI values, isolates SL2, SL19, and SL20 appeared to belong to the same strain, as did SL21 and SL24. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e growth by ocular surface commensal bacteria as determined by the agar slab method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOf the 52 isolates tested for inhibition against \u003cem\u003eMoraxella \u003c/em\u003espp.\u003cem\u003e \u003c/em\u003eusing the agar slab method, 15 showed a ZOI against \u003cem\u003eMoraxella bovis\u003c/em\u003e, ranging from 12.9 mm to 36.7 mm (Fig. 8; Table 3). There were also 13 isolates that inhibited the growth of \u003cem\u003eMoraxella bovoculi\u003c/em\u003e with a ZOI that ranged from 13 mm to 22.2 mm (Table 4). \u003cem\u003eBacillus pumilus\u003c/em\u003e (SL25 and 122H MRS-F) and \u003cem\u003eBacillus velezensis \u003c/em\u003e(42G MRS-A Aero) exhibited the strongest inhibition against \u003cem\u003eMoraxella \u003c/em\u003espp., while \u003cem\u003eLactiplantibacillus pentosus \u003c/em\u003e(SL27), \u003cem\u003eBacillus tequilensis\u003c/em\u003e (SL29), and \u003cem\u003eWeizmannia coagulans\u003c/em\u003e (SL35 and SL34) showed moderate inhibition. \u003cem\u003eLentilactobacillus buchneri \u003c/em\u003e(SL36) displayed only weak inhibition. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth-inhibitory effects against \u003cem\u003eMoraxella bovoculi\u003c/em\u003e in the presence of the culturable ocular microbiome as determined by qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the potential of the candidate commensal strains to prevent the proliferation of \u003cem\u003eMoraxella\u003c/em\u003e spp. growth within a microbial community, an experiment was designed to challenge the growth of \u003cem\u003eMoraxella bovoculi\u003c/em\u003e in the presence of selected bacterial strains and the ocular microbiome. The growth of \u003cem\u003eMoraxella\u003c/em\u003e over a 24-h period was measured using qPCR, indicating that the selected bacterial strains inhibited the growth of \u003cem\u003eMoraxella bovoculi\u003c/em\u003e within an ocular microbial community (Fig. 9). Among these isolates, \u003cem\u003eB. velezensis\u003c/em\u003e (42G MRS-A Aero) was most effective, reducing the concentration of \u003cem\u003eMoraxella bovoculi\u003c/em\u003e by 99.97% (Fig. 9). \u003cem\u003eB. pumilus \u003c/em\u003e(SL25) reduced \u003cem\u003eMoraxella bovoculi\u003c/em\u003e concentration by 99.95%, \u003cem\u003eW. coagulans \u003c/em\u003e(SL34) and \u003cem\u003eL. pentosus \u003c/em\u003e(SL27) by 99.90%, and \u003cem\u003eL. buchneri \u003c/em\u003e(SL36) by 99.86%. Additionally, a cocktail containing each of the aforementioned isolates lowered the concentration of \u003cem\u003eMoraxella\u003c/em\u003e by 99.59% after 24 h of co-incubation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe global cattle industry faces multifaceted challenges, including the growing issue of antimicrobial resistance in bovine bacterial pathogens. Antimicrobial resistance reduces the efficacy of antimicrobials routinely used to control infectious diseases in cattle, posing a significant threat to animal health and production and thereby emphasizing the need for alternative approaches. Advancements in high-throughput sequencing have improved our understanding of the taxonomic and functional features of the commensal microbiome across different cattle body sites. As a result, the microbiome has become a key target for preventing and mitigating infectious diseases, including those caused by antimicrobial-resistant bacteria (Wong and Santiago, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Relman and Lipsitch, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To leverage the ocular microbiome for improved resistance against IBK in cattle, the first logical step is to characterize and identify its taxonomic and functional signatures in both healthy and IBK-affected cattle using culture and metagenomic sequencing methods. Next, commensal isolates from the eyes of healthy cattle should be screened for their ability to inhibit the growth of IBK-associated \u003cem\u003eMoraxella\u003c/em\u003e spp. Synthetic ocular bacterial communities composed of beneficial commensal bacteria could then be developed to modulate the ocular microbiome and enhance resistance to colonization by IBK-associated pathogens.\u003c/p\u003e \u003cp\u003e Accordingly, in the present study, we isolated and identified a relatively large number of bacterial isolates (n\u0026thinsp;=\u0026thinsp;351) from the ocular surface of IBK-affected and healthy beef cattle using various growth media and conditions. As such, this is the first study to characterize the culturable fraction of the bovine ocular microbiome in cattle using both aerobic and anaerobic culturing with multiple growth media. An earlier study by Gafen et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) isolated bacteria from conjunctival swabs (n\u0026thinsp;=\u0026thinsp;387) obtained from 228 IBK-affected and 159 healthy eyes in cattle (dairy and beef) using 5% sheep blood agar and incubation with 10% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C for 48 h (Gafen et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In that study, \u003cem\u003eMoraxella bovis, Moraxella bovoculi, Moraxella osloensis, Trueperella pyogenes, Bacillus\u003c/em\u003e spp., and \u003cem\u003eProteus\u003c/em\u003e spp. were isolated at varying prevalences. Overall, the culturing results from Gafen and colleagues (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and our present study suggest that the bovine ocular surface harbors a relatively diverse community of culturable bacteria.\u003c/p\u003e \u003cp\u003eThis is further supported by culture-independent high-throughput sequencing studies. For example, a recent longitudinal study (139 days) using 16S rRNA gene sequencing of the ocular surface microbiota of 227 pre-weaned beef calves revealed the presence of a relatively rich and diverse bacterial microbiota (Bartenslager et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bacterial species within the \u003cem\u003eMoraxella\u003c/em\u003e, \u003cem\u003eMycoplasma, Pasteurella\u003c/em\u003e, and \u003cem\u003eAcinetobacter\u003c/em\u003e genera were noted to be the most relatively abundant (Bartenslager et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A similar taxonomic composition of the ocular microbiota has been reported in mature and older dairy and beef cattle. Using 16S rRNA gene (V4 region) sequencing, Gafen and colleagues characterized the conjunctiva of healthy (n\u0026thinsp;=\u0026thinsp;376) and IBK-affected cattle (n\u0026thinsp;=\u0026thinsp;228) across different states and farms (Gafen et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The dominant bacterial genera on the eyes of these mature adult cattle were similar to those reported in the pre-weaned calves.\u003c/p\u003e \u003cp\u003eIn addition, we previously characterized the ocular microbiota of neonatal beef calves and found that the ocular surface was already colonized at this age by a complex bacterial community dominated by \u003cem\u003eStreptococcus\u003c/em\u003e (57.6%) (Luecke et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, results from culturing and 16S rRNA gene sequencing-based studies provide strong evidence that the bovine ocular surface is colonized by a self-sustaining, diverse, and dynamic bacterial microbiota, with \u003cem\u003eMoraxella\u003c/em\u003e and \u003cem\u003eMycoplasma\u003c/em\u003e spp. being relatively abundant. The significant differences in the dominant bacterial genera identified by culturing and 16S rRNA sequencing highlight the necessity of using both culture-dependent (e.g. culturomics) and culture-independent methods for a comprehensive characterization of the ocular microbiome in cattle.\u003c/p\u003e \u003cp\u003eAlthough the sequencing-based studies discussed above, along with our extensive culturing, provided valuable insights into the taxonomic composition, diversity, and dynamics of the bovine ocular microbiota, amplicon-based sequencing lacks taxonomic resolution at the species level and beyond, and does not include functional information. Therefore, in the present study, we used shotgun metagenomic sequencing to characterize the ocular surface microbiome of IBK-affected and healthy cattle. A significant but relatively small difference was found in the microbial species composition of the two groups. This appeared to be driven by differences in the relative abundance of nine bacterial species, eight of which were enriched in the ocular microbiomes of healthy cattle. These enriched bacterial species included \u003cem\u003eB. globosum\u003c/em\u003e, commonly isolated from the rumen of cattle (Kelly et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and historically used as a probiotic in pigs (Apgar et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), as well as \u003cem\u003eB. licheniformis\u003c/em\u003e, which has been used to improve feed digestion and milk yield in cattle (Qiao et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOther bacterial species that were more abundant in healthy cattle included potentially pathogenic species. For example, \u003cem\u003eLawsonella clevelandensis\u003c/em\u003e has been reported to be a rare cause of abdominal, breast, spinal, and liver abscesses (Favila Menezes et al., 2018; Ramesh et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nour et al., 2023), and was recently reported to an emerging cause of vascular graft infection and vascular infection in humans (Ramesh et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nour et al., 2023). This species has not previously been found in bovine-associated microbial communities. In addition to \u003cem\u003eL. clevelandensis\u003c/em\u003e (often isolated from fresh water), \u003cem\u003eC. acnes\u003c/em\u003e, a member of human skin microbiome and considered to be both a beneficial commensal and opportunistic pathogen (Mayslich et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) were also enriched in the healthy cattle.\u003c/p\u003e \u003cp\u003eTwo uncharacterized \u003cem\u003eRuminococcus\u003c/em\u003e spp. (\u003cem\u003eR\u003c/em\u003e. sp900100595 and \u003cem\u003eR\u003c/em\u003e. sp900316555) were also more abundant in the eyes of healthy cattle. Species within the \u003cem\u003eRuminococcus\u003c/em\u003e genus are largely host-associated and play a key role in the cattle ruminal microbiome, primarily in the breakdown of forages (La Reau and Suen, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The only species significantly greater in abundance in the ocular microbiome of IBK-affected cattle was \u003cem\u003eA. luteus\u003c/em\u003e. There is limited information regarding its colonization niche and pathogenicity; however, \u003cem\u003eA. luteus\u003c/em\u003e has been reported to produce an endonuclease (AZuI), restriction enzyme that can attach and destroy viral DNA (Roberts et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). The relationship between \u003cem\u003eA. luteus\u003c/em\u003e and IBK pathogenesis therefore warrants further research. Surprisingly, the relative abundance of \u003cem\u003eMoraxella bovis\u003c/em\u003e or \u003cem\u003eMoraxella bovoculi\u003c/em\u003e was not significantly greater in IBK affected cattle as compared to healthy cattle. This suggests that the presence of these species alone is not sufficient to cause IBK or that there is inter-species diversity in terms of pathogenicity.\u003c/p\u003e \u003cp\u003eThe ocular resistome (all ARGs) did not differ between the IBK-affected and healthy cattle, although there was considerable inter-animal variability. Genes conferring resistance to the tetracyclines, macrolides, sulfonamides and aminoglycosides were most abundant in the metagenomes. Not surprisingly, these classes represent antimicrobials that are frequently used to treat cattle for various infectious or conditions. For example, tetracyclines and macrolides (e.g., tulathromycin) are used to treat IBK in North America. Sulfonamides, although not used for ocular infections, are administered to treat diarrhea in nursing calves and respiratory disease in cattle (Fossen et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Aminoglycosides are given to cattle for the treatment of various infections and are used topically in the ears and eyes and via intrauterine infusion to treat endometritis and occasionally may be infused into the udder to treat mastitis (Mercer, 2022). Although the ocular swabs in the present study were collected before antimicrobial treatment, our results highlight the persistence of ARGs even in the absence of direct exposure and could partially explain why certain antimicrobials (e.g., tetracycline and tulathromycin) are less effective against IBK.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eMoraxella bovis\u003c/em\u003e isolate genomes (n\u0026thinsp;=\u0026thinsp;5) and MAGs (n\u0026thinsp;=\u0026thinsp;2) recovered from the ocular metagenome appeared to represent unique strains based their ANI and phylogeny, although strains SL11 and SL18 from healthy animals and MAG OUG21 were closely related. Using this same criterion, there were at least 10 strains among the \u003cem\u003eMoraxella bovoculi\u003c/em\u003e isolates. This demonstrates the strain diversity of \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e on the bovine ocular surface. As such, this diversity presents a significant challenge in developing effective vaccines for IBK. The presence of different virulence factors within \u003cem\u003eMoraxella\u003c/em\u003e spp. strains may help explain their association with IBK-affected or healthy cattle. Certain virulence genes, such as \u003cem\u003efur\u003c/em\u003e, \u003cem\u003epilA\u003c/em\u003e, \u003cem\u003eplb\u003c/em\u003e, and \u003cem\u003etolC\u003c/em\u003e, were shared among all \u003cem\u003eMoraxella bovis\u003c/em\u003e genomes. However, the presence of others including the \u003cem\u003embxCABD\u003c/em\u003e, \u003cem\u003eflpA\u003c/em\u003e and \u003cem\u003eflpB\u003c/em\u003e genes varied among the five isolates and two MAGs. The \u003cem\u003eflpA\u003c/em\u003e and \u003cem\u003eflpB\u003c/em\u003e genes encode filamentous-haemagglutinin-like proteins that may facilitate adhesion to host cells and are typically located on a plasmid in \u003cem\u003eMoraxella bovis\u003c/em\u003e (Kakuda et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). While all \u003cem\u003eMoraxella bovoculi\u003c/em\u003e genomes carried \u003cem\u003efur\u003c/em\u003e and \u003cem\u003epilA\u003c/em\u003e, only six carried the \u003cem\u003embvCABD\u003c/em\u003e genes. The ubiquity of \u003cem\u003epilA\u003c/em\u003e and \u003cem\u003etolC\u003c/em\u003e in \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003epilA\u003c/em\u003e among \u003cem\u003eMoraxella bovoculi\u003c/em\u003e genomes is in agreement with previous work (Wynn et al., 2022). Although the hemolysin-encoding RTX operon genes, \u003cem\u003embxCABD\u003c/em\u003e (in \u003cem\u003eMoraxella bovis\u003c/em\u003e) and \u003cem\u003embvCABD\u003c/em\u003e (in \u003cem\u003eMoraxella bovoculi\u003c/em\u003e), are associated with pathogenesis (Loy and Brodersen, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Loy et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), they were detected in \u003cem\u003eMoraxella bovis\u003c/em\u003e isolates from both healthy and IBK-affected cattle.\u003c/p\u003e \u003cp\u003eNotably, 27% of the MAGs recovered from the ocular metagenomes were identified as \u003cem\u003eMesomycoplasma bovoculi\u003c/em\u003e, suggesting that this species is predominant in the cattle ocular microbial community. At the same time, the \u003cem\u003eMesomycoplasma bovoculi\u003c/em\u003e genome is relatively small (\u0026asymp;\u0026thinsp;700 Kbp) and thus less metagenomic sequencing depth is required to assemble. Although, we did not isolate \u003cem\u003eMesomycoplasma bovoculi\u003c/em\u003e or any related species within the \u003cem\u003eMesomycoplasma or Mycoplasma\u003c/em\u003e genera, this is likely due to the difficulty in culturing these species \u003cem\u003ein vitro\u003c/em\u003e (Pitt et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eMesomycoplasma bovoculi\u003c/em\u003e (formerly \u003cem\u003eMycoplasma bovoculi\u003c/em\u003e) has been reported to predispose cattle to IBK infection (Levisohn et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Schnee et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, characterizing the role of \u003cem\u003eMesomycoplasma bovoculi\u003c/em\u003e in ocular microbiome homeostasis, its interactions with \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e, and its role in IBK development deserve further research attention.\u003c/p\u003e \u003cp\u003eHuman studies suggest that the ocular surface microbiome is associated with ocular health (Matysiak et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mohamed et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and that a healthy ocular microbiome may provide resistance against colonization with infectious agents (Teweldemedhin et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chiang and Chern, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, we identified 15 bacterial isolates that inhibited the growth of \u003cem\u003eMoraxella bovis\u003c/em\u003e and 12 that inhibited \u003cem\u003eMoraxella bovoculi\u003c/em\u003e growth \u003cem\u003ein vitro\u003c/em\u003e. These isolates belonged to 10 different bacterial genera, including \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003ePaenibacillus, Lactiplantibacillus\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eWeissella\u003c/em\u003e, and \u003cem\u003eWeizmannia\u003c/em\u003e. \u003cem\u003eBacillus\u003c/em\u003e spp. produced the largest ZOI against \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e and members of this genus are commonly used as probiotics to inhibit pathogenic bacteria colonization through the production of antimicrobial compounds (e.g., lipopeptides and polyketides) or interference with signaling pathways (Piewngam et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fazle Rabbee and Baek, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDirect inhibition of pathogen growth by \u003cem\u003eL. pentosus\u003c/em\u003e (Behbahani et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e (Wang et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), L. \u003cem\u003ebuchneri\u003c/em\u003e (Rodr\u0026iacute;guez et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), L. \u003cem\u003ebrevis\u003c/em\u003e (Kwun et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)d \u003cem\u003eparamesenteroides\u003c/em\u003e (Wan et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) has also been reported. \u003cem\u003eWeizmannia coagulans\u003c/em\u003e is known for its broad-spectrum antimicrobial activity against foodborne pathogens (Kallur et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Maresca et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, the inhibition of \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e growth by these ocular bacterial species may be due to the production of antimicrobial substances such as lactic acid, bacteriocins, and hydrogen peroxide. Overall, these results suggest that the ocular surface environment is highly competitive with multiple bacteria capable of producing antimicrobial factors that can inhibit \u003cem\u003eMoraxella\u003c/em\u003e spp.\u003c/p\u003e \u003cp\u003eBacterial tolerance to antimicrobials can be affected by metabolic cross-feeding (Adamowicz et al., 2018; Aranda-D\u0026iacute;az et al., 2020). Consequently, the antimicrobial concentrations required to inhibit/kill target bacteria differ when they are grown in the presence of other bacteria rather than in monoculture, due to the interactions within the community (e.g., competition for nutrients, biofilm formation, and production of antimicrobial substances by other bacteria) (Aslam et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Since bacterial antimicrobial tolerance is influenced by the metabolic interdependence and interactions of different species within a microbial community, it is important to select bacterial strains that can inhibit the growth of IBK-associated pathogens in the presence of the bovine ocular microbiome. Here, we selected five isolates (\u003cem\u003eB. pumilus\u003c/em\u003e, \u003cem\u003eB. velezensis\u003c/em\u003e, \u003cem\u003eL. pentosus\u003c/em\u003e, \u003cem\u003eL. buchneri\u003c/em\u003e and \u003cem\u003eW. coagulans\u003c/em\u003e) that displayed relatively strong inhibition of \u003cem\u003eMoraxella bovis\u003c/em\u003e and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e. These isolates were then further evaluated for their ability to inhibit the growth of \u003cem\u003eMoraxella bovoculi\u003c/em\u003e in the presence of the bovine ocular microbiome. The growth of \u003cem\u003eMoraxella bovoculi\u003c/em\u003e co-cultured with the ocular microbiome was inhibited by all five strains, as well as a mixture of these strains. This suggests that the selected isolate strains have the potential to inhibit \u003cem\u003eMoraxella\u003c/em\u003e spp. on the ocular surface.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we characterized the ocular surface microbiome of IBK-affected and healthy cattle using culture-dependent and culture-independent methods. Eight bacterial species were relatively more abundant in the healthy cattle ocular microbiome based on metagenomic sequencing, including \u003cem\u003eB. globosum, B. licheniformis, Ruminococcus\u003c/em\u003e sp900316555, and \u003cem\u003eRuminococcus\u003c/em\u003e sp900100595. Using large-scale aerobic and anaerobic culturing, we identified 351 isolates representing 61 different genera. Of the 52 bacterial isolates screened for inhibition of \u003cem\u003eMoraxella bovoculi\u003c/em\u003e and \u003cem\u003eMoraxella bovis\u003c/em\u003e, 17 inhibited the growth of one of these species. Overall, our results provide deeper insight into the ocular microbiome in cattle. This knowledge is important for the development of antimicrobial alternative approaches for preventing and treating IBK in cattle.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the veterinarians and producers that assisted in sample collection. The authors are also appreciative of Kelli Maddock at the NDSU Veterinary Diagnostic Laboratory, and Garrett Havelka at the NDSU Beef Unit for assistance with sample collection. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceiving the idea, designing the study, and providing supervision: SA. Sample collection: SA, SML and GS. \u0026nbsp;Sample processing: SML and SA. Data processing, bioinformatics, and statistical analysis: DBH, KEG, AM, GS, SML, SA. Manuscript writing: SA. Manuscript review, editing, and finalizing: SA, DBH. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work in this study was funded by the North Dakota State Board of Agricultural Research and Education (Award numbers: 20-21-2022; 22-14-0231; 24-30-0265), and North Dakota Agricultural Experiment Station as part of a start-up package for S.A. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw sequence data and genome assemblies are available on NCBI under BioProject accession PRJNA1128155. Other data supporting the findings of this study are presented within the paper and in the supplementary information files. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAikman, J. G., E. M. Allan, and I. E. Selman. 1985. Experimental production of infectious bovine keratoconjunctivitis. Vet Rec 117(10):234-239. doi: 10.1136/vr.117.10.234\u003c/li\u003e\n \u003cli\u003eAmat, S., D. B. Holman, K. Schmidt, K. L. McCarthy, S. T. 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Eur J Biochem 61(1):119-138. doi: 10.1111/j.1432-1033.1976.tb10003.x\u003c/li\u003e\n \u003cli\u003eZegans, M. E., and R. N. Van Gelder. 2014. Considerations in understanding the ocular surface microbiome. Am J Ophthalmol 158(3):420-422. doi: 10.1016/j.ajo.2014.06.014\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eSummary of the ocular swab origin and bacterial isolates recovered from the ocular swab samples via culturing.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"622\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" rowspan=\"2\" valign=\"top\" style=\"width: 278px;\"\u003e\n \u003cp\u003eFour ND Veterinary Clinics, NDSU Beef Herd, NDSU VDL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of summer swabs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e110\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of winter swabs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSwab type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal number of swabs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAerobic isolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnaerobic isolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal isolate number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 46px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlood\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMRS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlood\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 37px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealthy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e281\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIBK\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e377\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubtotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e138\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e154\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e658\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eND: North Dakota; NDSU: North Dakota State University; VDL: veterinary diagnostic laboratory; IBK: infectious bovine keratoconjunctivitis; CB: Columbia blood agar plate; MRS: De Man, Rogosa Sharpe agar plate; MP: multi-sliced agar plate; Sharpe; WC: Wilkins-Chalgren\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eBacterial isolates (n= 351) isolated and identified from aerobic and anaerobic culturing of ocular swab samples obtained from IBK-affected and healthy cattle.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"429\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhylum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of isolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage of total isolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal phylum prevalence\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eActinomycetota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eCorynebacterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"11\" valign=\"bottom\" style=\"width: 69px;\"\u003e\n \u003cp\u003e5.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eActinomycetota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrueperella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eActinomycetota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eLuteococcus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eActinomycetota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eArthrobacter\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;Actinomycetota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eCellulosimicrobium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eActinomycetota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eStreptomyces\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eActinomycetota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eRothia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eActinomycetota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eNocardia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eActinomycetota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eCutibacterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eActinomycetota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eCurtobacterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eActinomycetota\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eMicrococcus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e25.90%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"41\" valign=\"bottom\" style=\"width: 69px;\"\u003e\n \u003cp\u003e82.20%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e11.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e11.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eMacrococcus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e4.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003ePaenibacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eCaldibacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eRummeliibacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eAerococcus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eDesemzia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eMammaliicoccus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.40%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eWeizmannia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eNiallia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eHeyndrickxia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eCarnobacterium\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eAlkalihalobacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eRobertmurraya\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.90%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003ePriestia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.90%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eEnterococcus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.90%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eCytobacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eBrevibacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003ePeribacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eLysinibacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eLentilactobacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eHelcococcus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eWeissella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eVirgibacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eTerribacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eRuoffia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003ePlanococcus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003ePeptoniphilus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eParaclostridium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eOceanobacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eLevilactobacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eLactiplantibacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eLacrimispora\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eGlobicatella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eFundicoccus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eFaecalicatena\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eFacklamia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eExiguobacterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eBacillota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eClostridium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003ePseudomonadota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eMoraxella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e9.40%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"9\" valign=\"bottom\" style=\"width: 69px;\"\u003e\n \u003cp\u003e13.50%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003ePseudomonadota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eSuttonella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003ePseudomonadota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eMannheimia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.90%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003ePseudomonadota.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eAcinetobacter\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003ePseudomonadota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003ePsychrobacter\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003ePseudomonadota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudoaxnthomonas\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003ePseudomonadota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003ePantoea\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003ePseudomonadota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eCitrobacter\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003ePseudomonadota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eAlysiella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 126px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e351\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 69px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eBacterial isolates that induced zones of inhibition (ZOI) against \u003cem\u003eMoraxella bovis\u0026nbsp;\u003c/em\u003eusing the agar slab method\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"101%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage ZOI*(mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus pumilus\u0026nbsp;\u003c/em\u003e122H-MRS-F-Ae\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e36.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus sonorensis\u0026nbsp;\u003c/em\u003eMVC02-G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e30.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus mojavensis\u0026nbsp;\u003c/em\u003e26Z-WC-C-An\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e29.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus velezensis\u0026nbsp;\u003c/em\u003e42G-MRS-A-Ae\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e29.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eWeissella paramesenteroides\u0026nbsp;\u003c/em\u003eSL16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e27.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eLactiplantibacillus pentosus\u0026nbsp;\u003c/em\u003eSL27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e27.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eLevilactobacillus brevis\u0026nbsp;\u003c/em\u003eSL28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e26.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus tequilensis\u0026nbsp;\u003c/em\u003eSL29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e26.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eLimosilactobacillus fermentum\u0026nbsp;\u003c/em\u003eATCC9338\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e22.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eWeizmannia coagulans\u0026nbsp;\u003c/em\u003eSL34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e21.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus australimaris\u0026nbsp;\u003c/em\u003eSL31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e16.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eDesemzia incerta\u0026nbsp;\u003c/em\u003e17G-WC-G-An\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e16.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus pluranimalium\u0026nbsp;\u003c/em\u003eSL30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e15.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eCaldibacillus hisashii\u0026nbsp;\u003c/em\u003e23D-WC-E-An\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e13.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eLentilactobacillus buchneri\u0026nbsp;\u003c/em\u003eSL36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 34px;\"\u003e\n \u003cp\u003e12.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*The mean ZOI was obtained from three replicates.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u0026nbsp;\u003c/strong\u003eBacterial isolates that induced zones of inhibition (ZOI) against \u003cem\u003eMoraxella bovoculi\u003c/em\u003e using the agar slab method\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage ZOI* (mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus pumilus\u0026nbsp;\u003c/em\u003e122H-MRS-F-Ae\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e22.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus velezensis\u0026nbsp;\u003c/em\u003e2G-MRS-A-Ae\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e21.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus tequilensis\u0026nbsp;\u003c/em\u003eSL29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e21.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eLactiplantibacillus pentosus\u0026nbsp;\u003c/em\u003eSL27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e20.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eLimosilactobacillus fermentum\u0026nbsp;\u003c/em\u003eATCC9338\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e19.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003ePaenibacillus polymyxa\u0026nbsp;\u003c/em\u003e42G-WC-F-An\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e19.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eWeizmannia coagulans\u0026nbsp;\u003c/em\u003eSL34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e18.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eLentilactobacillus buchneri\u0026nbsp;\u003c/em\u003eSL36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e18.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eLevilactobacillus brevis\u0026nbsp;\u003c/em\u003eSL28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e16.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus australimaris\u0026nbsp;\u003c/em\u003eSL31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e15.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eWeissella paramesenteroides\u0026nbsp;\u003c/em\u003eSL26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e15.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eMammaliicoccus sciuri\u0026nbsp;\u003c/em\u003eMVC05-MRS-A-Ae\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e14.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus pluranimalium\u0026nbsp;\u003c/em\u003eSL30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 36px;\"\u003e\n \u003cp\u003e13.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*The mean ZOI was obtained from three replicates.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"animal-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amic","sideBox":"Learn more about [Animal Microbiome](http://animalmicrobiome.biomedcentral.com)","snPcode":"42523","submissionUrl":"https://submission.nature.com/new-submission/42523/3","title":"Animal Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"pinkeye, ocular microbiome, infectious bovine keratoconjunctivitis, metagenomic sequencing, whole-genome sequencing, antimicrobial resistance, cattle","lastPublishedDoi":"10.21203/rs.3.rs-6253983/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6253983/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDespite widespread use of antimicrobials and vaccines, the incidence of infectious bovine keratoconjunctivitis (IBK), or pinkeye, continues to increase in North American beef cow-calf operations. Recent research suggests that there is potential for the commensal ocular microbiome to help mitigate IBK. Therefore, this study characterized the ocular microbiome of cattle with and without IBK using culturing and shotgun metagenomic sequencing and assessed the ability of commensal bacteria to inhibit \u003cem\u003eMoraxella\u003c/em\u003e spp. \u003cem\u003ein vitro\u003c/em\u003e. Ocular swabs (n\u0026thinsp;=\u0026thinsp;143) were collected from IBK-affected (n\u0026thinsp;=\u0026thinsp;102) and healthy cattle (n\u0026thinsp;=\u0026thinsp;41) before antimicrobial treatment from North Dakota herds. Bacteria were cultured aerobically and anaerobically on five different media and the isolates identified. A subset of swabs (37 IBK; 12 healthy) underwent shotgun metagenomic sequencing. The genomes of 31 isolates, including \u003cem\u003eMoraxella bovoculi\u003c/em\u003e, \u003cem\u003eMoraxella bovis\u003c/em\u003e, and commensal bacteria, were also sequenced. Fifty-two commensal isolates were screened for inhibition of \u003cem\u003eMoraxella\u003c/em\u003e spp. using an agar slab method, with five isolates further tested for inhibition in the presence of culturable ocular microbiome using qPCR.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe 351 bacterial isolates taxonomically identified represented 61 genera from three phyla. The majority of isolates belonged to \u003cem\u003eBacillus\u003c/em\u003e (25.9%), \u003cem\u003eStreptococcus\u003c/em\u003e (11.1%), \u003cem\u003eStaphylococcus\u003c/em\u003e (10.1%), and \u003cem\u003eMoraxella\u003c/em\u003e (9.4%) genera. Shotgun metagenomic analysis revealed significant differences in the ocular microbiome composition between IBK-affected and healthy cattle (R\u0026sup2; = 0.042; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.034). Dominant bacterial species included \u003cem\u003eCutibacterium acnes\u003c/em\u003e, \u003cem\u003eMannheimia pernigra\u003c/em\u003e, \u003cem\u003eMesomycoplasma bovoculi\u003c/em\u003e, \u003cem\u003eMoraxella bovis\u003c/em\u003e, and \u003cem\u003eMoraxella bovoculi\u003c/em\u003e. Eight bacterial species, including \u003cem\u003eBifidobacterium globosum\u003c/em\u003e and \u003cem\u003eBacillus licheniformis\u003c/em\u003e, were more abundant in healthy cattle, while \u003cem\u003eArthrobacter luteus\u003c/em\u003e was enriched in IBK cases. Thirty-seven high-quality metagenome-assembled genomes were also recovered, with 27% classified as \u003cem\u003eMesomycoplasma bovoculi\u003c/em\u003e. \u003cem\u003eMoraxella\u003c/em\u003e spp. genomes exhibited strain-specific antimicrobial resistance and virulence gene diversity. Seventeen commensal isolates inhibited \u003cem\u003eMoraxella\u003c/em\u003e, with \u003cem\u003eWeizmannia coagulans\u003c/em\u003e, \u003cem\u003eLentilactobacillus buchneri\u003c/em\u003e, and \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e showing strong activity. Selected isolates maintained inhibitory effects in co-culture with the ocular microbiome.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe ocular surface of beef cattle is inhabited by a diverse microbiome that includes several bacterial strains that have the potential to be used as therapeutics to inhibit IBK pathogens.\u003c/p\u003e","manuscriptTitle":"The Bovine Ocular Microbiome: A Multi-Approach Study of Composition and Antimicrobial Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-26 09:52:05","doi":"10.21203/rs.3.rs-6253983/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-01T03:24:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-23T22:47:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-18T04:33:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13815117116067065462192766092601028001","date":"2026-02-05T12:54:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30242335777166592624052298844878565878","date":"2026-02-02T14:11:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77623196870354287398258580810104012791","date":"2026-02-01T08:10:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215966802775503731265719085558126077602","date":"2026-02-01T04:14:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223738591034620813304906964328421884097","date":"2026-02-01T03:12:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-05T05:51:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-27T18:57:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-19T05:57:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Animal Microbiome","date":"2025-03-18T14:06:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"animal-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amic","sideBox":"Learn more about [Animal Microbiome](http://animalmicrobiome.biomedcentral.com)","snPcode":"42523","submissionUrl":"https://submission.nature.com/new-submission/42523/3","title":"Animal Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"381daab7-1432-47c1-916e-c178f43ad195","owner":[],"postedDate":"March 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-22T11:10:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-26 09:52:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6253983","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6253983","identity":"rs-6253983","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}