The Emergence of Antimicrobial and Phage Resistance in Piscine Lactococcus Isolates and Implications for Future Disease Management in Aquaculture.

The Emergence of Antimicrobial and Phage Resistance in Piscine Lactococcus Isolates and Implications for Future Disease Management in Aquaculture. | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 June 2025 V1 Latest version Share on The Emergence of Antimicrobial and Phage Resistance in Piscine Lactococcus Isolates and Implications for Future Disease Management in Aquaculture. Authors : Adam M. Blanchard 0000-0001-6991-7210 [email protected] , Bailey Secker , Samantha J. Windle , Robert Atterbury , Ha Thanh Dong , Le Thanh Dien , David Huchzermeyer , Bernard Hang'ombe , and Saengchan Senapin Authors Info & Affiliations https://doi.org/10.22541/au.174894333.36446684/v1 281 views 155 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Lactococcosis is a major bacterial disease impacting rainbow trout production in South Africa and Southeast Asia, particularly during the summer. This study analysed 15 bacterial isolates from affected aquaculture facilities, revealing that Lactococcus petauri (n=12) was the predominant species, rather than the traditionally recognised L. garvieae (n=3). This finding suggests a possible shift in the aetiology of lactococcosis, with potential implications for disease management. Genotypic analysis revealed that only L. garvieae isolates carried the adhesin gene (adh), which is critical for bacterial adhesion and colonisation. Most isolates possessed sortase-anchored proteins linked to iron uptake, adhesion, and stress resistance, with the LPxTG-6 subgroup unique to L. garvieae . Variations in the capsule operon, including transposase insertions, suggest ongoing horizontal gene transfer, possibly influencing immune evasion. Antimicrobial resistance (AMR) analysis identified efflux pumps ( mdtA, lsaD ) conferring resistance to macrolides and lincosamides, with additional genotypic resistance to erythromycin ( mefA, msrD ) and tetracyclines ( tetS, tetL ). The presence of plasmid-borne tetL raises concerns about potential gene transfer and the persistence of resistance in aquaculture systems. Prophage elements were identified in several isolates, potentially contributing to virulence, and immune modulation. Although phage therapy has shown promise in experimental settings, the presence of viral defence mechanisms may raise a unique challenge. These findings highlight the importance of monitoring pathogen evolution in aquaculture systems and suggest that ongoing genomic surveillance and treatment strategies may need to be adapted to account for the emerging role of L. petauri in lactococcosis outbreaks. Introduction Fish aquaculture is a rapidly growing sector of global food production, contributing significantly to food security, economic development, and livelihoods worldwide 1 . Among the diverse species farmed, economically important fish such as tilapia ( Oreochromis spp.), rainbow trout ( Oncorhynchus mykiss ), and Asian seabass ( Lates calcarifer ) have gained prominence due to their high market demand, nutritional value, and adaptability to various aquaculture systems 2–4 . Tilapia is a staple in both developing and developed countries, prized for its affordability and rapid growth rate. Rainbow trout is highly valued for its premium quality, appealing taste, and contribution to high-value markets. Similarly, Asian seabass is a highly valued species in the aquaculture sector, known for its versatility in farming environments, excellent growth performance, and economic value in both domestic and export markets. The increasing reliance on fish aquaculture to meet the protein needs of a growing human population highlights its importance. However, the sustainability and productivity of the industry are increasingly threatened by bacterial infections 5–7 , which result in significant economic losses and pose challenges to maintaining fish health and welfare. There are several common bacterial diseases affecting farmed fish, including streptococcosis, aeromonasis, columnaris, edwardsiellosis and lactococcosis. Among these, lactococcosis - caused by lactococcosis-causing bacteria (LCB) - is one of the emerging concerns in Africa and Southeast Asia. This disease affects a range of farmed fish, including tilapia and rainbow trout, resulting in economic losses for aquaculture operations 8 . The most common pathogen responsible for lactococcosis is Lactococcus garvieae , but other species such as Lactococcus petauri and Lactococcus formosensis have also been recently reported to contribute to the disease. Lactococcosis is typically characterized by clinical signs such as exophthalmos, acute haemorrhagic septicaemia, with mortality rates between 50-80% 9 . Lactococcosis is classed as an emerging global zoonotic pathogen with cases in marine and terrestrial mammals 10,11 , reptiles 12 , birds 13 , and also rarely in humans 14 . Outbreaks have occurred globally in aquaculture systems 15,16 which have a significant economic and welfare impact to the industry 17 . Outbreaks of disease are normally attributed to L. garvieae . However, classical microbiological identification techniques 18,19 often fail to differentiate this species from related aetiological agents such as L. petauri and L. formosensis. Additionally, pathogenesis is poorly defined and there is a lack of knowledge around virulence determinants in the three species, as most research is focused on clinical isolates from aquaculture 20 . Moreover, recent comparative genomics studies 21,22 have identified other homologous streptococcal virulence genes associated with adhesion 23 , iron transport 24 and capsular polysaccharide 25 . Encapsulation has long been considered a virulence factor in many streptococcal species as it masks the cell surface antigens from the immune system 26,27 . The capsule is immunogenic, and a target for antimicrobial drugs in many bacterial species. Shedding of the capsule on exposure to these compounds can be used as an evasion technique to evade autophagy-mediated killing in macrophages 28 . By comparing pathogenic and non-pathogenic strains of piscine L. garvieae the presence of a capsule has been shown to be crucial for virulence in fish 22 , however, it is sometimes not present, meaning encapsulation is not the sole requirement for virulence 29 . Adhesins are cell-surface components that facilitate the binding of bacteria to host cell receptors. L. garvieae has been found to produce three proteins (PsaA, PavA, and enolase) with high similarity to adhesion virulence factors in other streptococcal species, as well as adhesin clusters 1 and 2 ( adhCI and adhCII ) and adhesin ( adh ) 23,29 . Sortase- anchored proteins contribute to the establishment and persistence of disease, and as such are likely to be virulence factors 30 . Antibiotics have long been deployed to control Lactococcus spp and Streptococcus spp. generally in aquaculture. Most commonly these include erythromycin, oxytetracycline, amoxicillin and low-level doxycycline 31 . However, resistance to erythromycin, oxytetracycline and lincomycin is growing due to the presence of ermB and tetS 32 . Outbreaks are becoming more frequent and control through medicated feed often fails due to a combination of inappetence and antimicrobial resistance (AMR) 15 . This is the first study to date which has analysed Lactococcal genomes isolated from outbreaks of lactococcosis at aquaculture facilities in Zambia, South Africa, Vietnam and Thailand. We have provided a deeper understanding of antimicrobial resistance, virulence factors, abundance of phage resistance mechanisms and the implications for the use of phage therapy in future disease management. Bacterial isolates Lactococcus spp. isolates from Thailand (n=3), Vietnam (n=5), Zambia (n=5), and South Africa (n=2), previously obtained from diseased fish species including tilapia, Asian seabass, and rainbow trout were employed for whole genome sequencing in this study (Table 1). These isolates were previously identified as L. garvieae through 16S rDNA sequencing. Virulence test results from experimental infections using some of these isolates are also presented in Table 1. not-yet-known not-yet-known not-yet-known unknown DNA Extraction DNA extraction of Lactococcus spp. was performed following previously optimised methods33 with some modifications as follows. A single colony was cultured overnight at 30°C in 1.5 mL of tryptic soy broth (Difco). Cells were harvested by centrifugation at 9,000 × g for 5 minutes, followed by washing with 1 mL of distilled water and subsequent collection by centrifugation. The cell pellet was resuspended in 300 µL of EDTA-saline solution (0.01 M EDTA, 0.15 M NaCl, pH 8.0) and thoroughly vortexed. Then, 50 µL of 110 mg/mL lysozyme (SERVA) and 10 µL of 20 mg/mL RNase A (Thermo Fisher Scientific) were added, mixed by vortexing, and incubated at 37°C for 2 hours, with vortexing every 15 minutes. After incubation, 160 µL of 20% SDS and 20 µL of 5 mg/ml proteinase K (Merck) were added, followed by vortexing and incubation at 65°C for 30 minutes. Next, 0.5 volume of 5 M NaCl was added and briefly mixed. An equal volume of phenol (1:1, v/v) was added, vortexed, and centrifuged at 12,000 × g for 15 minutes. The upper aqueous phase was transferred to a new tube, and this extraction step was repeated once more. Then, 2 µL of 20 mg/mL RNase A was added and incubated for 15 minutes, followed by a final phenol extraction. The upper phase was transferred to a new tube. To precipitate the DNA, 0.1 volume of 3 M sodium acetate (pH 5.2) was added and mixed gently, followed by the addition of 2 volumes of cold absolute ethanol. The mixture was incubated at -20°C for 2 hours and then centrifuged for 10 minutes to collect the DNA pellet. The DNA pellet was washed with cold 70% ethanol, air-dried, and resuspended in 20 µl of RNase- and DNase-free water. DNA quality and quantity were measured using a Nanodrop spectrophotometer. Table 1: Provenance of the strains used in the study Thailand Oct 22 Kanchanaburi L. garvieae Red Tilapia 3749 No 34 Thailand May 22 Chachoengsao L. petauri Asian sea bass 3750 No 34 Thailand May 22 Chachoengsao L. petauri Asian sea bass 3752 N/A Zambia Mar 17 Lake Kariba L. petauri Nile tilapia 3816 N/A Zambia Oct 17 Lake Kariba L. petauri Nile tilapia 3817 N/A Zambia Feb 18 Lake Kariba L. petauri Nile tilapia 3818 N/A Zambia Oct 18 Lake Kariba L. petauri Nile tilapia 3819 N/A Zambia Nov 18 Lake Kariba L. petauri Nile tilapia 3820 N/A South Africa Nov 15 Unknown L. petauri Rainbow trout 3821 N/A South Africa May 16 Unknown L. petauri Rainbow trout 3822 N/A Vietnam Aug 22 Ben Tre L. petauri Red Tilapia 3830 Low Vietnam Aug 22 Ben Tre L. garvieae Red Tilapia 3831 Medium Vietnam Aug 22 Ben Tre L. garvieae Red Tilapia 3832 High Vietnam Jan 22 Tien Giang L. petauri Red Tilapia 3833 Medium Vietnam Jan 22 Tien Giang L. petauri Red Tilapia 3834 Low *Species level taxonomic assignment of the 15 Lactococcus genomes is based on digital DNA:DNA hybridisation determined in this study. Sequencing Bacterial DNA samples (1.0 µg each) were sent for whole genome sequencing to BGI Hong Kong Tech Solution NGS Lab via Bangkok Genomics Innovation Co., Ltd. (Thailand). DNA quantity and quality were verified using microplate reading and gel electrophoresis before library preparation. The DNA libraries were then prepared using the short-insert library method and sequenced with DNBseq technology at a PE150 read length generating 4 million reads per sample. Data filtering, including the removal of adaptor sequences and low-quality reads, was performed by the company. Data Analysis Assemblies were created using NF-core Bacass 35 v2.2.0 under short read mode. Briefly, the reads are assessed for quality using FastQC v0.12.0 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and trimmed using FastP 36 v0.23.4. Clean and high quality reads are assembled using Unicycler 37 v0.5.0 and the resulting contigs were checked and filtered for taxonomic assignment using Kraken2 38 v2.1.3. Genome assemblies have been deposited in National Library of Medicine (NCBI) GenBank under the BioProject PRJNA1264975. Taxonomic classification of genomes was calculated using digital DNA:DNA hybridisation with GGDC 39 using reference genomes L. garvieae GCA_016026695, L. petauri GCA_023499275 and L. formosensis GCA_037892375. Relatedness of bacterial genomes was tested using Sourmash 40 in average nucleotide identity (ANI) mode and heatmaps were created in R v4.3.2 41 using Pheatmap (https://github.com/raivokolde/pheatmap). The genomes were parsed for genotypic antimicrobial resistance using the AMR++ and the MegaRes3 database 42 . Virulence gene presence was determined using abricate with a custom database of L. garvieae specific virulence genes as described by Lin et al 21 . Presence of prophage regions was determined using three tools; geNomad v1.8.0 43 , PhageBoost v0.1.7 44 and VIBRANT v1.2.1 45 . The predictions were then consolidated to identify the longest possible region using Biopython 46 . The resulting sequences were processed using CheckV v1.0.3 47 , retaining only sequences identified as ‘medium quality’ or higher. Additionally, where host contamination was identified by CheckV, the trimmed sequence was kept. Sequences from the same host were dereplicated using CheckV supporting code. Finally, the sequences were annotated using Pharokka 48 v1.7.5 followed by phold v0.2.0 (https://github.com/gbouras13/phold). Taxonomic assignment was completed using taxmyPHAGE 49 and proteomic trees were generated using Viral Proteomic Tree (ViPTree). Genomic level comparisons of the prophage were performed using CheckV supporting ANI and clustering code, default clustering cutoffs of 95% ANI over 85% alignment fraction were used as described by Roux et al . 50 . Genomic organisation and protein clustering was performed using LoVis4u v0.1.1 51 , loci were reorientated, and functional annotation was performed for defence, anti-defence, virulence and antimicrobial resistance genes. Prophage were considered ‘intact’ if they contained an integrase, structural proteins and lysis modules in the conserved gene order as assessed by manual curation 52,53 . Finally, the termini of prophage regions of interest were investigated using PhageTermVirome v4.1 54 . Results Genome Assembly The quality control data achieved a mean Phred quality of 39 and de novo assembled following the Bacass 35 short read pipeline to approximately 2.0 Mbp (min 2.0 Mbp max 2.3 Mbp) with a mean N50 of 756 Kbp. Based on Kraken2 38 the final assemblies where classified as Lactococcus species. Further taxonomic assignment was generated through dDDH which yielded a species level classification with probability scores ≥93% to reference species L. garvieae GCA_016026695 and L. petauri GCA_023499275, corresponding to 3 and 12 isolates, respectively (Table 1). Interestingly, all of these isolates had previously been identified as L. garvieae through 16S rDNA sequencing. Genome Comparison Based on average nucleotide identity scores (Figure 1), the isolates seem to form two clusters based on species classification. Cluster 1 is the L. garvieae isolates, where clusters 2, 3 and 4 are all L. petauri where they split into country of origin. Cluster 2 contains isolates from Zambia and South Africa whereas cluster 3 is Vietnamese isolates and cluster 4 is Thai isolates. Figure 1. Average Nucleotide Identify comparison of all isolates. The nucleotide identity score is between 0.94 and 1, representing 94% and 100% similarity between all isolates. The Y axis is coloured by country of origin, and clustered using a Euclidean approach, and the X axis shows host species. Antimicrobial resistance genotype All isolates showed multi-drug genotypic resistance with the presence of mdtA (drug, biocide and metal RND efflux pumps) and lsaD (multi-drug ABC efflux pumps). Two isolates (3833 and 3834) had mefA (MLS erythromycin resistance MFS efflux pumps) and msrD (MLS erythromycin resistance ABC efflux pumps). Only two isolates (3821 and 3822) had plasmid borne tetracycline genotypic resistance with the presence of tetL (accession NF012176.0) and three isolates (3750, 3821 and 3822) had tetS. Also, two isolates (3833 and 3834) had lnuC (lincosamide) genotypic resistance (Supp Table 1). Overall, the isolates could be ascribed to 4 resistance profiles, the most common being MLS efflux pumps (Table 2). Table 2. Antimicrobial Resistance Profiles for the isolates 3749 • • L. garvieae Thailand 3750 • • • L. petauri Thailand 3752 • • L. petauri Thailand 3816 • • L. petauri Zambia 3817 • • L. petauri Zambia 3818 • • L. petauri Zambia 3819 • • L. petauri Zambia 3820 • • L. petauri Zambia 3821 • • • • L. petauri S Africa 3822 • • • • L. petauri S Africa 3830 • • L. petauri Vietnam 3831 • • L. garvieae Vietnam 3832 • • L. garvieae Vietnam 3833 • • • • • L. petauri Vietnam 3834 • • • • • L. petauri Vietnam not-yet-known not-yet-known not-yet-known unknown Gene names and linked function: mdtA Drug and biocide and metal RND efflux pumps, lsaD Multi-drug ABC efflux pumps, lnuC Lincosamide nucleotidyltransferases, mefA MLS erythromycin resistance MFS efflux pumps, msrD MLS erythromycin resistance MFS efflux pumps, tetL Tetracycline resistance MFS efflux pumps, and tetS Tetracycline resistance ribosomal protection proteins. Full data in Supplementary Table 1. Virulence genotype Based on a cutoff of ≥95 identity, all isolates had the three haemolysin genes, the nine iron uptake genes and adhesion genes (enolase, pavA and psaA ) but only three isolates (3749, 3831 and 3832) had adhesin. All isolates had GAPDH, NADH oxidase, phosphoglucomutase, superoxide dismutase and collagenase genes. Only 2 isolates had a complete capsule operon of all 12 loci (3830, 3832), while 2 isolates 3833 and 3834 had 11 genes from the operon, each missing the 1p transposase. All isolates had srtA and LPxTG-1, and nine isolates (3861, 3817, 3818, 3819, 3820, 3821, 3822, 3831 and 3832 had LPxTG-3 and LPxTG-6 while 3749 only had LPxTG-6. Only 2 isolates (3821 and 3832) had LPxTG-2 and LPxTG-4 (Supplementary Table 2). Prophage A total of 144 putative prophage regions were identified in the isolates, of these, 22 were retained after filtering with CheckV. Only two (2/15, 13%) of the Lactococcus genomes did not contain any prophage regions, and between 1-3 prophage regions were identified in the remaining genomes (13/15, 87%). The prophage regions were between 13-69 kbp in length and contained between 29-129 coding sequences. taxmyPHAGE assigned all the prophage to novel genera, however, 3830 prophage 5, 3833 prophage 1, and 3834 prophage 1 were taxonomically similar to Siphovirus -like phage genus Piorkowskivirus . Comparison of the regions using a proteomic tree did not identify clustering based on host species or country of origin (Figure 2). Additionally, when viewed on a proteomic tree with 3,399 sequences from the ViPTree viral database, the prophage were not closely related. However, they did all fall within the area of the tree with viruses of Bacillota (previously Firmicutes ; Supplementary Figure 1). The majority (59%;13/22) of the prophage were similar to other Lactococcus phage, the remaining regions clustered with phage that infect Thermoanaerobacterium , Streptococcus , Enterococcus , and Lactobacillus . Figure 2. Prophage proteomic phylogenetic tree. VipTree was used to generate a proteomic tree of the prophage regions identified as part of the present study and visualised using iToL. The scale represents 1 base difference per 10 bases. The colour bars represent the country of origin and host species. As the prophage regions could not be taxonomically classified, the genomic diversity of the prophage was investigated using pairwise ANI, followed by clustering using thresholds of 95% ANI over 85% alignment fraction, this identified 11 putative virus operational taxonomic units (vOTUs) (Supplementary Figure 2). Similarly, when the annotated prophage were visualised with LoVis4u, protein clustering supported 11 vOTUs (Supplementary Figure 3). This revealed that the proteins within prophage regions were highly conserved within vOTUs. This is particularly interesting for prophage identified in the genomes of L. petauri 3816-20 as these samples were collected from infected Nile tilapia found in Lake Kariba across a period of almost two years suggesting the persistence of L. petauri within the tilapia population over this period. When the prophage regions were interrogated for the presence of antimicrobial resistance and virulence determinants, a number of genes were identified. However, despite the comprehensive methods used to identify prophage regions, only 45% (10/22) were found to contain protein sequences indicative of an intact prophage region. No virulence or AMR genes were identified within an intact prophage region (Figure 3). Despite this, several defence genes were identified associated with intact prophage regions, this would be an important consideration were bacteriophage treatment investigated as an alternative to antibiotics. Specifically, a coding sequence identified as AbiD was found in two prophage regions. Abi systems have been previously reported on lactococcal plasmids 55 . To confirm that AbiD was within the prophage region, PhageTermVirome was used to identify the termini of prophage 3822 prophage 3 and 3821 prophage 1. The termini of 3822 prophage 3 could not be determined however the termini of 3821 prophage 1 was identified and included the coding sequence of AbiD. Restriction-modification (R-M) systems are ubiquitous among bacterial chromosomes and plasmids, these function by degrading DNA while protecting self-DNA with modification 56 . Coding sequences identified as type II DNA methyltransferases and endonucleases were present in multiple prophage regions. Although involved in other phage processes, these are thought to protect the prophage from host R-M systems during the lytic phase 57 . Figure 3. Genomic organisation of the identified intact prophage regions. The genomes are reorientated and homology between coding sequences is shown. Coding sequences are coloured based on presumed function. Discussion This study characterised the genomes of 15 Lactococcus strains isolated from piscine disease outbreaks in Asia and Africa. This revealed the presence of up to five antimicrobial resistance genes and three novel prophage regions per strain. Virulence gene characterisation found that only L. garvieae isolates carried the adhesin gene adh which is critical for bacterial colonisation. Although the prophage regions did not harbour any known virulence or antimicrobial resistance genes, they did contain several genes encoding phage defence systems such as (AbiD and R-M systems). This information is important when considering the potential development of phage therapy for piscine lactococcosis. Interestingly, the isolates from cases of lactococcosis in the present study were not all L. garvieae (n=3), the most commonly recognised aetiological cause of the disease, but a majority were L. petauri (n=12). The predominant detection of L. petauri in this study, supported by recent reports 18,58 , suggests that this species may have been either previously misidentified as L. garvieae , due to their high phenotypic and 16S rRNA gene sequence similarity, or previously overlooked in past surveillance efforts. These findings underscore the importance of implementing both active and retrospective surveillance strategies for Lactococcus spp. in fish populations to improve species-level identification and enhance disease management strategies. Only three isolates (3749, 3831 and 3832) had the adhesin gene ( adh ), which seems specific to the L. garvieae isolates. Adhesins are necessary for the ability of the bacteria to bind to the host cell surface and are required for colonisation and pathogenesis. Despite the essentiality of these loci, it has been documented that some strains of L. garvieae do not possess all three loci adh, pav and psaA 29 and these variations may hint to host specificity 59 . Sortase Proteins linked to Increased Virulence Most of the isolates investigated possessed multiple sortase-anchored proteins, however, the low virulence isolate, 3749 only had LPxTG-6 and isolates 3821 and the high virulence isolate 3832 had LPxTG-2 and LPxTG-4. These sortase anchored proteins in particular are highly associated with the iron uptake operon, adhesion and haemolysis 21 . This may aid in colonisation and stress resistance, enabling adaptation and survival in nutrient-poor or hostile environments. The LPxTG-6 subgroup is unique to isolates 3749, 3821 and 3822 which came from two different locations. There is a dearth of information on the specific functional implications of different sortase anchored protein variants, but due to the high co-occurrence of LPxTG-6 to the iron uptake operon, function could be suggested to play an important role in nutrient uptake, whereas LPxTG-4 seems to be more associated with psaA, which could hint at a roll in initial colonisation 21 . Only isolates 3833 and 3834 had an incomplete complement of 12 genes from the capsule operon, each missing the 1p transposase. The feature of genes encoding transposases or phage-related proteins within a capsule operon may suggest evidence of transposition or horizontal gene transfer. It is likely they alter production of the capsule related proteins rather than complete disruption, serving as a method of immune evasion. This has been documented in other Gram positive bacteria, where the transposase loci in Streptococcus pneumoniae differentiates between serotypes in serogroup 12 60 . Development of Antimicrobial Resistance to Commonly used Antibiotics All isolates processed the efflux pumps mdtA which are associated with macrolide resistance , and lsaD associated with lincosamide resistance. These resistances are commonly found generally in Gram positive bacteria and would not be of cause for concern in first line treatment of infections. However, two isolates showed genotypic resistance to erythromycin ( mefA and msrD ). While not uncommon in Gram positive bacteria, erythromycin has been used in prophylactic feed based medication in aquaculture, which has driven the selective pressure for resistant strains of Lactococcus 61 . Finally, three isolates showed genotypic resistance to tetracyclines with the presence of tetS and two additionally showed plasmid-borne tetracycline resistance from tetL . This resistance is also prevalent in isolates from aquacultural systems, and has been detected more frequently since the increased use of tetracycline for treatment of lactococcosis from 2014 62 . There might be a greater concern from the plasmid-borne nature of tetS and the potential of its ability to transfer if stock is imported from different brood stock providers. Identification of Phage Defence Mechanisms The rapid development of antimicrobial resistance has focused attention on alternative treatments such as bacteriophage therapy, bacterial viruses that infect and replicate within bacterial cells, as an alternative to antibiotics. Only one study has attempted phage therapy against L. garvieae infections in yellowtail fish, which reported significant reductions in mortality in fish treated with phage either injected peritoneally, or through feed 63 . However, timing of phage delivery was critical, with mortality appreciably higher when treatment was delayed to 24 h after bacterial challenge. Despite promising results, there are some limitations of bacteriophage therapy, namely the development of resistance which has been discussed in detail elsewhere (https://doi.org/10.1038/nrmicro2315). One mechanism by which bacteria can be resistant to bacteriophage infection is related to prophage, temperate bacterial viruses which are incorporated in the bacterial genome which can confer phenotypic advantages in environmental niches. These advantages may include increased resistance to acid or antimicrobials, enhanced biofilm production, higher growth rates, and modulation of eukaryotic host immune response 64 . Prophage-encoded proteins of Streptococcus mitis 65 and Enterococcus faecalis 66 facilitate adhesion to human platelets which is crucial for the development of endocarditis, and may be relevant to L. garvieae pathophysiology. Temperate phage are also associated with horizontal gene transfer, via transduction, and conferring superinfection immunity to closely related phage. In the present study, AbiD was identified within the one of the prophage regions. Abortive infection (Abi) systems are diverse in Lactococcus lactis and generally function by causing death of the bacterial host 67 , consequently, the presence of Abi systems may have unexpected effects if bacteriophage were used to treat these infections. Typically, for these reasons, temperate phage are considered unsuitable for use as direct therapeutic agents. However, in cases where strictly-lytic phage are rare or impossible to find, prophage can be used as the basis for developing effective treatments 68 . Most commonly, this includes identifying sequences encoding hydrolytic enzymes (usually endolysins) phage use to escape from infected cells after replication, then expressing and purifying the proteins. Endolysins are particularly effective against Gram positive bacterial pathogens which lack an outer membrane to defend their cell wall from digestion 69 . Lytic and temperate phage which infect L. garvieae have been isolated 42,43 . However, only minimal data is available on the lytic spectrum of these phage – which indicates a very limited host range - so it is difficult to determine if they would be useful against the broad diversity of L. garvieae strains causing disease. Although tilapia is native to Africa, it has been domesticated and cultivated in over 140 countries worldwide. Seed production and aquaculture technologies have advanced significantly in Asia. African countries have a long history of importing tilapia seed from Asian nations. Movement of live fish, which may carry pathogens through subclinical infections for aquaculture, could contribute to the translocation of these pathogens, similar to the dynamics observed with viruses such as TiLV and ISKNV 73,74 . The genomic analysis in this study suggest that the African isolates and Asian isolates form different unique clusters, suggesting their diversification in genetic evolution. Nonetheless, this study provides some fundamental knowledge of genomic characterization of the piscine Lactococcus spp., which is useful for further study on distribution and tracking genomic epidemiology as reported earlier with other pathogens 75 . In summary, the predominance of L. petauri among isolates from lactococcosis outbreaks in fish farms in South Africa and Southeast Asia indicates a potential shift in the etiological landscape of this disease. Comparative genomic analyses revealed distinct differences between L. petauri and L. garvieae in terms of virulence-associated factors, antimicrobial resistance genes, and prophage content. These findings underscore the need for enhanced taxonomic resolution in diagnostic protocols and reinforce the importance of ongoing genomic surveillance. Adaptation of current disease management and treatment strategies is warranted to address the emerging role of L. petauri in aquaculture, particularly in light of its potential for antimicrobial resistance and horizontal gene transfer. Acknowledgements This research was supported by the Budget Bureau and the National Science and Technology Development Agency. We are grateful to Dr Evelien Adriaenssens and Dr Ryan Cook for their helpful discussions regarding analysis of prophage in this study. The authors would like to thank Janchai Wongkaew for her technical assistance. References 1. The State of World Fisheries and Aquaculture 2024 . (FAO, 2024). doi:10.4060/cd0683en.2. Biology and Culture of Asian Seabass Lates Calcarifer . (CRC Press, 2013). doi:10.1201/b15974.3. D’Agaro, E., Gibertoni, P. & Esposito, S. Recent Trends and Economic Aspects in the Rainbow Trout (Oncorhynchus mykiss) Sector. Applied Sciences 12 , 8773 (2022).4. Pullin, R. S. V. 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Clustering shown on the left was performed using cutoffs of 95% ANI over 85% alignment fraction. ANI and clustering was performed using CheckV supporting code. Supplementary Figure 3 Genomic organisation of the 22 prophage regions identified as part of the present study. The genomes are reorientated and homology between coding sequences is shown. Coding sequences are coloured based on presumed function. Supplementary Tables Supplementary Table 1 Antimicrobial resistance genes Supplementary Table 2 Virulence Associated Genes Information & Authors Information Version history V1 Version 1 03 June 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords antibiotic resistance bacteriophages food-borne pathogens genomics Authors Affiliations Adam M. Blanchard 0000-0001-6991-7210 [email protected] University of Nottingham School of Veterinary Medicine and Science View all articles by this author Bailey Secker University of Nottingham School of Veterinary Medicine and Science View all articles by this author Samantha J. Windle University of Nottingham School of Veterinary Medicine and Science View all articles by this author Robert Atterbury University of Nottingham School of Veterinary Medicine and Science View all articles by this author Ha Thanh Dong Asian Institute of Technology School of Environment Resources and Development View all articles by this author Le Thanh Dien Van Lang University View all articles by this author David Huchzermeyer North-West University View all articles by this author Bernard Hang'ombe University of Zambia School of Veterinary Medicine View all articles by this author Saengchan Senapin National Center for Genetic Engineering and Biotechnology View all articles by this author Metrics & Citations Metrics Article Usage 281 views 155 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Adam M. Blanchard, Bailey Secker, Samantha J. Windle, et al. 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