Antimicrobial potential of class II bacteriocins on gut microbiota species

Antimicrobial potential of class II bacteriocins on gut microbiota species | 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 Antimicrobial potential of class II bacteriocins on gut microbiota species Carola Elisa Heesemann Rosenkilde, Ruben Vazquez-Uribe, Ditte Olsen Lützhøft, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3282788/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : Bacteriocins are antimicrobial peptides with properties making them applicable for food preservation and pathogen control. However, their impact on the gut microbiota remains understudied. In this study we sought to examine the inhibitory spectrum of 75 class II bacteriocins against 49 representative species of the human gut microbiota including 21 Biosafety level 2 organisms. The bacteriocins were cloned and expressed in Escherichia coli and evaluated in vitro, ex vivo and in vivo. Results : In vitro spot assays using Escherichia coli BL21-AI expressing these bacteriocins revealed that 22 bacteriocins inhibited at least one species, with greater efficacy against Gram-positive than Gram-negative species. Two bacteriocins, Actifencin and Bacteroidetocin A, were selected based on their broad spectrum of inhibition for further characterization and applied to murine feces ex vivo . Application of these bacteriocins led to substantial modifications in the composition of the microbial community of murine feces. Yet, these findings could not be replicated in vivo when bacteriocin producing Escherichia coli Nissle strains were dosed to mice. Conclusions : This study evaluate the potential and expands our knowledge of the inhibitory spectrum of class II bacteriocins against a large and representative collection of bacterial isolates from the gut microbiota and underscores that further optimization is needed to use bacteriocins in vivo for modulating microbial community composition. Class II bacteriocins gut microbiota novel antimicrobial peptides gut modulation strategies Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The increasing concerns surrounding food safety, antibiotic resistance, and the intricate relationship between the gut microbiota and human health have prompted extensive research on bacteriocins. These naturally occurring antimicrobial peptides, produced by a diverse range of bacteria, have garnered interest for their ability to target closely related species (Zacharof and Lovitt 2012 ). Bacteriocins exhibit both narrow and broad-spectrum activities and possess desirable characteristics such as nanomolar range efficacy, diverse modes of action, and high specificity compared to traditional antibiotics (Auke Johan van Heel, Montalban-Lopez, and Kuipers 2011; Rea et al. 2010 ; Chikindas et al. 2018 ). Additionally, their proteinaceous nature allows for engineering to enhance specificity and stability (Kheadr et al. 2010 ; Field et al. 2019 ). Based on structural and functional attributes, bacteriocins are classified into three major types (Hernández-González et al. 2021). Among these types, class II bacteriocins, predominantly derived from Lactic Acid Bacteria (LAB), have been extensively studied in vitro for their potential as bio-preservatives in food, particularly against undesirable pathogens such as Listeria spp. (Dabour et al. 2009 ; Fatima 2013 ). Apart from their role in food preservation, class II bacteriocins have also been explored as alternative antibiotics for medical and veterinary applications, including combating C. difficile infections (Cotter, Hill, and Ross 2005; Soltani et al. 2021 ; Rea et al. 2010 ). In vivo investigations of class II bacteriocins have demonstrated their ability to eliminate target bacterial strains without significantly affecting the overall microbiome composition, often employing the natural bacteriocin producer strain (Dabour et al. 2009 ; Dobson et al. 2011 ; Umu et al. 2016 ). However, limited studies have examined the impact of bacteriocins on the overall composition of the gut microbiota. Understanding the mechanisms underlying bacteriocin-mediated microbiota modulation is crucial for their effective utilization as antimicrobial agents (Heilbronner et al. 2021). Systematic and high-throughput studies of bacteriocins can be challenging when relying solely on natural bacteriocin producers. Therefore, utilizing heterologous hosts offers advantages for in vitro screening and controlled in vivo delivery of bacteriocins. This approach circumvents the need for specific regulatory conditions in the native producer and facilitates large-scale production required for commercial viability in the food and pharmaceutical industries (Mesa-Pereira et al. 2018 ). Escherichia coli (E. coli) , extensively characterized as a production host for bacteriocins and proteins, is commonly employed due to its well-established features (Mesa-Pereira et al. 2018 ; Jia and Jeon 2016). Notably, a previous study demonstrated heterologous expression of 80 different biosynthetic gene clusters in E. coli , showcasing their activity against ESKAPE pathogens (Ayikpoe et al. 2022 ). A typical bacteriocin gene cluster comprises a leader peptide, bacteriocin gene, immunity gene, modifier gene, and a secretion machinery such as an ABC transporter (Walsh et al. 2015 ). However, for class II bacteriocins, which possess a simpler structure, expression solely using the bacteriocin gene itself is feasible. By utilizing a tight and inducible expression system, the need for an innate immunity gene can often be eliminated (Mesa-Pereira et al. 2017). Moreover, employing a native signal peptide for E. coli , such as OmpA, obviates the requirement for a specific bacteriocin leader peptide and transporter, as the bacteriocin can be secreted using the native E. coli secretion system (L. H. Zhang et al. 1995 ). This study assessed the impact of 75 class II bacteriocins on a collection of 49 representative gut microbiota strains. Our study encompassed common gut species including relevant pathogenic species and probiotic bacteria. Additionally, we tested 2 bacteriocins on a complex microbiota ex vivo and in vivo . The findings from this research provide valuable insights into the target range of class II bacteriocins, thereby contributing to their potential future application. Results In silico identification of class II bacteriocin genes and cloning into E. coli . To obtain a non-redundant list of bacteriocin sequences, all class II bacteriocins available in the BAGEL3 (Auke J. van Heel et al. 2013) and Bactibase (Hammami et al. 2010) databases were downloaded, resulting in a total of 233 non-redundant sequences (Supplementary Table 1). Subsequently, 84 sequences were selected based on being single component (the presence of one bacteriocin is sufficient to observe an effect), as well as successful verification through a blast search. 75 bacteriocins were successfully cloned and expressed in E. coli BL21-AI. Among the cloned bacteriocins, three originated from Gram-negative species, while the remaining 72 originated from Gram-positive species. The native bacteriocin producers span 5 different phyla ( Bacillota being the most prevalent) as well as 19 different genera with Lactobacillus and Enterococcus being the most prevalent (Supplementary Table 2). The bacteriocin genes were inserted into the pMUT vector, which is native to E. coli Nissle and has been previously used as an expression vector (Vaaben, Vazquez-Uribe, and Sommer 2022). The insertion was performed downstream of the E. coli native OmpA signal sequence and fused with a gfp reporter gene. The expression of the inserted genes was controlled by an arabinose-inducible T7 promoter (Figure 1A). Building the collection of representative gut microbiota strains To compile a comprehensive list of species associated with the representative gut microbiota a non-redundant list was generated based on a literature search encompassing four studies. The selected studies included species of the core microbiome (Shetty et al. 2017), gut microbiome strains in relation to their susceptibility for non-antibiotic drugs (Maier and Typas 2017; Maier et al. 2018), and gut strains with the ability to grow on the modified gut media mGAM (Tramontano et al. 2018). To validate the inclusion of the selected strains as part of the core microbiome, their prevalence in human stool samples was assessed using the assembled metagenomic species (MGS) identified from 396 stool samples obtained from the Human Microbiome Project (HMP), as reported by Nielsen et al. (Nielsen et al. 2014). A total of 56 strains were deemed suitable for inclusion in the representative gut microbiota collection. Among these, 49 demonstrated growth capability in either mGAM, MRS, or BHI media. The selected strains consisted of 24 Gram-negative and 25 Gram-positive species, encompassing a range of phyla including Bacteroides (11 species), Firmicutes (27 species), Proteobacteria (7 species), Actinomycetes (2 species), Fusobacteria (1 species), and Actinobacteria (1 species). For a detailed list of the selected strains, refer to Supplementary Table 2. In vitro screening of bacteriocins on the collection of representative gut microbiota strains The inhibition spectrum of each bacteriocin was assessed by employing the engineered bacteriocin-producing strains and conducting an overlay spot assay against the representative species of the gut microbiota (Suppl. Table 3). Inhibition was assessed through visual inspection of the plates and measurement of the inhibition halo size. A strain was considered inhibited if any inhibition zone was observed and exceeded the negative control (pMUT0). Among the 75 bacteriocins tested, 22 (29%) demonstrated inhibitory activity against at least one species in the assay and will henceforth be referred to as the inhibitory bacteriocins (Figure 1B). Of the inhibitory bacteriocins, 21 originated from Gram-positive species and predominantly inhibited other Gram-positive species. However, actifencin, naturally produced by Actinomyces ruminicola DPC 7226, displayed inhibition against the Gram-negative strain Bacteroides eggerthii DSM 20697. Notably, 5 out of the 24 Gram-negative strains were inhibited by at least one bacteriocin. Bacteroidetocin A, derived from the Gram-negative Bacteroides vulgatus , exclusively inhibited other Gram-negative species (4 species), including B. vulgatus itself (DSM 1447). The species most frequently inhibited by the bacteriocins were Lactobacillus sakei ATCC 15521 (21 bacteriocins), Enterococcus faecalis ATCC 19433 (18 bacteriocins), and Listeria innocua DSM 20649 (13 bacteriocins). Actifencin exhibited the highest inhibitory activity against the tested species, affecting 11 out of 49 strains. To elucidate the observed variations in the inhibitory spectra among the bacteriocins, a multiple sequence alignment of their sequences was performed using COBALT multiple alignment tool (Table 1). Additionally, the structure prediction tool alpha-fold was utilized to visually analyze differences between the bacteriocins (Figure 2). The analysis identified that the consensus sequence xYGNGV, known to be conserved among class IIa bacteriocins (Coyne et al. 2019) was present in a majority of the examined bacteriocins. In addition, these bacteriocins shared highly similar structural characteristics, comprising a helix, a beta-sheet, and in certain instances, a leader sequence that also adopted a helical conformation. It was also observed that bacteriocins deviating from this consensus sequence exhibited distinct structural differences (refer to the top 6 sequences in Table 1 and the top row in Figure 2). Table 1: Multiple alignment of the bacteriocin sequences reveal highly conserved amino acids. Bacteriocin Class Sequence 25_EJ97enterocin D MLAKIKAMIKKFPNPYTLAAKLTTYEINWY-KQQYGRY--------PWERPVA 13_actifencin A GFGCNL--------ITSNPYQCSNHCKSV--------GYRG------GYCKLRTVCTCY 33_garvieacinQ A EYHLMNGANGYLTR-VNGKYVYRVTKDPVSAVFGVISN--------GWGSAGAG----FGPQH 42_Bovicin_255_variant D GKGY-------CKPVYYAANGYSCRYSSGEWGYVVTKGTFQATTDVIATGWVSSLGGG--Y 46_Bovicin_255_peptide D GKGY-------CKPVYYAANGYSCRYSNGEWGYVVTKGAFQATTDVIANGWVSSLGGG--YFGKP 22_bacteroidetocin A A KFWGSRVECRV-QANGH---------CMCRKVYYRFGIRSYGGWWPAN---PSQC 12_ubericin-A A KTV NYGNG LYCNQ-KKC WV --NWSETATT IV NNSIMNGLTGGNAGWHSGGRA 10_Leucocin_C A ---K NYGNG VHCTK-KGCS V --DWGYAWTN IA NNSVMNGLTGGNAGWHN 18_Mundticin A ---K YYGNG VSCNK-KGCS V -- - DWGKAIG ---- II GNNSAANLATGGAAGWSK 70_Sakacin_5XSakX A ---K YYGNG LSCNK-SGCS V — - DWSKAI ----- SII GNNAVANLTTGGAAGWKS 8_sakacin_A A -AR SYGNG VYCNN-KKC WV —NRGEATQ -- SII GGMIS-------GWASGLAGM 58_Eenterocin_P-like D ATR SYDNG IYCNN-SKC WV -- - NWGEAKEN - IA GIVIS--------GWASGLAGM----GH 63_EnterocinP A ATR SYGNG VYCND-DKC WV --NWNEANQQ IA GIVIS--------GWASGLAGM--- - GH 53_Enterocin_SE-K4 A --AT YYGNG VYCNK-QKC WV —DWSRARSE - II DRGVK-------AYVNGFTKVLGGIGGR 6_Hiracin_JM79 A --AT YYGNG LYCNK-EKC WV —DWNQAKGE - I GKIIVN--------GWVNHG-----PWAPRR 52_Bacteriocin_31 A -AT YYGNG LYCNK-QKC WV — - DWNKASRE -- I GKIIVN-------GWVQHG-----PWAPR 38_Penocin_A A ---K YYGNG VHCGK-KTCY V — -- DWGQATAS - I GKIIVN--------GWTQHG-----PWAHR 50_Divercin_V41 A -TK YYGNG VYCNS-KKC WV -- -- DWGQASGC - I GQTVVG------GWLGGAI— -- PGKC 35_Leucocin_A A ---K YYGNG VHCTK-SGCS V -- -- NWGEAFSAG V HRLAN--------GGN------GFW 36_Mesentericin A ---K YYGNG VHCTK-SGCS V -- -- NWGEAASAG I HRLAN--------GGN-------GFW 31_Plantaricin_423 A ---K YYGNG VTCGK-HSCS V- - -- NWGQAFSCS V SHLAN--------FGH-------GKC 32_sakacin_G_skgA2 A ---K YYGNG VSCNS-HGCS V- -NWGQAWTCG V NHLAN-------GGH-------GVC Table 1: highly conserved amino acids are highlighted in red. The top 6 bacteriocins does not contain these conserved regions (multiple alignment tool: https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi). The six bacteriocins exhibiting deviations from both the common structure and sequence patterns typical for class II bacteriocins: #25_E97enterocin (class IId), #13_actifencin (class IIa), #33_garvieacinQ (class IIa), #42_bovicin_255_variant (class IId), #46_Bovicin_255_peptide (class IId), and #22_bacteroidetocin_A (class IIa) also displayed differences in their species inhibition pattern. For instance, #46_Bovicin_255_peptide specifically targeted the Gram-negative species Bacteroides fragilis DSM2151 and Collinsella aerofaciens DSM3979 . In contrast, its closely related sister sequence, #42_bovicin_255_variant, did not exhibit inhibition against these two species. This highlights that even slight variations in the sequence can lead to differences in the target spectrum of the bacteriocins, which is generally observed for class IIa bacteriocins (Ennahar et al. 2000). These findings emphasize the importance of determining the target specificity of bacteriocins for even very similar sequences and provide insights into the molecular basis of their inhibitory activities against specific bacterial strains. To further evaluate the potential of the bacteriocins examined in this study as novel antibiotics or targeted therapeutics, we compiled a table of species associated with diseases or disorders(Table 2). Table 2: Inhibited strains in our assay and the strains association to disease or spoilage. Strain Disease Inhibited by bacteriocins Reference Collinsella aerofaciens DSM 3979 * Increased in psoriasis patients’ stool, increased in coronary artery disease (CAD) 13,46 (Shapiro et al. 2019; Liu et al. 2019) Enterococcus fecalis Leading causes of highly antibiotic-resistant, hospital-acquired infection, Dental Root Canal Pathogen 6, 8, 10, 12, 18, 25, 31, 32, 33, 35, 38, 46, 50, 52, 53, 58, 63, 70 (Shankar, Baghdayan, and Gilmore 2002; Alghamdi et al. 2020) Eubacterium rectale DSM 17629 * E. rectale functions as a ‘driver’ bacterium and contributes to cancer initiation via promoting inflammation. 13 (Wang et al. 2021) Prevotella copri DSM 18205 High prevalence of the bacteria Prevotella copri in patients with rheumatoid arthritis (RA) 22 (Bernard 2014) Phocaeicola dorei DSM 17855 Bloodstream infection 22 (Cobo et al. 2022) Bacteroides vulgatus DSM 1447 Involved in colitis, both positive and negative depending on the strain 22 (Li et al. 2021) Bacteroides eggerthii DSM 20697 * Enhancing the severity of DSS induced colitis in mice ( B . eggerthii 12986) 13 (Dziarski et al. 2016) Listeria inoccua DSM 20649 Closely related to the foodborne pathogen L. monocytogenes 6, 8, 10, 12, 18, 31, 35, 38, 50, 52, 53, 58, 63, 70 (Buchrieser et al. 2003) Lactic Acid Bacteria Rare infections, especially in diabetic and immunocompromised individuals and with increased microbial ethanol production leading to non-alcoholic fatty liver disease (NAFLD) All the inhibitory bacteriocins were able to inhibit at least one LAB except #22 (Meijnikman et al. 2022; Rossi et al. 2022). (*) indicates that this species not before has been characterized to be inhibited by this bacteriocin. As an example Collinsella aerofaciens , a biosafety level 2 organism, has been implicated in psoriasis (Shapiro et al. 2019) and coronary artery disease (CAD) (Liu et al. 2019). It is also commonly found in the human gut, present in approximately 86% of the 396 stool samples from the Human Microbiome Project (Nielsen et al. 2014). In our study, we observed that two of the tested bacteriocins, #13_actifencin and #46_Bovicin_255_peptide, targeted this particular strain. Considering the specific targeting of Collinsella aerofaciens in the gut, the administration of bacteriocin #46_Bovicin_255_peptide could potentially lead to the elimination of this unwanted species. Notably, #46_Bovicin_255_peptide targets only five species from the representative gut microbiota collection meaning that administration of this bacteriocin most likely would not lead to overall disruption of the microbiota. Other optional targets could be to target the gut strain Bacteroides vulgatus involved in colitis, both positive and negative depending on the strain (Li et al. 2021). These findings suggest that bacteriocins such as #13_ actifencin, #46_bovicin_255_peptide and #22_bacteroidetocin A among others holds promise for selectively targeting specific unwanted strains within the gut microbiota, offering a potential strategy for modulating microbial composition while minimizing disturbances to the overall ecosystem. Selective alteration of fecal-derived microbial communities in vitro Based on the observations during screening, we hypothesized that we could selectively target and remove certain species within a complex microbial community by expressing the bacteriocins in situ. For this characterization 3 bacteriocins were selected: #12_ubericinA (narrow spectrum against Gram-positive bacteria), #13_actifencin (broader spectrum against both Gram-positive and Gram-negative bacteria), and #22_bacteroidetocin A (narrow spectrum against Gram-negative bacteria). To assess the effect of the selected bacteriocins murine fecal samples were plated on selection plates containing each of the different bacteriocins. Using 16S rRNA amplicon sequencing we characterized the microbial community composition for the different bacteriocin and media combinations (Figure 3A). The number of colony-forming units (CFU) observed on the different conditions varied substantially. The CFU counts of #22_bacteroidetocinA + mGAM and #22_bacteroidetocinA + mGAM-vk were significantly lower compared to the control (Figure 3B). Beta-diversity analysis of these samples revealed distinct clusters corresponding to the selection media and the different bacteriocins used (Figure 3C). A heatmap was generated to visualize the abundance of Amplicon Sequence Variants (ASVs) with at least 5% abundance in at least 5 of the samples. The results showed a clear reduction in ASVs associated to the genus Lactobacillus selected on the #13_actifencin + mGAM media, but not with the #13_actifencin + MRS media. Additionally, there appeared to be a decrease in ASVs mapped to the genus Bacteroides , particularly Bacteroides vulgatus , on the #22_bacteroidetocin + mGAM and #22_bacteroidetocin + mGAM-vk selection plates (Figure 4) Relative abundance plots were generated to analyze the distribution of ASVs across different media and bacteriocin conditions. The plots revealed distinct patterns of ASVs mapped to specific genera. On the mGAM media, the majority of ASVs were associated with the genus Lactobacillus , except in the #13_actifencin + mGAM condition where a higher abundance of ASVs mapped to Bacteroides was observed (Figure 3B). This difference was found to be statistically significant (Figure 3D), with a significant decrease in an unclassified Lactobacillus species and the overall genus Lactobacillus . Concurrently, an increase in the Escherichia-Shigella genus was observed (Figure 3D). Notably, Bacteroides faecichinchillae showed high abundance (indicated in yellow) on the plot, although statistical significance could not be established. On the MRS selection plates, ASVs primarily mapped to the genus Lactobacillus , but no significant differences were observed among the different groups (Figure 2B). In the case of the #22_bacteroidetocinA + mGAM selection plates, a significant decrease in Bacteroides and specifically Bacteroides vulgatus was observed (Figure 3B and 3C). These results indicate that #22_bacteroidetocinA is indeed capable of inhibiting naturally occurring microbiota strains of Bacteroides vulgatus in a complex community. Evaluation of bacteriocin-producing E. coli Nissle strains in mice To investigate the impact of bacteriocins on a complete microbiome in mice, we chose to express the two bacteriocins #13_actifencin and #22_bacteroidetocin A, which were found to substantially modulate the microbiota community composition ex vivo . For the in vitro and ex vivo studies we utilized the E. coli production strain BL21-AI. However, BL21-AI express bacteriocins under an inducible promoter, and is not optimized for in vivo growth. Therefore, we chose to engineer Escherichia coli Nissle (EcN) to produce bacteriocins under a constitutive promoter. EcN was selected as the production strain due to its favorable characteristics as a safe probiotic organism (GRAS status) with robust growth capabilities under anaerobic conditions. To ensure the suitability of these engineered strains, we evaluated their growth rates and confirmed their functional properties through an overlay spot assay against indicator strains (Supplementary figure 1). The engineered strains EcN_ pMUT13_actifencin and EcN_ pMUT22_bacteroidetocinA exhibited an average doubling time that was 50% and 37.5% higher than that of the EcN_ WT_GFP strain. The EcN_ pMUT0_control strain showed an increase in doubling time of 15% (Supplementary Figure 1) The in vivo study spanned a period of 7 consecutive days, during which the mice received daily oral doses of the respective strains followed by a 7-day washout period. The study design allowed us to investigate the colonization potential of the bacteriocin-producing strains compared to the control group and confirm the elimination of the EcN strains from the gut over time (Figure 5A). Colonization of the EcN cultures was observed at approximately 10 5 -10 6 colony-forming units (CFUs) per gram of feces over the 7-day oral delivery period. By day 9, the CFUs decreased to approximately 10 4 , and no surviving EcN colonies could be detected on days 11 or 14 (Figure 5C). The functionality of EcN strains from fecal samples was confirmed using an overlay spot assay with indicator strains L. mali DSM 20444 and B. vulgatus DSM 1447 . Detection of small inhibitory zones around colonies validated the bacteriocin production of EcN_ pMUT22_bacteroidetocinA. (Figure 5B) and restreaks of EcN from the fecal samples were compared to the EcN strain before entering the mouse gut as well as a negative EcN control, showing distinct inhibition zones around the four EcN colonies tested from the fecal samples. Nine colonies of both EcN _pMUT13_actifencin and EcN _pMUT22_bacteroidetocinA underwent colony PCR and Sanger sequencing, confirming their identity as the correct EcN strains after having been through the murine gut with no mutations detected in the bacteriocin gene or promoter region. Fecal samples were collected on study day 0 (prior to the first oral delivery), day 7, and day 14 for sequencing analysis. Despite the ex vivo findings indicating differential abundance of certain bacterial genera and species, no discernible differences in ASVs were detected in the fecal samples from any of the mouse study groups. Specifically, we focused on examining the overall abundance of Lactobacillus and Bacteroides vulgatus , the species that exhibited differential abundance in the ex vivo study. In addition, we analyzed and graphically represented the prevalence of ASVs that corresponded to the genus Escherichia-Shigella , observing their presence within the murine gut. Nevertheless, no distinct difference emerged between the experimental groups receiving gavage and the control group treated with PBS (Figure 5E). Given that the CFUs in feces approximated ~10 6 , and assuming a colon density of 10 11 , the abundance of EcN in the colon would be 10 6 /10 11 = 10 -5 or 0.00001% of the total bacterial population. Given this relatively low abundance, we do not anticipate quantifying EcN in the gut using 16S rRNA amplicon sequencing. Discussion Bacteriocins and their native producers have been extensively used in the food industry to combat food pathogens. Recent studies have explored their potential as novel antimicrobial agents, particularly against antibiotic-resistant pathogens, due to their specificity compared to traditional antibiotics and their ability to not disturb the overall composition of the microbiome. These traits in particular highlights their potential and benefits as a novel group of antibiotics (Dabour et al. 2009; Rea et al. 2010; Heilbronner et al. 2021). In this study, we aimed to investigate the effects of class II bacteriocins on a selection of representative gut microbiota strains to further asses their target spectrum. We performed in vitro screening of 75 class II bacteriocins against 49 species, including pathogens such as C. difficile and Salmonella enterica subsp. Enterica DSM 5569 . Consistent with previous studies, most class II bacteriocins generally did not affect Gram-negative species (Umu et al. 2016; Rea et al. 2010). The only exception was #13_actifencin and the Gram negatively produced bacteriocin #22_bacteroidetocinA. #13_actifencin is natively produced by Actinomyces ruminicola DPC 7226 and has been characterized by Sugrue et al. (Sugrue et al. 2020). In our study actifencin was seen to target gut species associates with various diseases and disorders, as well as a broad range of LAB species both in vitro and ex vivo . Typically LAB species are recognized for their beneficial properties as probiotics, but have also been associated with rare infections, especially in diabetic and immunocompromised individuals (Rossi et al. 2022). Furthermore, they have been associated with increased microbial ethanol production leading to non-alcoholic fatty liver disease (NAFLD) (Meijnikman et al. 2022; Kuraji et al. 2023). Actifencin is believed to be part of a new class of bacteriocins produced by the Actinomyces genus. Sugure et al. report that 47 of 161 genomes were seen to exhibit at least one actifencin-related bacteriocin with a high sequence diversity amongst the genes. Based on the findings in our study we hypothesize that this new group of bacteriocins inhabit a very interesting potential as future antimicrobials that should be explored further. The other bacteriocin showing interesting inhibition patterns both in vitro and ex vivo , was #22_bacteroidetocinA, a compound first characterized by Coyne et al. (Coyne et al. 2019), and natively produced by Bacteroides vulgatus . Our analysis discerned the ability of bacteroidetocin A to impede four species: Prevotella copri DSM 18205 , Bacteroides vulgatus DSM 1447 , Bacteroides clarus DSM 22519 , and Bacteroides stercoris DSM 19555 . To back this up our ex vivo sequencing analysis revealed the complete depletion of ASVs assigned to the Bacteroides genus, including Bacteroides vulgatus . Conversely, an increase in ASVs from the Tannerellaceae family, belonging to the order Bacteroidales , was observed, suggesting the presence of Bacteroides strains not targeted by #22_bacteroidetocinA. Interestingly, our in vitro discoveries diverged considerably from those reported by Coyne et al., in which bacteroidetocin A was shown to inhibit Bacteroides thetaomicron , Parabacteroides merdae DSM 19495, and Bacteroides fragilis DSM 2151. These species, however, did not exhibit inhibition in our study. One reason could be the media used in the inhibition assay, where in our study was used mGAM, Coyne et. al tested the susceptibility of Bacteroides strains in BHI media. Media type has before been shown to be important for the susceptibility of bacteria against class II bacteriocins (Carl et al. 2004). This discordance emphasized the importance of studying bacteriocins in relation to the environment in where they are intended to function. Furthermore, it could imply the likelihood of bacteroidetocin A operating through a strain-specific targeting mechanism or that unidentified immunity mechanisms are present in the resistant strains. Considering the strain-dependent involvement of B. vulgatus in colitis, as indicated by S. Li et al (Li et al. 2021), our results emphasize the necessity to broaden our understanding of the target specificity of bacteroidetocin A. This pertains not only to better characterization of the implicated B. vulgatus strains in colitis, but also to the identification of potentially harmful variants and the establishment of their respective inhibitory profiles. The exact mechanism of action of bacteroidetocin A still remains elusive (Coyne et al. 2019), underscoring the need for further investigation and continual research to both fully comprehend and potentially exploit the antibacterial capabilities of bacteroidetocin A. Significant alterations in species composition observed in the ex vivo study did not extend to the in vivo experiment. Nevertheless, EcN did colonize the murine gut during the delivery phase, although it was entirely eliminated within 3-4 days post-delivery cessation. These observations align with the prevailing understanding that EcN can transiently colonize the mammalian gut but fails to persist without sustained delivery (Martinson and Walk 2020). The resilience of the EcN producers was evaluated using an overlay spot assay, which confirmed their ability to inhibit target strains post-transit through the mouse gut. Potential reasons for the observed lack of significant gut microbiota shifts could include suboptimal bacteriocin production by the EcN strains or a metabolic burden from bacteriocin expression (as suggested by our assessment of growth rates of bacteriocin producers to be 50% and 37.5% higher than the EcN _WT_GFP strain for #13_actifencin and #22_bacteroidetocinA producers, respectively). Other reasons could be the rapid in vivo digestion of bacteriocins, as well as the spatial separation between the producers and target strains. Considering the differential gut colonization patterns of LABs and EcN - with the former mostly populating the upper intestine (Walter 2008) and the latter largely colonizing the colon (Grauke et al. 2002), as well as the ability of B. vulgatus and other Bacteroides members to produce biofilm and to colonize the intestinal mucosa (Béchon and Ghigo 2022), complicates delivery with bacteriocins further. This discordance between in vitro , ex vivo and in vivo efficacy underscores the complex, context-dependent character of bacteriocin activity and the hurdles inherent in extrapolating in vitro results to in vivo applications. Nevertheless, our in vitro and ex vivo and results accentuate the potential of bacteriocins as precision modulators of the gut microbiota. Enhancing in vivo bacteriocin effectiveness may be achieved via strategies such as strengthening protein stability while maintaining efficacy, as demonstrated by Field et al., 2019 with nisin (Field et al. 2019). An alternative approach could involve shifting the choice of production host to Lactococcus lactis (Song et al. 2017) or Lactobacillus plantarum (N. Zhang et al. 2020). Our study, through the utilization of a heterologous host, facilitated high-throughput screening of class II bacteriocins, an approach that could extend to other bacteriocin classes, like the novel actifencin-like group. Furthermore, this methodology opens avenues to facile host optimization via engineering, thereby addressing the challenge of effective in vivo delivery. Future research should focus on optimizing bacteriocin production and delivery, thereby maximizing their potential as novel antimicrobial agents. This could involve identifying optimal gut-colonizing hosts or improve bacteriocin stability for optimized efficacy. Materials and methods Selection of bacteriocin genes All class II bacteriocins from the Bagel3 database (Auke J. van Heel et al. 2013) (now known as Bagel4: http://bagel4.molgenrug.nl/) as well as all class II bacteriocins available from Bactibase https://bactibase.hammamilab.org/main.php bactibase.hammamilab.org was downloaded (date of download: 5 th of February 2020) and used to create a non-redundant list of all bacteriocin sequences. Each sequence was verified in the original literature, the original secretion taq was identified in the sequence (often before a GG double glycine sequence) and only the pro-peptide was used for expression. The DNA sequence was codon optimized for production in E. coli K-12 from IDT online codon optimization tool (https://eu.idtdna.com/CodonOpt). 84 of the ~ 200 available Class II sequences available in the databases were selected for cloning based on the following criteria: having a unique sequence, being 1 compartment, and having a sequence that has a 100% identity match when using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). 2 bacteriocins: Actifencin(Sugrue et al. 2020) and Bacteroidetocin A(Coyne et al. 2019) was added to the collection as well. In vitro screening: Selection of gut strains to build the representative gut strain catalogue: The gut strains were selected based their ability to grow on mGAM as well as their prevalence in the human gut: (Maier and Typas 2017; Shetty et al. 2017; Tramontano et al. 2018; Maier et al. 2018; Forster et al. 2019) . Furthermore a script was used to create a list of the prevalence of strains found in the study: (Nielsen et al. 2014). Most strains were ordered from the Leibniz institute (DSMZ-German Collection of Microorganisms and Cell Cultures). Supplementary Table 2 contains an extensive list of the microorganisms, their prevalence, source, and growth medium. Plasmid and strain construction for expression in E. coli BL21-AI: Primers, and plasmids in Supplementary Table 3. Primers and gBlocks were ordered from Integrated DNA Technologies (IDT). gBlocks containing the bacteriocin sequences were ordered from Twist Bioscience. List of bacteriocin sequences can be found in Supplementary Table 1. Native E. coli Nissle plasmid pMUT-kanR-Hok/Sok was used as the vector to produce the bacteriocins. The native signal sequence for E. coli OmpA (Freudl, Klose, and Henning 1990) was placed immediately in front of the bacteriocin gene. A T7 promoter sequence was used to facilitate arabinose induction from the BL21-AI strain. All plasmid assemblies were conducted with Gibson assembly (Gibson et al. 2009) and transformed into Escherichia coli One Shot TOP10 (Thermo Fisher Scientific) via electroporation. Cells were recovered in SOC + 0.5ml 1M Mg2Cl2 + 2ml 1M glucose, for 1 hour at 37C with shake, then plated on LB agar plates containing 50ug/ml kanamycin (Roth Art-Nr: T832.3) and incubated at 37C ON. All E. coli were grown in lysogeny broth (LB) (Sigma Aldrich) unless something else is specified. Colony-PCR using OneTaq (Thermo Scientific ™ ) confirmed the plasmid assembly. PCR product was Sanger Sequenced using Eurofinsgenomics. Plasmids were extracted using plasmid extraction kit (Machery-Nagel - Nucleospin plasmid easy pure - 740725.250) and transformed into Invitrogen TM BL21-AI TM Oneshot ® Chemically Competent E. coli according to suppliers’ protocol (Thermo Fisher Scientific 11540146) and selected on LB-kanamycin plates. Colony-PCR using OneTaq was performed again to verify integration into BL21-AI. Evaluations of optimal expression conditions for the bacteriocins: Optimal growth conditions were examined using the target strain Lactobacillus mali DSM 20444 using 8 different bacteriocins. The following growth conditions were tested: incubation temperatures of the plates (from the time BL21-AI was spotted on the plates to the end of the study) at 25, 30, or 37C, preculture overnight growth of BL21-AI in either mGAM of 2-YT, 6 hours of growth versus 18 hours of growth of the spotted BL21-AI culture on the plate before pouring the top agar, 0.2% vs 1% of arabinose concentration in the culture plates. The inhibition zones of the bacteriocins were used to evaluate the optimal growth conditions (which were very comparable through all the groups. The following protocol produced the largest inhibition zones for all bacteriocins tested. Expression of bacteriocins for spot assay: All bacteriocin expression for spot assays was performed in the following manner unless something else is stated. The bacteriocin producer was streaked on LB-kanamycin plates from a -80 cryo-stock and incubated at 37C ~14-18 hours. One colony was inoculated into 2-YT media containing 50ug/ml of kanamycin and incubated with shake for ~14-18 hours. Cultures were spun down and resuspended in PBS, then spun down again and resuspend in 2-YT to make sure all kanamycin was gone from the media. 10ul of the culture was spotted on mGAM square plates containing 1% arabinose (for induction of the bacteriocin gene via the T7 promoter). Plates were placed in aerobic conditions at 37C for ~20 hours. After 20 hours the plates that were used to screen anaerobic strains were transferred to ANO boxes and incubated further at 37C ON (~20hours) to pre-reduce the plates. Plates that were used to screen aero-tolerant strains were poured with top agar after the initial 20 hours of incubation. As a negative control was used BL21-AI with a pMUT plasmid present with no bacteriocin gene. Cultivation of target strains for spot assay: Target anaerobic gut strains were grown in mGAM where possible. Aerobic Lactic acid bacteria were grown in MRS, E. faecalis and L. innocua were grown in BHI. Anaerobic strains were streaked from -80C cryo-stocks under anaerobic conditions (Whitley A95 Workstation - Don Whitley Scientific); gas mixture, 95% N 2 and 5% H 2 . Lactic acid strains were streaked from -80C in aerobic conditions on MRS and transferred to anerobic boxes. Strains growing on BHI were streaked aerobically and incubated aerobically. All strains were incubated at 37C ~ 18 hours. One colony was used to inoculate 1ml of the respective media and incubated for additionally 18-20 hours prior to the spot assay. Strains with Biosafety level2 were handled in the same manner, except that a Coy Laboratory Products Vinyl; gas mixture, 95% N 2 and 5% H 2 was used to make anaerobic conditions. Overlay agar for spot assay: For each square plate (Thermo fisher scientific omnitray w/lid Non treated sterile #264728) in total of 12ml pre-reduced top agar was used (0.5% agar) (Milipore # 69964) + 100ul of the target strain adjusted to OD ~0.5. Aerobic strains: Strains were mixed with top agar and poured over the plates. The plates were transferred to anaerobic boxes and incubated for 1-2 days at 37C. Anerobic strains: Agar plates with spots of BL21-AI bacteriocin producers and the liquid cultures containing the target strains, were transferred from anaerobic conditions into an aerobic laf bench. Top agar (~40C) was mixed with each of the target strains and poured over the respective plates and left to dry for ~5-10 mins before being transferred back to the anaerobic chamber and incubated in anaerobic boxes at 37C for 1-2 days. After 1 and 2 days of growth the plates were checked for inhibition zones. All strains were tested in at least duplicates. Halosize in mm can be found in Supplementary Table 4. Data analysis: Heatmap with inhibited species against the bacteriocins with at least 1 target strain was generated using R (phyloseq). NCBI common taxonomy tree (“Common Taxonomy Tree” n.d.) was used to build a phylogenetic tree of the target species, and the heatmap was ordered according to that. Clustal Omega (Sievers et al. 2011) was used to create a multiple alignment of the protein sequences of the bacteriocins, and the heatmap was sorted according to that. Ex vivo screening study Growth media preparation: The following plates were used in the study: MRS containing 25% of E. coli BL21-AI supernatant from either BL21-pMUT0-no-bacteriocin, BL21-pMUT12-ubericinA, BL21-pMUT13-actifencin, to select for a variety of LAB strains. mGAM agar plates containing 25% of supernatant from BL21-pMUT0-no-bacteriocin, BL21-pMUT12-ubericinA, BL21-pMUT13-Actifencin, BL21-pMUT22-bacteriodetocinA to select for gut strains in general. mGAM-vancomycin (5ug/ml)-kanamycin (50ug/ml) containing 25% of supernatant from BL21-pMUT0-no-bacteriocin, BL21-pMUT22-bacteroidetocinA, to select for a variety of Bacteroides strains. Supernatant for creating the agar plates were produced in the following manner: BL21-AI cultures were streaked on LB-kanamycin (50ug/ml) agar plates from -80C cryostocks and incubated at 37C for ~ 20hours. 1 colony was inoculated into 5ml 2YT-kanamycin(50ug/ml) and incubated with shake ~18 hours at 37C. Cultures were diluted 1:100 into fresh 200ml 2YT media in 1L shake flasks – without antibiotics and incubated with shake at 37C until OD reaches 0.4-0.5. Cultures were then induced with 1% arabinose and incubated with shake for 6 hours at 30C. After 6 hours the cultures were centrifuged for 5 mins in 50ml falcon tubes at 4500xG at 4C, 1 tablet of protease inhibitor (Roche - cOmplete ULTRA Tablets, Mini, EDTA-free #05892791001) was added to every 50ml of supernatant. The supernatant was sterile filtered with 200ml filter cups (Biofil - FPV213500). Supernatant was immediately used to make agar plates by mixing with 60C freshly prepared 1.25X concentrated media. mGAM and mGAM-vancomycin-kanamycin plates were moved to anaerobic environment for prereduction for 24 hours. MRS plates were stored aerobically at 5C. Feces collection: Feces from 3 male C57BL/6nTAC mice that had received CHOW diet, and no antibiotics was collected and transferred to anaerobic environment within ~ 10 mins. Pellets were resuspended in 1ml pre-reduced 1% PBS. After resuspension the tubes were left for ~ 20 mins to allow sediment to form. 2x 200ul of the samples were transferred to 1.5ml Eppendorf tubes and centrifuged at 10.000xG for 10 mins. Supernatant was removed and the pellet was stored at -20C, for sequencing. 100ul of the fecal samples was used to make serial dilutions in 1% PBS down to 10 -8 . 100ul of the three independant biological replicates were plated of the dilutions 10 -4 -10 -8 on mGAM and mGAM-vancomycin-kanamycin plates. Plates were incubated anaerobically at 37C. For the MRS plates, selecting for lactic acid bacteria, the dillutions 10 -3 – 10 - ⁷ dilutions were used. MRS plates were incubated aerobically, to allow further selection for lactic acid strains, at 37C. Colonies were counted every day for 5 days – until no new colonies appeared on the plates. Three dilutions, consisting of the countable dilution (between 25 and 250 colonies) and the dilutions above and below it, were chosen for sequencing. None of the plate dilutions utilized represented a situation where a lawn was formed or the plate would not be able to be counted. 2ml 1XPBS where administered onto the plates and a spatula was used to mix the colonies on the plates. ~1.2ml was collected in 1.5ml eppendorph tubes, and centrifuged 15min 12000XG. Supernatant was removed at pellets were frozen at -20 untill DNA extraction. The pelleted samples were then extracted as individual dilutions using ZymoBiomics DNA Kit (D4300- zymoresearch). DNA preparation for and 16S rRNA MiSeq sequencing: All DNA samples were prepared for sequencing using the following protocol: “16S Metagenomic Sequencing Library Preparation (“16s-Metagenomic-Library-Prep-Guide-15044223-b.Pdf” n.d.)”. In short: Extracted DNA was diluted to 5 ng/ml for all samples to keep quantities constant for the amplification step. PCR was performed using KAPA PCR Master Mix (Roche) and tagged Illumina primers (10 mM concentration) in 25 ml reactions targeting the hypervariable V3-V4 (341F - 785R) region (primer are listed in Supplementary Table 3). Illumina overhangs (100 mM concentration) were attached in a second PCR reaction by combining barcoded samples in equal amounts as template for amplifying multiple 50 ml reactions. Thermocycling conditions for both PCR steps were as follows, except 25 cycles in the first PCR and 8 cycles in the second PCR: initial denaturation 95 °C for 3 min, followed by 25 or 8 cycles of: 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, and a final elongation at 72 °C for 10 min. PCR product sizes were confirmed at each step and the final PCR product was purified using AMPure XP bead (Beckman coulter). The samples were measured with qubit and normalized to 10nM/ul (diluted in Tris_HCL pH:8.5), then run on Agilent 2100 Bioanalyzer (Covaris) to verify the size of the fragments, and sequenced on an Illumina MiSeq system. Sequence quality control and processing: Fastq files were downloaded from Basespace.illumina.com. Qiime2 was used to process the fastq files to count matrices, followed by downstream data analysis using R. The following tutorial was used to perform the analysis using Qiime2 (“Metadata in QIIME 2 — QIIME 2 2022.8.3 Documentation” n.d.). In short: fastq files were imported into qiime2 as paired end with input phred33. Next quality filtering, chimera checking, and paired- end read joining of the sequence data was perform with DADA2 (Callahan et al. 2016) through the q2-dada2 plugin. For the ex vivo study: reads were truncated when the quality score became approximately below 25 (forward reads at 285bp, reverse reads at 240bp). Reads were filtered from each sample (between 55.49% and 89.85% with a mean of 85.32% - reads per sample were between 4621 and 633583 with a mean of 89248 reads). For the in vivo study: reads were truncated when the quality score became approximately below 25 (forward reads at 260bp, reverse reads at 240bp). Reads were filtered from each sample with a mean of 57% - reads per sample were between 401 and 104.077 with a mean of 70.255 reads). For both the ex vivo and in vivo study: A feature table and feature data were generated using the command “qiime feature-table” describing the ASVs that were observed in each sample and how many times it was observed. To assign taxonomic information to the ASV sequences a trained classifier for the V3-V4 region based on the “SILVA release_139 nr99” SSU database, which uses 99% similarity to assign species to an ASV. The classifier was downloaded from Github: https://github.com/anweshmaile/silva-138_classifiers. The command “qiime feature-classifier” was used for this analysis, outputting a count matrix used for further processing in R using the phyloseq package. In R the further data processing was handled. ASVs were removed if they had less than 2 counts in at least 10% of samples. This reduced ASVs from 4541 to 332 taxa in the ex vivo study and removing ASVs with less than 2 counts in 5% of samples reduced the number of taxa from 4600 to 2293 in the in vivo study. Rarefaction curves were made for both studies with curves showing max species at ~2000 species in both cases, therefore rarefaction was performed using 2000 species per sample. This removed five samples from the ex vivo data, to leave in total 76 samples, and one sample in the in vivo data to leave in total 71 samples. Beta-diversity was examined using Bray-Curtis method quantifying the difference between the overall taxonomic composition between samples. In vivo study: Plasmid and strain construction for expression in E. coli Nissle: Primers, promoters and sequences are listed in the Supplementary Table 3. Primers and geneblocks were ordered from Integrated DNA Technologies (IDT). The probiotic strain used in this study, EcN _GFP, StrepR: E. coli Nissle 1917 is a modified version of the wild-type E. coli Nissle 1917 (tradename Mutaflor, Ardeypharm, Germany) strain(Jacobi and Malfertheiner 2011). Plasmid pMUT (Zainuddin, Bai, and Mansell 2019)-kanR-HokSok was used as the vector. EcN _GFP was used as production host using a strong constitutive promotor (#1.7 from the Schantzetta library (Armetta et al. 2021)). The bacteriocins were secreted using the OmpA signal sequence (Freudl, Klose, and Henning 1990). The ribosomal binding site (RBS) of the bacteriocin gene was measured with salislab.net (Salis, Mirsky, and Voigt 2009). Strength was ~5000 compared to the RBS in the pMUT plasmid for in vitro expression of bacteriocins which was ~10.000. The following plasmids were cloned and expressed in EcN _GFP: EcN- pMUT0-no-bacteriocin, EcN- pMUT12-ubericinA, EcN- pMUT13-actifencin, EcN- pMUT22-bacteroidetocinA, and EcN- pMUT52_Bacteriocin31. Cloning and transformation, plasmid evaluation and purification was performed similar to the plasmid construction for E. coli BL21-AI expression. Generation of competent EcN : EcN _GFP was made competent for electroporation in the following way: Culture was streaked from -80C cryo-stock on LB-streptomycin (50ug/ml) agar plates and incubated at 37C for ~20 hours. Then, 1 colony was used to inoculate 5ml 2YT-streptomycin (50ug/ml) and incubated at 37C for ~20hours with shake. Cultures were diluted 1:100 and incubated at 37C with shake until OD reached 0.3-0.5 (~2 hours). When desired OD was reached cultures were placed on ice for 15mins, then centrifuged for 10 mins at 4C and 4500xG. Supernatant was removed and pellet was resuspended in 1ml MQ water+10% glycerol (4C). Cultures were centrifuged for 3 mins at 6500RPM at 4C, this step was repeated 3 times. After the last wash cells were resuspended in 50ul MQ water+10% glycerol (4C). 1ul of the purified plasmid was used for electroporation of the 50ul competent EcN _GFP cells. Cells were recovered in 1ml fortified SOC for 1 hour at 37C with shake, then plated on LB agar plates containing 50ug/ml kanamycin and incubated at 37C for 14-18 hours. Colony PCR and gel electrophoresis was used to identify clones. Plasmids were extracted and subjected to whole plasmid sequencing using plasmidsaurus.com. Growth rate of the clones were evaluated using a plate reader (Synergy H1 - Holm and Halby). Two clones of each of the four EcN _GFP strains were inoculated 1:100 in 3 replicates into 100ul LB-kanamycin (50ug/ml) in a 96-well plate. Growth was followed for 24hours in a plate reader (continuous shake 37C) (Synergy H1 - Holm and Halby). Data was extracted and doubling time was calculated using R. Animal experiment: Ethics: The animal experiment was conducted according to the Danish Animal Experiments Act on protection of animals used for scientific purpose (LBK 1107 from 02/07/2022) and Directive 2012/63/EU of the European Parliament. In addition, the protocol was licensed accordingly by the Animal Experimentation Committee under the Ministry of Food, Fishing, and Agriculture (license number 2020-15-0201-00405). The study was carried out in accordance with the ARRIVE guidelines (Percie du Sert et al. 2020). Animal study design: Twenty-four male C57BL/6NTac (Taconic Biosciences, Lille Skensved, Danmark) mice aged 5 weeks went through 7 days of acclimatization before being divided into four groups (n=6) based on weight stratification. Hereafter, the mice received 1 daily oral dosing of 100 µl either containing PBS, E. Coli Nissle pMUT-empty (CFU 10 11 ), E. coli Nissle pMUT13_actifencin (CFU 10 11 ) or E. coli Nissle pMUT22_bacteroidetocinA (CFU 10 11 ). CFU was measured based on OD600 measurements and standard curves created by spotting different dilutions of the gavage. After 7 days the mice went through a washout period of 7 more days. Colonization was investigated by fecal sampling on days: 0, 1,2,3,4,5,6,7, 9, 11, and 14. The mice were co-housed 3 per cage in individually ventilated cages (IVC). All mice were housed at 22 °C ± 2 °C, light cycle was 6 am to 6 pm, and the mice were given ad libitum access to water and chow diet (Safe Diets, A30). At end- study the mice were euthanized by CO 2 sedation and cervical dislocation. Content from small intestine, cecum and colon collected in 1xPBS to be tested for colonization (CFU count) immediately after collection. Colonization: The faeces were collected in pre-weighed 2.0 mL Eppendorf tubes including 1 mL of 1x PBS. After fecal samples had been added to the tubes these were weighed again to determine the faecal weight. All sample preparation for assessing CFU numbers was kept on ice and followed the same practice. The faecal samples were homogenised by vortexed at ~2400 rpm for 20 min. The samples were then spun down at 100xG for 30 seconds, followed by a dilution series, where 5 μL of each dilution was plated on LB supplemented with 50mg/ml kanamycin and 50mg/ml streptomycin. (Sigma Aldrich). After 24 hours CFU was determined by counting. Samples were then spun down for 20mins at 11000xG, supernatant was removed, and pellet was stored at -20C until DNA samples were extracted using DNeasy powersoil HTP 96 kit (qiagen Cat. No. 12955-4) DNA preparation for MiSeq 16S rRNA amplicon sequencing and sequence quality control was performed in the same manner as described for the ex vivo samples. Verification of EcN strains from mouse feces: 2-3 Fecal pellets from mice at study day 6 from the #13_actifencin and #22_bacteroidetocinA groups were resuspended in 200ml 1X PBS and serial diluted to 10^-3. 100ul of each dilution was plated on LB-kanamycin plates and incubated for 20 hours at 37C. EcN _pMUT22_bacteroidetocinA plates were transferred to ANO boxes and placed at 5C for 24 hours before performing the spot assay. 4 colonies from 2 replicate mouse feces of EcN _pMUT13_actifencin was re-streaked on LB plates containing no antibiotics and incubated for additional 20hours before performing the spot assay. Plates containing EcN _pMUT22_bacteroidetocinA was tested using the indicator strain Bacteroides vulgatus DSM 1447. EcN _pMUT13_actifencin was tested using the indicator strain: Lactobacillus mali DSM 20444. Indicator strains were cultured similar to what is described in the in vitro spot assay section. After pouring the top agar on the respective plates these were incubated ANO in the case for EcN _pMUT22_bacteroidetocinA and AE in the case for EcN _pMUT13_actifencin at 37C. After ~20 hours the plates were inspected for inhibition zones. 9 colonies of each of the EcN strains were subjected to colony PCR to verify that no mutations had occurred in the promotor or gene region of the plasmid. PCR product was Sanger sequenced with eurofinsgenomics. Media and antibiotic concentrations used and antibiotic references: Fortified SOC medium recipe: 100ml SOC + 0.5ml 1M Mg2Cl2 + 2ml 1M glucose, Tween80: Sigma-Aldrich 102578383, kanamycin sulfate: Roth Art-Nr: T832.3 (concentration used: 50ug/ml), streptomycin sulfate salt: Sigma-Aldrich - Merck Life Science 9137, Vancomycin: Sigma-Aldrich - Merck Life Science -94747-1G, arabinose: Sigma Aldrich - L-(+)-Arabinose W325501, MRS deMan, Rogosa, Sharpe media: Milipore # 69966, MRS deMan, Rogosa, Sharpe agar: Milipore # 69964, mGAM media and agar (modified Gifu Anaerobic Media – Nissui pharmaceutical CO.,LTD), BHI (Brain Heart Infusion, Merck #53286) Declarations Ethics approval and Consent to participate The animal experiment was conducted according to the Danish Animal Experiments Act on protection of animals used for scientific purpose (LBK 1107 from 02/07/2022) and Directive 2012/63/EU of the European Parliament. In addition, the protocol was licensed accordingly by the Animal Experimentation Committee under the Ministry of Food, Fishing, and Agriculture (license number 2020-15-0201-00405). The study was carried out in accordance with the ARRIVE guidelines (Percie du Sert et al. 2020) Consent for publication Not applicable Availability of data and materials The datasets supporting the conclusions of this article are available in the NCBI repository. Ex vivo data: BioProject ID PRJNA1007568 (“ID 1007563 - BioProject - NCBI” n.d.). The In vivo dataset supporting the conclusions of this article are available in the NCBI repository: BioProject ID: PRJNA1007563 (“ID 1007568 - BioProject - NCBI” n.d.) The datasets supporting the conclusions of this article are included within the article and its additional files: Supplementary files: Supplementary Table 1: List of the bacteriocins cloned and expressed in this study. Supplementary Table 2: list of representative gut microbiota strains. Supplementary Figure 1: Growth rate assesment for the EcN strains and spot assay for the EcN 13 and 22 Supplementary Table 3: Primers, promoters, and sequences Supplementary Table 4: List of screened bacteriocins against representative gut strains including halo-size of inhibition. Competing interests The authors declares that they have no competing interests Funding This work received funding from The Novo Nordisk Foundation under NNF grant number: NNF20CC0035580 and The Novo Nordisk Foundation, Challenge programme, CaMiT under grant agreement: NNF17CO0028232 Authors’ contributions MS: provided the initial and overall idea of the project. CR: designed the concepts for the in vitro experiment, CR & RV: designed the concepts for the ex vivo experiment, CR & DL: designed and conducted the in vivo study, CR: analyzed the data, CR: wrote the manuscripts with input from MS, RV, & DL. Acknowledgements Not applicable References “16s-Metagenomic-Library-Prep-Guide-15044223-b.Pdf.” n.d. 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Supplementary Files SupplementaryFigure1GrowthrateassesmentfortheEcNstrainsandspotassayfortheEcN13and22.docx SupplementaryTable1Listofthebacteriocinsclonedandexpressedinthisstudy.xlsx SupplementaryTable2listofrepresentativegutmicrobiotastrains..xlsx SupplementaryTable3Primerspromotersandsequences.xlsx SUPPLEMENTARYTABLE4.LISTOFSCREENEDBACTERIOCINSAGAINSTREPRESENTATIVEGUTSTRAINSINCLUDINGHALOSIZEOFINHIBITION..xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-3282788","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":228530704,"identity":"38eb4a2b-87be-4b08-937b-9fcc32d4adf9","order_by":0,"name":"Carola Elisa Heesemann Rosenkilde","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYDACCSD+2CAB5RkA8QEg5jmAXwvjTIgWxgawlmNEaGHmbWCAamEgQgv/7OaDj213WMjJz0h//uBHwR0Gvvs9BgxvzuCx5M6xZOPcMxLGBjdyDBt7DJ4xSB7jMWCccwO3FgOJHDPp3DaJxA0SOYwNPAaHGQyAWph5PuDTkv/9tyVQy/wZ6Q8b/xCnJYeNmRGopeFGgmEzwhY8DpO4kWYs2Qvyy5k3hrNlDA7zSB5LKzg4B4/3+WckP/zwc0ednHx7+oOPb/4cluM7fHjjgzfHcGvBADwg4gAJGkbBKBgFo2AUYAEAA4hVdQWHAkwAAAAASUVORK5CYII=","orcid":"","institution":"Technical University of Denmark","correspondingAuthor":true,"prefix":"","firstName":"Carola","middleName":"Elisa Heesemann","lastName":"Rosenkilde","suffix":""},{"id":228530705,"identity":"f6301e53-90e8-45da-92fd-8d653b8fbb98","order_by":1,"name":"Ruben Vazquez-Uribe","email":"","orcid":"","institution":"Technical University of Denmark","correspondingAuthor":false,"prefix":"","firstName":"Ruben","middleName":"","lastName":"Vazquez-Uribe","suffix":""},{"id":228530706,"identity":"37014d3c-63fd-4b91-b4db-94311bc358da","order_by":2,"name":"Ditte Olsen Lützhøft","email":"","orcid":"","institution":"Technical University of Denmark","correspondingAuthor":false,"prefix":"","firstName":"Ditte","middleName":"Olsen","lastName":"Lützhøft","suffix":""},{"id":228530707,"identity":"524455b8-0962-41d9-9ffe-6378f69a0ddc","order_by":3,"name":"Morten Otto Alexander Sommer","email":"","orcid":"","institution":"Technical University of Denmark","correspondingAuthor":false,"prefix":"","firstName":"Morten","middleName":"Otto Alexander","lastName":"Sommer","suffix":""}],"badges":[],"createdAt":"2023-08-21 14:14:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3282788/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3282788/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42264931,"identity":"76436912-92d9-433f-92c5-9ca4319df865","added_by":"auto","created_at":"2023-08-28 19:44:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":572031,"visible":true,"origin":"","legend":"\u003cp\u003eA) Graphical abstract of the \u003cem\u003ein vitro\u003c/em\u003e screening process. B) Heatmap showing inhibited species based on spot assay of 49 common core gut species. The heatmap is colored according to the halo-size of the \u003cem\u003ein vitro\u003c/em\u003e assay. White tiles indicate NA values. The phylogenetic relationship of bacteriocins and target strains are displayed on top and to the right of the plot, respectively.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3282788/v1/2d720de4ed7272237823a14f.png"},{"id":42265233,"identity":"1085d50e-a875-4fff-beb1-fa2d765fa791","added_by":"auto","created_at":"2023-08-28 19:52:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":363861,"visible":true,"origin":"","legend":"\u003cp\u003eThe predicted bacteriocin structures for the inhibitory bacteriocins. Structures were predicted using alpha-fold (Jumper et al. 2021), only the pro-peptide was used for the analysis. On the structure model, confidence is colored according to: Dark blue: Very high (pLDDT \u0026gt; 90) Light blue: Confident (90 \u0026gt; pLDDT \u0026gt; 70), Yellow: Low (70 \u0026gt; pLDDT \u0026gt; 50), Red: Very low (pLDDT \u0026lt; 50). ♱ No leader sequence; * Lower helix constitute the leader sequence. Structures could not be predicted for the following bacteriocins: #13_actifencin, #42_bovicin_255_variant, #63_enterocinP, #52_bacteriocin31, #38_penocinA.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3282788/v1/f2127ae13e9dfef46f018c04.png"},{"id":42265438,"identity":"b77a8ed7-ff5d-4aeb-82a7-8334796dd364","added_by":"auto","created_at":"2023-08-28 20:00:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":446211,"visible":true,"origin":"","legend":"\u003cp\u003eA) Graphical abstract of the ex vivo study setup. B) Relative abundance plot for strains isolated on mGAM, mGAM-vancomycin-kanamycin, and MRS media with different bacteriocin supernatants. On top is plotted the CFU isolated on the plates that was sequenced. P-values refer to the differences in CFU under the different conditions. Taxonomy is shown for the lowest taxonomy accounted. C) Beta-diversity plot showing that some samples group together based on media and bacteriocin selection criteria. D) Significant species found on the selection plates: mGAM+#13_actifencin, mGAM+#22_bacteroidetocinA, and mGAM-VK+#22 bacteroidetocin A. Significance is based on LogFC compared to control plates. (Significance level, padj \u0026lt; 0.05). Below 0 indicates less abundance on the bacteriocin plates compared to control. (All tests were made with DAtest and EdgeR - Quasi likelihood test).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3282788/v1/d09e8efa4201034f04ddef8e.png"},{"id":42264933,"identity":"9185357a-6926-4e69-86cc-a2cc3af0382c","added_by":"auto","created_at":"2023-08-28 19:44:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":506267,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap showing the ASVs with at least 5% abundance in at least 5 samples in all the different selection media. The headers refer to the different bacteriocin supernatants mixed with the various media (x-axis). Notably the abundance of \u003cem\u003eLactobacillus\u003c/em\u003e is almost zero on the mGAM media mixed with supernatant from #13 actifencin, whereas the abundance of \u003cem\u003eBacteroides\u003c/em\u003eis clearly enriched (Blue boxes) The reverse patterns can be visualized on the mGAM media mixed with supernatant from #22_bacteroidetocinA (green boxes)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3282788/v1/ee538406297eb689ccaf50af.png"},{"id":42264932,"identity":"11ccc36e-9893-4dd3-96e1-2fb05a19a1fc","added_by":"auto","created_at":"2023-08-28 19:44:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":389786,"visible":true,"origin":"","legend":"\u003cp\u003eA) graphical representation of the \u003cem\u003ein vivo\u003c/em\u003estudy showing the gavage and fecal selection. B) Overlay spot assay of \u003cem\u003eEcN\u003c/em\u003eisolated from fecal samples of mice orally dosed with \u003cem\u003eEcN\u003c/em\u003e_pmut13_actifencin and \u003cem\u003eEcN_\u003c/em\u003epMUT22_bacteroidetocinA\u003cem\u003e. \u003c/em\u003eIndicator strain used: \u003cem\u003eL. mali\u003c/em\u003e DSM 20444 and \u003cem\u003eB. vulgatus \u003c/em\u003eDSM 1447 respectively. Inhibition zones can be seen around the growing cultures. pMUT0 refers to the strain producing no bacteriocin, pMUT13 refers to the strain that has not been through the murine gut, \u003cem\u003eEcN\u003c/em\u003e 13.a-d refers to individual colonies restreaked on LB plates from the fecal samples. #22_bacteroidetocinA was assessed directly from the plating of feces on selection plates using top agar containing \u003cem\u003eB. vulgatus\u003c/em\u003e. Inhibition zones can be detected around the colonies (yellow circles), indicating that bacteriocins are being produced and secreted. C) Plot showing the Log10 (CFU/g of feces) at the different days. D) Bray-Curtis beta diversity plot showing no clear grouping between samples. E) abundance plots for the genera: \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eEscherichia-Shigella\u003c/em\u003eshowing no significant difference in abundance between the days.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3282788/v1/49e39a90947832db28719aa4.png"},{"id":43796903,"identity":"65490c90-f01d-47a4-b1eb-d0bbd689ce77","added_by":"auto","created_at":"2023-09-27 23:07:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2884290,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3282788/v1/7b4a940a-de65-4915-91ef-19c5716441a6.pdf"},{"id":42264939,"identity":"ece416c0-5136-430a-b969-9ce6a0ed7099","added_by":"auto","created_at":"2023-08-28 19:44:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":307851,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1GrowthrateassesmentfortheEcNstrainsandspotassayfortheEcN13and22.docx","url":"https://assets-eu.researchsquare.com/files/rs-3282788/v1/8c9e339e08cfdce7e076893e.docx"},{"id":42264938,"identity":"b585f0d4-0583-4f94-8ff4-7015c9ef7204","added_by":"auto","created_at":"2023-08-28 19:44:25","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":62158,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1Listofthebacteriocinsclonedandexpressedinthisstudy.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3282788/v1/415af4571546a27d360cc235.xlsx"},{"id":42265231,"identity":"53a3a6f5-e98b-4d69-94d3-d3b196888398","added_by":"auto","created_at":"2023-08-28 19:52:25","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14949,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2listofrepresentativegutmicrobiotastrains..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3282788/v1/28757c3697688df1abc68543.xlsx"},{"id":42265232,"identity":"cd9b52bf-2017-4ca4-a20d-5c20b542d0df","added_by":"auto","created_at":"2023-08-28 19:52:25","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9339,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3Primerspromotersandsequences.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3282788/v1/db770f8a2480a6c042bfce92.xlsx"},{"id":42264937,"identity":"1bf37cf6-b1ec-4ed2-923a-ed55b48e1d7f","added_by":"auto","created_at":"2023-08-28 19:44:25","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":23332,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYTABLE4.LISTOFSCREENEDBACTERIOCINSAGAINSTREPRESENTATIVEGUTSTRAINSINCLUDINGHALOSIZEOFINHIBITION..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3282788/v1/3e3f389231e2509e12fd9372.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antimicrobial potential of class II bacteriocins on gut microbiota species","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe increasing concerns surrounding food safety, antibiotic resistance, and the intricate relationship between the gut microbiota and human health have prompted extensive research on bacteriocins. These naturally occurring antimicrobial peptides, produced by a diverse range of bacteria, have garnered interest for their ability to target closely related species (Zacharof and Lovitt \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Bacteriocins exhibit both narrow and broad-spectrum activities and possess desirable characteristics such as nanomolar range efficacy, diverse modes of action, and high specificity compared to traditional antibiotics (Auke Johan van Heel, Montalban-Lopez, and Kuipers 2011; Rea et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Chikindas et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, their proteinaceous nature allows for engineering to enhance specificity and stability (Kheadr et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Field et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on structural and functional attributes, bacteriocins are classified into three major types (Hern\u0026aacute;ndez-Gonz\u0026aacute;lez et al. 2021). Among these types, class II bacteriocins, predominantly derived from Lactic Acid Bacteria (LAB), have been extensively studied \u003cem\u003ein vitro\u003c/em\u003e for their potential as bio-preservatives in food, particularly against undesirable pathogens such as \u003cem\u003eListeria\u003c/em\u003e spp. (Dabour et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Fatima \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Apart from their role in food preservation, class II bacteriocins have also been explored as alternative antibiotics for medical and veterinary applications, including combating \u003cem\u003eC. difficile\u003c/em\u003e infections (Cotter, Hill, and Ross 2005; Soltani et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rea et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). \u003cem\u003eIn vivo\u003c/em\u003e investigations of class II bacteriocins have demonstrated their ability to eliminate target bacterial strains without significantly affecting the overall microbiome composition, often employing the natural bacteriocin producer strain (Dabour et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Dobson et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Umu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, limited studies have examined the impact of bacteriocins on the overall composition of the gut microbiota.\u003c/p\u003e \u003cp\u003eUnderstanding the mechanisms underlying bacteriocin-mediated microbiota modulation is crucial for their effective utilization as antimicrobial agents (Heilbronner et al. 2021). Systematic and high-throughput studies of bacteriocins can be challenging when relying solely on natural bacteriocin producers. Therefore, utilizing heterologous hosts offers advantages for \u003cem\u003ein vitro\u003c/em\u003e screening and controlled \u003cem\u003ein vivo\u003c/em\u003e delivery of bacteriocins. This approach circumvents the need for specific regulatory conditions in the native producer and facilitates large-scale production required for commercial viability in the food and pharmaceutical industries (Mesa-Pereira et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eEscherichia coli (E. coli)\u003c/em\u003e, extensively characterized as a production host for bacteriocins and proteins, is commonly employed due to its well-established features (Mesa-Pereira et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jia and Jeon 2016). Notably, a previous study demonstrated heterologous expression of 80 different biosynthetic gene clusters in \u003cem\u003eE. coli\u003c/em\u003e, showcasing their activity against ESKAPE pathogens (Ayikpoe et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA typical bacteriocin gene cluster comprises a leader peptide, bacteriocin gene, immunity gene, modifier gene, and a secretion machinery such as an ABC transporter (Walsh et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, for class II bacteriocins, which possess a simpler structure, expression solely using the bacteriocin gene itself is feasible. By utilizing a tight and inducible expression system, the need for an innate immunity gene can often be eliminated (Mesa-Pereira et al. 2017). Moreover, employing a native signal peptide for \u003cem\u003eE. coli\u003c/em\u003e, such as OmpA, obviates the requirement for a specific bacteriocin leader peptide and transporter, as the bacteriocin can be secreted using the native \u003cem\u003eE. coli\u003c/em\u003e secretion system (L. H. Zhang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study assessed the impact of 75 class II bacteriocins on a collection of 49 representative gut microbiota strains. Our study encompassed common gut species including relevant pathogenic species and probiotic bacteria. Additionally, we tested 2 bacteriocins on a complex microbiota \u003cem\u003eex vivo\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The findings from this research provide valuable insights into the target range of class II bacteriocins, thereby contributing to their potential future application.\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003eIn silico identification of class II bacteriocin genes and cloning into \u003cem\u003eE. coli\u003c/em\u003e.\u003c/h3\u003e\n\u003cp\u003eTo obtain a non-redundant list of bacteriocin sequences, all class II bacteriocins available in the BAGEL3 (Auke J. van Heel et al. 2013) and Bactibase (Hammami et al. 2010) databases were downloaded, resulting in a total of 233 non-redundant sequences (Supplementary Table 1). Subsequently, 84 sequences were selected based on being single component (the presence of one bacteriocin is sufficient to observe an effect), as well as successful verification through a blast search. 75 bacteriocins were successfully cloned and expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21-AI. Among the cloned bacteriocins, three originated from Gram-negative species, while the remaining 72 originated from Gram-positive species. The native bacteriocin producers span 5 different phyla (\u003cem\u003eBacillota\u003c/em\u003e being the most prevalent) as well as 19 different genera with \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e being the most prevalent (Supplementary Table 2). The bacteriocin genes were inserted into the pMUT vector, which is native to \u003cem\u003eE. coli\u003c/em\u003e Nissle and has been previously used as an expression vector (Vaaben, Vazquez-Uribe, and Sommer 2022). The insertion was performed downstream of the \u003cem\u003eE. coli\u003c/em\u003e native OmpA signal sequence and fused with a \u003cem\u003egfp\u003c/em\u003e reporter gene. The expression of the inserted genes was controlled by an arabinose-inducible T7 promoter (Figure 1A).\u003c/p\u003e\n\u003ch3\u003eBuilding the collection of representative gut microbiota strains\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eTo compile a comprehensive list of species associated with the representative gut microbiota a non-redundant list was generated based on a literature search encompassing four studies. The selected studies included species of the core microbiome (Shetty et al. 2017), gut microbiome strains in relation to their susceptibility for non-antibiotic drugs (Maier and Typas 2017; Maier et al. 2018), and gut strains with the ability to grow on the modified gut media mGAM (Tramontano et al. 2018). To validate the inclusion of the selected strains as part of the core microbiome, their prevalence in human stool samples was assessed using the assembled metagenomic species (MGS) identified from 396 stool samples obtained from the Human Microbiome Project (HMP), as reported by Nielsen et al. (Nielsen et al. 2014). A total of 56 strains were deemed suitable for inclusion in the representative gut microbiota collection. Among these, 49 demonstrated growth capability in either mGAM, MRS, or BHI media. The selected strains consisted of 24 Gram-negative and 25 Gram-positive species, encompassing a range of phyla including \u003cem\u003eBacteroides\u003c/em\u003e (11 species), \u003cem\u003eFirmicutes\u003c/em\u003e (27 species), \u003cem\u003eProteobacteria\u003c/em\u003e (7 species), \u003cem\u003eActinomycetes\u003c/em\u003e (2 species), \u003cem\u003eFusobacteria\u003c/em\u003e (1 species), and \u003cem\u003eActinobacteria\u003c/em\u003e (1 species). For a detailed list of the selected strains, refer to Supplementary Table 2.\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eIn vitro\u003c/em\u003e screening of bacteriocins on the collection of representative gut microbiota strains\u003c/h3\u003e\n\u003cp\u003eThe inhibition spectrum of each bacteriocin was assessed by employing the engineered bacteriocin-producing strains and conducting an overlay spot assay against the representative species of the gut microbiota (Suppl. Table 3). Inhibition was assessed through visual inspection of the plates and measurement of the inhibition halo size. A strain was considered inhibited if any inhibition zone was observed and exceeded the negative control (pMUT0). Among the 75 bacteriocins tested, 22 (29%) demonstrated inhibitory activity against at least one species in the assay and will henceforth be referred to as the inhibitory bacteriocins (Figure 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOf the inhibitory bacteriocins, 21 originated from Gram-positive species and predominantly inhibited other Gram-positive species. However, actifencin, naturally produced by \u003cem\u003eActinomyces ruminicola\u003c/em\u003e DPC 7226, displayed inhibition against the Gram-negative strain \u003cem\u003eBacteroides eggerthii\u003c/em\u003e DSM 20697. Notably, 5 out of the 24 Gram-negative strains were inhibited by at least one bacteriocin.\u003c/p\u003e\n\u003cp\u003eBacteroidetocin A, derived from the Gram-negative \u003cem\u003eBacteroides vulgatus\u003c/em\u003e, exclusively inhibited other Gram-negative species (4 species), including \u003cem\u003eB. vulgatus\u003c/em\u003e itself (DSM 1447). The species most frequently inhibited by the bacteriocins were \u003cem\u003eLactobacillus sakei\u0026nbsp;\u003c/em\u003eATCC 15521 (21 bacteriocins), \u003cem\u003eEnterococcus faecalis\u003c/em\u003e ATCC 19433 (18 bacteriocins), and \u003cem\u003eListeria innocua\u003c/em\u003e DSM 20649 (13 bacteriocins). Actifencin exhibited the highest inhibitory activity against the tested species, affecting 11 out of 49 strains.\u003c/p\u003e\n\u003cp\u003eTo elucidate the observed variations in the inhibitory spectra among the bacteriocins, a multiple sequence alignment of their sequences was performed using COBALT multiple alignment tool (Table 1). Additionally, the structure prediction tool alpha-fold was utilized to visually analyze differences between the bacteriocins (Figure 2).\u003c/p\u003e\n\u003cp\u003eThe analysis identified that the consensus sequence xYGNGV, known to be conserved among class IIa bacteriocins (Coyne et al. 2019) was present in a majority of the examined bacteriocins. In addition, these bacteriocins shared highly similar structural characteristics, comprising a helix, a beta-sheet, and in certain instances, a leader sequence that also adopted a helical conformation. It was also observed that bacteriocins deviating from this consensus sequence exhibited distinct structural differences (refer to the top 6 sequences in Table 1 and the top row in Figure 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1: Multiple alignment of the bacteriocin sequences reveal highly conserved amino acids.\u003c/p\u003e\n\u003ctable style=\"width:467.55pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;line-height:200%;font-size:15px;font-family:\"Calibri\",sans-serif;margin-top:0in;margin-bottom:10.0pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12px;line-height:200%;color:black;\"\u003eBacteriocin\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.4pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;line-height:200%;font-size:15px;font-family:\"Calibri\",sans-serif;margin-top:0in;margin-bottom:10.0pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12px;line-height:200%;color:black;\"\u003eClass\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 325.15pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;line-height:200%;font-size:15px;font-family:\"Calibri\",sans-serif;margin-top:0in;margin-bottom:10.0pt;'\u003e\u003cstrong\u003e\u003cspan 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325.15pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;line-height:200%;font-size:15px;font-family:\"Calibri\",sans-serif;margin-top:0in;margin-bottom:10.0pt;'\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003e---K\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:red;'\u003eYYGNG\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003eVHCTK-SGCS\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:red;'\u003eV\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003e--\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri 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style=\"font-size:12px;line-height:200%;color:black;\"\u003e31_Plantaricin_423\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.4pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;line-height:200%;font-size:15px;font-family:\"Calibri\",sans-serif;margin-top:0in;margin-bottom:10.0pt;'\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003eA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 325.15pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;line-height:200%;font-size:15px;font-family:\"Calibri\",sans-serif;margin-top:0in;margin-bottom:10.0pt;'\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003e---K\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:red;'\u003eYYGNG\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003eVTCGK-HSCS\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:red;'\u003eV-\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003e-\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003e--\u003c/span\u003e\u003cspan style='font-size:12px;line-height: 200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003eNWGQAFSCS\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:red;'\u003eV\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003eSHLAN--------FGH-------GKC\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;line-height:200%;font-size:15px;font-family:\"Calibri\",sans-serif;margin-top:0in;margin-bottom:10.0pt;'\u003e\u003cspan style=\"font-size:12px;line-height:200%;color:black;\"\u003e32_sakacin_G_skgA2\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;line-height:200%;font-size:15px;font-family:\"Calibri\",sans-serif;margin-top:0in;margin-bottom:10.0pt;'\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003eA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 325.15pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;line-height:200%;font-size:15px;font-family:\"Calibri\",sans-serif;margin-top:0in;margin-bottom:10.0pt;'\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003e---K\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:red;'\u003eYYGNG\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003eVSCNS-HGCS\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:red;'\u003eV-\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003e-NWGQAWTCG\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:red;'\u003eV\u003c/span\u003e\u003cspan style='font-size:12px;line-height:200%;font-family:\"Calibri Light\",sans-serif;color:black;'\u003eNHLAN-------GGH-------GVC\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 1: highly conserved amino acids are highlighted in red. The top 6 bacteriocins does not contain these conserved regions (multiple alignment tool: https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe six bacteriocins exhibiting deviations from both the common structure and sequence patterns typical for class II bacteriocins: #25_E97enterocin (class IId), #13_actifencin (class IIa), #33_garvieacinQ (class IIa), #42_bovicin_255_variant (class IId), #46_Bovicin_255_peptide (class IId), and #22_bacteroidetocin_A (class IIa) also displayed differences in their species inhibition pattern. For instance, #46_Bovicin_255_peptide specifically targeted the Gram-negative species \u003cem\u003eBacteroides fragilis DSM2151\u003c/em\u003e and \u003cem\u003eCollinsella aerofaciens DSM3979\u003c/em\u003e. In contrast, its closely related sister sequence, #42_bovicin_255_variant, did not exhibit inhibition against these two species. This highlights that even slight variations in the sequence can lead to differences in the target spectrum of the bacteriocins, which is generally observed for class IIa bacteriocins (Ennahar et al. 2000). These findings emphasize the importance of determining the target specificity of bacteriocins for even very similar sequences and provide insights into the molecular basis of their inhibitory activities against specific bacterial strains.\u003c/p\u003e\n\u003cp\u003eTo further evaluate the potential of the bacteriocins examined in this study as novel antibiotics or targeted therapeutics, we compiled a table of species associated with diseases or disorders(Table 2). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2: Inhibited strains in our assay and the strains association to disease or spoilage.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.465890183028286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDisease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.62728785357737%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInhibited by bacteriocins\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.96173044925125%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.465890183028286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eCollinsella aerofaciens DSM 3979\u003c/em\u003e\u003cem\u003e\u0026nbsp;*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eIncreased in psoriasis patients\u0026rsquo; stool,\u0026nbsp;increased in coronary artery disease (CAD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.62728785357737%\" valign=\"top\"\u003e\n \u003cp\u003e13,46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.96173044925125%\" valign=\"top\"\u003e\n \u003cp\u003e(Shapiro et al. 2019; Liu et al. 2019)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.465890183028286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eEnterococcus fecalis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eLeading causes of highly antibiotic-resistant, hospital-acquired infection,\u0026nbsp;Dental Root Canal Pathogen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.62728785357737%\" valign=\"top\"\u003e\n \u003cp\u003e6, 8, 10, 12, 18, 25, 31, 32, 33, 35, 38, 46, 50, 52, 53, 58, 63, 70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.96173044925125%\" valign=\"top\"\u003e\n \u003cp\u003e(Shankar, Baghdayan, and Gilmore 2002; Alghamdi et al. 2020)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.465890183028286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eEubacterium\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003erectale DSM 17629\u003c/em\u003e\u003cem\u003e\u0026nbsp;*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eE. rectale\u003c/em\u003e functions as a \u0026lsquo;driver\u0026rsquo; bacterium and contributes to cancer initiation via promoting inflammation.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.62728785357737%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.96173044925125%\" valign=\"top\"\u003e\n \u003cp\u003e(Wang et al. 2021)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.465890183028286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePrevotella\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003ecopri DSM 18205\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eHigh prevalence of the bacteria \u003cem\u003ePrevotella copri\u003c/em\u003e in patients with rheumatoid arthritis (RA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.62728785357737%\" valign=\"top\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.96173044925125%\" valign=\"top\"\u003e\n \u003cp\u003e(Bernard 2014)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.465890183028286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePhocaeicola dorei DSM 17855\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eBloodstream infection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.62728785357737%\" valign=\"top\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.96173044925125%\" valign=\"top\"\u003e\n \u003cp\u003e(Cobo et al. 2022)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.465890183028286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBacteroides\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003evulgatus DSM 1447\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eInvolved in colitis, both positive and negative depending on the strain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.62728785357737%\" valign=\"top\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.96173044925125%\" valign=\"top\"\u003e\n \u003cp\u003e(Li et al. 2021)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.465890183028286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBacteroides\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eeggerthii DSM 20697\u003c/em\u003e\u003cem\u003e\u0026nbsp;*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eEnhancing\u0026nbsp;the severity of\u0026nbsp;DSS induced\u0026nbsp;colitis\u0026nbsp;in mice\u0026nbsp;(\u003cem\u003eB\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;eggerthii\u003c/em\u003e 12986)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.62728785357737%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.96173044925125%\" valign=\"top\"\u003e\n \u003cp\u003e(Dziarski et al. 2016)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.465890183028286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eListeria inoccua DSM 20649\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eClosely related to the foodborne pathogen \u003cem\u003eL. monocytogenes\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.62728785357737%\" valign=\"top\"\u003e\n \u003cp\u003e6, 8, 10, 12, 18, 31, 35, 38, 50, 52, 53, 58, 63, 70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.96173044925125%\" valign=\"top\"\u003e\n \u003cp\u003e(Buchrieser et al. 2003)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.465890183028286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eLactic Acid Bacteria\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eRare infections, especially in diabetic and immunocompromised individuals and with increased microbial ethanol production leading to non-alcoholic fatty liver disease (NAFLD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.62728785357737%\" valign=\"top\"\u003e\n \u003cp\u003eAll the inhibitory bacteriocins were able to inhibit at least one LAB except #22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.96173044925125%\" valign=\"top\"\u003e\n \u003cp\u003e(Meijnikman et al. 2022; Rossi et al. 2022).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e(*) indicates that this species not before has been characterized to be\u0026nbsp;inhibited by this bacteriocin.\u003c/p\u003e\n\u003cp\u003eAs an example \u003cem\u003eCollinsella aerofaciens\u003c/em\u003e, a biosafety level 2 organism, has been implicated in psoriasis (Shapiro et al. 2019) and coronary artery disease (CAD) (Liu et al. 2019). It is also commonly found in the human gut, present in approximately 86% of the 396 stool samples from the Human Microbiome Project (Nielsen et al. 2014). In our study, we observed that two of the tested bacteriocins, #13_actifencin and #46_Bovicin_255_peptide, targeted this particular strain. Considering the specific targeting of \u003cem\u003eCollinsella aerofaciens\u003c/em\u003e in the gut, the administration of bacteriocin #46_Bovicin_255_peptide could potentially lead to the elimination of this unwanted species. Notably, #46_Bovicin_255_peptide targets only five species from the representative gut microbiota collection meaning that administration of this bacteriocin most likely would not lead to overall disruption of the microbiota. Other optional targets could be to target the gut strain \u003cem\u003eBacteroides\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003evulgatus\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003einvolved in colitis, both positive and negative depending on the strain (Li et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings suggest that bacteriocins such as\u0026nbsp;#13_ actifencin,\u0026nbsp;#46_bovicin_255_peptide\u0026nbsp;and #22_bacteroidetocin A among others\u0026nbsp;holds\u0026nbsp;promise for selectively targeting specific unwanted strains within the gut microbiota, offering a potential strategy for modulating microbial composition while minimizing disturbances to the overall ecosystem.\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eSelective alteration of fecal-derived microbial communities in vitro\u0026nbsp;\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eBased on the observations during screening, we hypothesized that we could selectively target and remove certain species within a complex microbial community by expressing the bacteriocins in situ. For this characterization 3 bacteriocins were selected: #12_ubericinA (narrow spectrum against Gram-positive bacteria), #13_actifencin (broader spectrum against both Gram-positive and Gram-negative bacteria), and #22_bacteroidetocin A (narrow spectrum against Gram-negative bacteria). To assess the effect of the selected bacteriocins murine fecal samples were plated on selection plates containing each of the different bacteriocins. Using 16S rRNA amplicon sequencing we characterized the microbial community composition for the different bacteriocin and media combinations (Figure 3A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;number of\u0026nbsp;colony-forming units (CFU)\u0026nbsp;observed on the\u0026nbsp;different conditions\u0026nbsp;varied substantially.\u0026nbsp;The\u0026nbsp;CFU counts of\u0026nbsp;#22_bacteroidetocinA + mGAM and #22_bacteroidetocinA + mGAM-vk\u0026nbsp;were\u0026nbsp;significantly lower\u0026nbsp;compared to\u0026nbsp;the control (Figure\u0026nbsp;3B).\u0026nbsp;Beta-diversity analysis of these\u0026nbsp;samples revealed distinct clusters corresponding to the selection media and the different bacteriocins used (Figure\u0026nbsp;3C).\u003c/p\u003e\n\u003cp\u003eA heatmap was generated to visualize the abundance of Amplicon Sequence Variants (ASVs) with at least 5% abundance in at least 5 of the samples. The results showed a clear reduction in ASVs associated to the genus \u003cem\u003eLactobacillus\u003c/em\u003e selected on the #13_actifencin + mGAM media, but not with the #13_actifencin + MRS media. Additionally, there appeared to be a decrease in ASVs mapped to the genus \u003cem\u003eBacteroides\u003c/em\u003e, particularly \u003cem\u003eBacteroides vulgatus\u003c/em\u003e, on the #22_bacteroidetocin + mGAM and #22_bacteroidetocin + mGAM-vk selection plates\u0026nbsp;(Figure 4)\u003c/p\u003e\n\u003cp\u003eRelative abundance plots were generated to analyze the distribution of ASVs across different media and bacteriocin conditions. The plots revealed distinct patterns of ASVs mapped to specific genera. On the mGAM media, the majority of ASVs were associated with the genus \u003cem\u003eLactobacillus\u003c/em\u003e, except in the #13_actifencin + mGAM condition where a higher abundance of ASVs mapped to \u003cem\u003eBacteroides\u003c/em\u003e was observed (Figure 3B). This difference was found to be statistically significant (Figure 3D), with a significant decrease in an unclassified \u003cem\u003eLactobacillus\u003c/em\u003e species and the overall genus \u003cem\u003eLactobacillus\u003c/em\u003e. Concurrently, an increase in the \u003cem\u003eEscherichia-Shigella\u003c/em\u003e genus was observed (Figure 3D). Notably, \u003cem\u003eBacteroides faecichinchillae\u003c/em\u003e showed high abundance (indicated in yellow) on the plot, although statistical significance could not be established.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the MRS selection plates, ASVs primarily mapped to the genus \u003cem\u003eLactobacillus\u003c/em\u003e, but no significant differences were observed among the different groups (Figure 2B). In the case of the #22_bacteroidetocinA + mGAM selection plates, a significant decrease in \u003cem\u003eBacteroides\u003c/em\u003e and specifically \u003cem\u003eBacteroides vulgatus\u003c/em\u003e was observed (Figure 3B and 3C). These results indicate that #22_bacteroidetocinA is indeed capable of inhibiting naturally occurring microbiota strains of \u003cem\u003eBacteroides vulgatus\u003c/em\u003e in a complex community.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEvaluation of bacteriocin-producing E. coli Nissle strains in mice\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the impact of bacteriocins on a complete microbiome in mice, we chose to express the two bacteriocins #13_actifencin and #22_bacteroidetocin A, which were found to substantially modulate the microbiota community composition \u003cem\u003eex vivo\u003c/em\u003e. For the \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand \u003cem\u003eex vivo\u003c/em\u003e studies we utilized the \u003cem\u003eE. coli\u003c/em\u003e production strain BL21-AI. However, BL21-AI express bacteriocins under an inducible promoter, and is not optimized for \u003cem\u003ein vivo\u003c/em\u003e growth. Therefore, we chose to engineer \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eNissle\u003cem\u003e\u0026nbsp;(EcN)\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eto produce bacteriocins under a constitutive promoter. \u003cem\u003eEcN\u003c/em\u003e was selected as the production strain due to its favorable characteristics as a safe probiotic organism (GRAS status) with robust growth capabilities under anaerobic conditions.\u003c/p\u003e\n\u003cp\u003eTo ensure the suitability of these engineered strains, we evaluated their growth rates and confirmed their functional properties through an overlay spot assay against indicator strains (Supplementary figure 1). The engineered strains \u003cem\u003eEcN_\u003c/em\u003epMUT13_actifencin and \u003cem\u003eEcN_\u003c/em\u003epMUT22_bacteroidetocinA exhibited an average doubling time that was 50% and 37.5% higher than that of the \u003cem\u003eEcN_\u003c/em\u003eWT_GFP \u0026nbsp;strain. The \u003cem\u003eEcN_\u003c/em\u003epMUT0_control\u003cem\u003e\u0026nbsp;\u003c/em\u003estrain showed an increase in doubling time of 15% (Supplementary Figure 1)\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e study spanned a period of 7 consecutive days, during which the mice received daily oral doses of the respective strains followed by a 7-day washout period. The study design allowed us to investigate the colonization potential of the bacteriocin-producing strains compared to the control group and confirm the elimination of the \u003cem\u003eEcN\u003c/em\u003e strains from the gut over time (Figure 5A). Colonization of the \u003cem\u003eEcN\u003c/em\u003e cultures was observed at approximately 10\u003csup\u003e5\u003c/sup\u003e-10\u003csup\u003e6\u003c/sup\u003e colony-forming units (CFUs) per gram of feces over the 7-day oral delivery period. By day 9, the CFUs decreased to approximately 10\u003csup\u003e4\u003c/sup\u003e, and no surviving \u003cem\u003eEcN\u003c/em\u003e colonies could be detected on days 11 or 14 (Figure 5C).\u003c/p\u003e\n\u003cp\u003eThe functionality of \u003cem\u003eEcN\u003c/em\u003e strains from fecal samples was confirmed using an overlay spot assay with indicator strains \u003cem\u003eL. mali DSM 20444\u003c/em\u003e and \u003cem\u003eB. vulgatus DSM 1447\u003c/em\u003e. Detection of small inhibitory zones around colonies validated the bacteriocin production of \u003cem\u003eEcN_\u003c/em\u003epMUT22_bacteroidetocinA. (Figure 5B) and restreaks of \u003cem\u003eEcN\u003c/em\u003e from the fecal samples were compared to the \u003cem\u003eEcN\u003c/em\u003e strain before entering the mouse gut as well as a negative \u003cem\u003eEcN\u003c/em\u003e control, showing distinct inhibition zones around the four \u003cem\u003eEcN\u003c/em\u003e colonies tested from the fecal samples. Nine colonies of both \u003cem\u003eEcN\u003c/em\u003e_pMUT13_actifencin and \u003cem\u003eEcN\u003c/em\u003e_pMUT22_bacteroidetocinA underwent colony PCR and Sanger sequencing, confirming their identity as the correct \u003cem\u003eEcN\u003c/em\u003e strains after having been through the murine gut with no mutations detected in the bacteriocin gene or promoter region.\u003c/p\u003e\n\u003cp\u003eFecal samples were collected on study day 0 (prior to the first oral delivery), day 7, and day 14 for sequencing analysis. Despite the \u003cem\u003eex vivo\u003c/em\u003e findings indicating differential abundance of certain bacterial genera and species, no discernible differences in ASVs were detected in the fecal samples from any of the mouse study groups. Specifically, we focused on examining the overall abundance of \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBacteroides\u003c/em\u003e \u003cem\u003evulgatus\u003c/em\u003e, the species that exhibited differential abundance in the \u003cem\u003eex vivo\u003c/em\u003e study. In addition, we analyzed and graphically represented the prevalence of ASVs that corresponded to the genus \u003cem\u003eEscherichia-Shigella\u003c/em\u003e, observing their presence within the murine gut. Nevertheless, no distinct difference emerged between the experimental groups receiving gavage and the control group treated with PBS (Figure 5E). Given that the CFUs in feces approximated ~10\u003csup\u003e6\u003c/sup\u003e, and assuming a colon density of 10\u003csup\u003e11\u003c/sup\u003e, the abundance of \u003cem\u003eEcN\u003c/em\u003e in the colon would be 10\u003csup\u003e6\u003c/sup\u003e/10\u003csup\u003e11\u003c/sup\u003e = 10\u003csup\u003e-5\u003c/sup\u003e or 0.00001% of the total bacterial population. Given this relatively low abundance, we do not anticipate quantifying \u003cem\u003eEcN\u003c/em\u003e in the gut using 16S rRNA amplicon sequencing.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBacteriocins and their\u0026nbsp;native producers\u0026nbsp;have been extensively used in the food industry to combat food pathogens.\u0026nbsp;Recent studies have explored their potential as novel\u0026nbsp;antimicrobial agents, particularly against antibiotic-resistant pathogens, due to their specificity compared to traditional antibiotics\u0026nbsp;and their ability to not\u0026nbsp;disturb\u0026nbsp;the overall composition of the microbiome. These traits in particular highlights their potential and benefits as a novel group of antibiotics\u0026nbsp;(Dabour et al. 2009; Rea et al. 2010; Heilbronner et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we aimed to investigate the effects of\u0026nbsp;class II bacteriocins on a\u0026nbsp;selection of representative gut microbiota strains to further asses their target spectrum. We performed \u003cem\u003ein vitro\u003c/em\u003e screening of 75 class II bacteriocins against 49 species, including pathogens such as \u003cem\u003eC. difficile\u003c/em\u003e and \u003cem\u003eSalmonella enterica subsp. Enterica DSM 5569\u003c/em\u003e.\u0026nbsp;Consistent with previous studies, most\u0026nbsp;class II bacteriocins\u0026nbsp;generally\u0026nbsp;did not affect Gram-negative species\u0026nbsp;(Umu et al. 2016; Rea et al. 2010). The only exception was #13_actifencin and the Gram negatively produced bacteriocin #22_bacteroidetocinA.\u003c/p\u003e\n\u003cp\u003e#13_actifencin\u0026nbsp;is natively\u0026nbsp;produced by \u003cem\u003eActinomyces ruminicola DPC 7226\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eand has been characterized by Sugrue et al.\u0026nbsp;(Sugrue et al. 2020). In our study\u0026nbsp;actifencin\u0026nbsp;was seen to target gut species associates with various diseases and disorders, as well as a broad range of LAB species both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e. Typically LAB species are\u0026nbsp;recognized for their beneficial\u0026nbsp;properties\u0026nbsp;as probiotics,\u0026nbsp;but have also\u0026nbsp;been associated with rare infections, especially in diabetic and immunocompromised individuals\u0026nbsp;(Rossi et al. 2022). Furthermore, they have been associated with\u0026nbsp;increased microbial ethanol production leading to non-alcoholic fatty liver disease (NAFLD)\u0026nbsp;(Meijnikman et al. 2022; Kuraji et al. 2023). Actifencin is believed to be part of a new class of bacteriocins produced by the \u003cem\u003eActinomyces\u003c/em\u003e genus. Sugure et al. report that 47 of 161 genomes were seen to exhibit at least one actifencin-related bacteriocin with a high sequence diversity amongst the genes. Based on the findings in our study we hypothesize that this new group of bacteriocins inhabit a very interesting potential as future antimicrobials that should be explored further.\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;other\u0026nbsp;bacteriocin\u0026nbsp;showing interesting inhibition patterns both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e, was\u0026nbsp;#22_bacteroidetocinA, a compound first characterized by Coyne et al. (Coyne et al. 2019), and\u0026nbsp;natively\u0026nbsp;produced by \u003cem\u003eBacteroides vulgatus\u003c/em\u003e. Our analysis discerned the ability of bacteroidetocin A to impede four species: \u003cem\u003ePrevotella\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003ecopri DSM 18205\u003c/em\u003e, \u003cem\u003eBacteroides\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003evulgatus DSM 1447\u003c/em\u003e, \u003cem\u003eBacteroides clarus DSM 22519\u003c/em\u003e, and \u003cem\u003eBacteroides stercoris DSM 19555\u003c/em\u003e.\u0026nbsp;To back this up our \u003cem\u003eex vivo\u003c/em\u003e sequencing analysis revealed the complete depletion of ASVs assigned to the \u003cem\u003eBacteroides\u003c/em\u003e genus, including \u003cem\u003eBacteroides vulgatus\u003c/em\u003e. Conversely, an increase in ASVs from the \u003cem\u003eTannerellaceae\u003c/em\u003e family, belonging to the order \u003cem\u003eBacteroidales\u003c/em\u003e, was observed, suggesting the presence of \u003cem\u003eBacteroides\u003c/em\u003e strains not targeted by\u0026nbsp;#22_bacteroidetocinA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, our \u003cem\u003ein vitro\u003c/em\u003e discoveries diverged\u0026nbsp;considerably from those reported by Coyne et al., in which bacteroidetocin A was shown to inhibit \u003cem\u003eBacteroides thetaomicron\u003c/em\u003e, \u003cem\u003eParabacteroides merdae\u003c/em\u003e DSM 19495, and \u003cem\u003eBacteroides fragilis\u003c/em\u003e DSM 2151. These species, however, did not exhibit inhibition in our\u0026nbsp;study.\u0026nbsp;One reason could be the media used in the inhibition assay, where in our study was used mGAM, Coyne et. al tested the susceptibility of \u003cem\u003eBacteroides\u003c/em\u003e strains in BHI media. Media type has before been shown to be important for the susceptibility of bacteria against class II bacteriocins\u0026nbsp;(Carl et al. 2004).\u0026nbsp;This discordance\u0026nbsp;emphasized the importance of studying bacteriocins in relation to the environment in where they are intended to function. Furthermore, it could imply\u0026nbsp;the likelihood of bacteroidetocin A operating through a strain-specific targeting mechanism or\u0026nbsp;that\u0026nbsp;unidentified immunity mechanisms\u0026nbsp;are present\u0026nbsp;in the resistant strains. Considering the strain-dependent involvement of \u003cem\u003eB. vulgatus\u003c/em\u003e in colitis, as indicated by S. Li et al\u0026nbsp;(Li et al. 2021), our results emphasize the necessity to\u0026nbsp;broaden\u0026nbsp;our understanding of the target specificity of bacteroidetocin A. This pertains not only to better characterization of the implicated \u003cem\u003eB. vulgatus\u003c/em\u003e strains in colitis, but also to the identification of potentially harmful variants and the establishment of their respective inhibitory profiles.\u0026nbsp;The exact mechanism of action of bacteroidetocin A\u0026nbsp;still\u0026nbsp;remains elusive\u0026nbsp;(Coyne et al. 2019), underscoring the need for further investigation\u0026nbsp;and\u0026nbsp;continual research\u0026nbsp;to both fully comprehend and potentially exploit the antibacterial capabilities of bacteroidetocin A.\u003c/p\u003e\n\u003cp\u003eSignificant\u0026nbsp;alterations\u0026nbsp;in species composition observed in the \u003cem\u003eex vivo\u003c/em\u003e study did not extend to the \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eexperiment. Nevertheless, \u003cem\u003eEcN\u003c/em\u003e did colonize the murine gut during the delivery phase, although it was entirely eliminated within 3-4 days post-delivery cessation. These observations align with the prevailing understanding that \u003cem\u003eEcN\u003c/em\u003e can transiently colonize the mammalian gut but fails to persist without sustained delivery\u0026nbsp;(Martinson and Walk 2020).\u003c/p\u003e\n\u003cp\u003eThe resilience of the \u003cem\u003eEcN\u003c/em\u003e producers was evaluated using an overlay spot assay, which\u0026nbsp;confirmed\u0026nbsp;their ability to inhibit target strains post-transit through the mouse gut. Potential reasons for the observed lack of significant gut microbiota shifts could include suboptimal bacteriocin production by the \u003cem\u003eEcN\u003c/em\u003e strains\u0026nbsp;or a\u0026nbsp;metabolic burden from bacteriocin expression (as suggested by our assessment\u0026nbsp;of growth rates of bacteriocin producers\u0026nbsp;to be\u0026nbsp;50% and 37.5% higher than the \u003cem\u003eEcN\u003c/em\u003e_WT_GFP strain for\u0026nbsp;#13_actifencin and\u0026nbsp;#22_bacteroidetocinA producers, respectively). Other reasons could be the\u0026nbsp;rapid \u003cem\u003ein vivo\u003c/em\u003e digestion of bacteriocins,\u0026nbsp;as well as the\u0026nbsp;spatial separation between the producers and target\u0026nbsp;strains.\u003c/p\u003e\n\u003cp\u003eConsidering the differential gut colonization patterns of LABs and \u003cem\u003eEcN\u003c/em\u003e - with the former mostly populating the upper intestine\u0026nbsp;(Walter 2008)\u0026nbsp;and the latter largely colonizing the colon\u0026nbsp;(Grauke et al. 2002),\u0026nbsp;as well as the ability of\u0026nbsp;\u003cem\u003eB. vulgatus\u003c/em\u003e and other \u003cem\u003eBacteroides\u003c/em\u003e members\u0026nbsp;to produce\u0026nbsp;biofilm and\u0026nbsp;to colonize the\u0026nbsp;intestinal mucosa\u0026nbsp;(B\u0026eacute;chon and Ghigo 2022), complicates delivery with bacteriocins further.\u0026nbsp;This discordance between \u003cem\u003ein vitro\u003c/em\u003e, \u003cem\u003eex vivo\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e efficacy underscores the complex, context-dependent character of bacteriocin activity and the hurdles inherent in extrapolating \u003cem\u003ein vitro\u003c/em\u003e results to \u003cem\u003ein vivo\u003c/em\u003e applications. Nevertheless, our\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e and\u0026nbsp;\u003cem\u003eex vivo\u003c/em\u003e and results accentuate the potential of bacteriocins as precision modulators of the gut microbiota.\u003c/p\u003e\n\u003cp\u003eEnhancing \u003cem\u003ein vivo\u003c/em\u003e bacteriocin effectiveness may be achieved via strategies such as strengthening protein stability while maintaining efficacy, as demonstrated by Field et al., 2019 with nisin\u0026nbsp;(Field et al. 2019). An alternative approach could involve shifting the choice of production host to\u0026nbsp;\u003cem\u003eLactococcus lactis\u003c/em\u003e (Song et al. 2017)\u0026nbsp;or \u003cem\u003eLactobacillus plantarum\u003c/em\u003e (N. Zhang et al. 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur study, through the utilization of a heterologous host, facilitated high-throughput screening of class II bacteriocins, an approach that could extend to other bacteriocin classes, like the novel actifencin-like group. Furthermore, this methodology opens avenues to facile host optimization via engineering, thereby addressing the challenge of effective \u003cem\u003ein vivo\u003c/em\u003e delivery.\u003c/p\u003e\n\u003cp\u003eFuture research should focus on optimizing bacteriocin production and delivery, thereby maximizing their potential as novel antimicrobial agents. This could involve identifying optimal gut-colonizing hosts or improve bacteriocin stability for optimized efficacy.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch2\u003eSelection of bacteriocin genes\u003c/h2\u003e\n\u003cp\u003eAll class II bacteriocins from the Bagel3 database (Auke J. van Heel et al. 2013) (now known as Bagel4: http://bagel4.molgenrug.nl/) as well as all class II bacteriocins available from Bactibase https://bactibase.hammamilab.org/main.php bactibase.hammamilab.org was downloaded (date of download: 5\u003csup\u003eth\u003c/sup\u003e of February 2020) and used to create a non-redundant list of all bacteriocin sequences. Each sequence was verified in the original literature, the original secretion taq was identified in the sequence (often before a GG double glycine sequence) and only the pro-peptide was used for expression. The DNA sequence was codon optimized for production in \u003cem\u003eE. coli\u003c/em\u003e K-12 from IDT online codon optimization tool (https://eu.idtdna.com/CodonOpt). 84 of the ~ 200 available Class II sequences available in the databases were selected for cloning based on the following criteria: having a unique sequence, being 1 compartment, and having a sequence that has a 100% identity match when using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). 2 bacteriocins: Actifencin(Sugrue et al. 2020) and Bacteroidetocin A(Coyne et al. 2019) was added to the collection as well.\u003c/p\u003e\n\u003ch2\u003eIn vitro screening:\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of gut strains to build the representative gut strain catalogue:\u003c/strong\u003e The gut strains were selected based their ability to grow on mGAM as well as their prevalence in the human gut:\u0026nbsp;(Maier and Typas 2017; Shetty et al. 2017; Tramontano et al. 2018; Maier et al. 2018; Forster et al. 2019)\u0026nbsp;.\u0026nbsp;Furthermore a script was used to create a list of the prevalence of strains found in the study:\u0026nbsp;(Nielsen et al. 2014). Most strains were ordered from the Leibniz institute (DSMZ-German Collection of Microorganisms and Cell Cultures). Supplementary Table 2 contains an extensive list of the microorganisms, their prevalence, source, and growth medium.\u003cbr\u003e\u0026nbsp;\u003cstrong\u003ePlasmid and strain construction for expression in E. coli BL21-AI:\u003c/strong\u003e Primers, and plasmids in Supplementary Table 3. Primers and gBlocks were ordered from Integrated DNA Technologies (IDT). gBlocks containing the bacteriocin sequences were ordered from Twist Bioscience. List of bacteriocin sequences can be found in Supplementary Table 1. Native \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eNissle plasmid pMUT-kanR-Hok/Sok was used as the vector to produce the bacteriocins. The native signal sequence for \u003cem\u003eE. coli\u003c/em\u003e OmpA\u0026nbsp;(Freudl, Klose, and Henning 1990)\u0026nbsp;was placed immediately in front of the bacteriocin gene. A T7 promoter sequence was used to facilitate arabinose induction from the BL21-AI strain. All plasmid assemblies were conducted with Gibson assembly\u0026nbsp;(Gibson et al. 2009)\u0026nbsp;and transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e One Shot TOP10 (Thermo Fisher Scientific) via electroporation. Cells were recovered in SOC\u0026nbsp;+ 0.5ml 1M Mg2Cl2 + 2ml 1M glucose, for 1 hour at 37C with shake, then plated on LB agar plates containing 50ug/ml kanamycin (Roth Art-Nr: T832.3) and incubated at 37C ON. All \u003cem\u003eE. coli\u003c/em\u003e were grown in lysogeny broth (LB) (Sigma Aldrich) unless something else is specified.\u0026nbsp;Colony-PCR using OneTaq (Thermo Scientific\u003csup\u003e\u0026trade;\u003c/sup\u003e) confirmed the\u0026nbsp;plasmid assembly. PCR product was Sanger Sequenced using Eurofinsgenomics. Plasmids were extracted using plasmid extraction kit (Machery-Nagel - Nucleospin plasmid easy pure - 740725.250) and transformed into Invitrogen\u003csup\u003eTM\u003c/sup\u003e BL21-AI\u003csup\u003eTM\u003c/sup\u003e Oneshot \u0026reg; Chemically Competent\u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e according to suppliers\u0026rsquo; protocol (Thermo Fisher Scientific 11540146) and selected on LB-kanamycin plates. Colony-PCR using OneTaq was performed again to verify integration into BL21-AI.\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eEvaluations of optimal expression conditions for the bacteriocins:\u003c/strong\u003e Optimal growth conditions were examined using the target strain\u0026nbsp;\u003cem\u003eLactobacillus mali\u003c/em\u003e DSM 20444 using 8 different bacteriocins. The following growth conditions were tested: incubation temperatures of the plates (from the time BL21-AI was spotted on the plates to the end of the study) at 25, 30, or 37C, preculture overnight growth of BL21-AI in either mGAM of 2-YT, 6 hours of growth versus 18 hours of growth of the spotted BL21-AI culture on the plate before pouring the top agar, 0.2% vs 1% of arabinose concentration in the culture plates. The inhibition zones of the bacteriocins were used to evaluate the optimal growth conditions (which were very comparable through all the groups. The following protocol produced the largest inhibition zones for all bacteriocins tested.\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eExpression of bacteriocins for spot assay:\u003c/strong\u003e All bacteriocin expression for spot assays was performed in the following manner unless something else is stated. The bacteriocin producer was streaked on LB-kanamycin plates from a -80 cryo-stock and incubated at 37C ~14-18 hours. One colony was inoculated into 2-YT media containing 50ug/ml of kanamycin and incubated with shake for ~14-18 hours. Cultures were spun down and resuspended in PBS, then spun down again and resuspend in 2-YT to make sure all kanamycin was gone from the media. 10ul of the culture was spotted on mGAM square plates containing 1% arabinose (for induction of the bacteriocin gene via the T7 promoter). Plates were placed in aerobic conditions at 37C for ~20 hours. After 20 hours the plates that were used to screen anaerobic strains were transferred to ANO boxes and incubated further at 37C ON (~20hours) to pre-reduce the plates. Plates that were used to screen aero-tolerant strains were poured with top agar after the initial 20 hours of incubation. As a negative control was used BL21-AI with a pMUT plasmid present with no bacteriocin gene.\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eCultivation of target strains for spot assay:\u003c/strong\u003e Target anaerobic gut strains were grown in mGAM where possible. Aerobic Lactic acid bacteria were grown in MRS, \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eL. innocua\u003c/em\u003e were grown in BHI. Anaerobic strains were streaked from -80C cryo-stocks under anaerobic conditions (Whitley A95 Workstation - Don Whitley Scientific); gas mixture,\u0026nbsp;95% N\u003csub\u003e2\u003c/sub\u003e and 5% H\u003csub\u003e2\u003c/sub\u003e\u003csub\u003e.\u0026nbsp;\u003c/sub\u003eLactic acid strains were streaked from -80C in aerobic conditions on MRS and transferred to anerobic boxes. Strains growing on BHI were streaked aerobically and incubated aerobically. All strains were incubated at 37C ~ 18 hours. One colony was used to inoculate 1ml of the respective media and incubated for additionally 18-20 hours prior to the spot assay. Strains with Biosafety level2 were handled in the same manner, except that a\u0026nbsp;Coy Laboratory Products Vinyl; gas mixture, 95% N\u003csub\u003e2\u003c/sub\u003e and 5% H\u003csub\u003e2\u003c/sub\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003ewas used to make anaerobic conditions.\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eOverlay agar for spot assay:\u003c/strong\u003e For each square plate (Thermo fisher scientific omnitray w/lid Non treated sterile #264728) in total of 12ml pre-reduced top agar was used (0.5% agar) (Milipore # 69964) + 100ul of the target strain adjusted to OD ~0.5. Aerobic strains: Strains were mixed with top agar and poured over the plates. The plates were transferred to anaerobic boxes and incubated for 1-2 days at 37C. Anerobic strains: Agar plates with spots of BL21-AI bacteriocin producers and the liquid cultures containing the target strains, were transferred from anaerobic conditions into an aerobic laf bench. Top agar (~40C) was mixed with each of the target strains and poured over the respective plates and left to dry for ~5-10 mins before being transferred back to the anaerobic chamber and incubated in anaerobic boxes at 37C for 1-2 days. After 1 and 2 days of growth the plates were checked for inhibition zones. All strains were tested in at least duplicates. Halosize in mm can be found in Supplementary Table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis:\u003c/strong\u003e Heatmap with inhibited species against the bacteriocins with at least 1 target strain was generated using R (phyloseq). NCBI common taxonomy tree\u0026nbsp;(\u0026ldquo;Common Taxonomy Tree\u0026rdquo; n.d.)\u0026nbsp;was used to build a phylogenetic tree of the target species, and the heatmap was ordered according to that. Clustal Omega\u0026nbsp;(Sievers et al. 2011)\u0026nbsp;was used to create a multiple alignment of the protein sequences of the bacteriocins, and the heatmap was sorted according to that.\u003c/p\u003e\n\u003ch2\u003eEx vivo screening study\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth media preparation:\u003c/strong\u003e The following plates were used in the study: MRS containing 25% of \u003cem\u003eE. coli\u003c/em\u003e BL21-AI supernatant from either BL21-pMUT0-no-bacteriocin, BL21-pMUT12-ubericinA, BL21-pMUT13-actifencin, to select for a variety of LAB strains. mGAM agar plates containing 25% of supernatant from BL21-pMUT0-no-bacteriocin, BL21-pMUT12-ubericinA, BL21-pMUT13-Actifencin, BL21-pMUT22-bacteriodetocinA to select for gut strains in general.\u0026nbsp;mGAM-vancomycin (5ug/ml)-kanamycin (50ug/ml) containing 25% of supernatant from BL21-pMUT0-no-bacteriocin, BL21-pMUT22-bacteroidetocinA, to select for a variety of\u0026nbsp;\u003cem\u003eBacteroides\u003c/em\u003e strains. Supernatant for creating the agar plates were produced in the following manner: BL21-AI cultures were streaked on LB-kanamycin (50ug/ml) agar plates from -80C cryostocks and incubated at 37C for ~ 20hours. 1 colony was inoculated into 5ml 2YT-kanamycin(50ug/ml) and incubated with shake ~18 hours at 37C. Cultures were diluted 1:100 into fresh 200ml 2YT media in 1L shake flasks \u0026ndash; without antibiotics and incubated with shake at 37C until OD reaches 0.4-0.5. Cultures were then induced with 1% arabinose and incubated with shake for 6 hours at 30C. After 6 hours the cultures were centrifuged for 5 mins in 50ml falcon tubes at 4500xG at 4C, 1 tablet of protease inhibitor (Roche - cOmplete ULTRA Tablets, Mini, EDTA-free #05892791001) was added to every 50ml of supernatant. The supernatant was sterile filtered with 200ml filter cups (Biofil - FPV213500). Supernatant was immediately used to make agar plates by mixing with 60C freshly prepared 1.25X concentrated media. mGAM and mGAM-vancomycin-kanamycin plates were moved to anaerobic environment for prereduction for 24 hours. MRS plates were stored aerobically at 5C.\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eFeces collection:\u003c/strong\u003e Feces from 3 male C57BL/6nTAC mice that had received CHOW diet, and no antibiotics was collected and transferred to anaerobic environment within ~ 10 mins. Pellets were resuspended in 1ml pre-reduced 1% PBS. After resuspension the tubes were left for ~ 20 mins to allow sediment to form. 2x 200ul of the samples were transferred to 1.5ml Eppendorf tubes and centrifuged at 10.000xG for 10 mins. Supernatant was removed and the pellet was stored at -20C, for sequencing. 100ul of the fecal samples was used to make serial dilutions in 1% PBS down to 10\u003csup\u003e-8\u003c/sup\u003e.\u0026nbsp;100ul of the three independant biological replicates were plated of the dilutions\u0026nbsp;10\u003csup\u003e-4\u003c/sup\u003e -10\u003csup\u003e-8\u0026nbsp;\u003c/sup\u003eon mGAM and mGAM-vancomycin-kanamycin\u0026nbsp;plates. Plates were incubated anaerobically at 37C. For the MRS plates, selecting for lactic acid bacteria,\u0026nbsp;the dillutions 10\u003csup\u003e-3\u003c/sup\u003e \u0026ndash; 10\u003csup\u003e-\u003c/sup\u003e⁷ dilutions\u0026nbsp;were used. MRS\u0026nbsp;plates were incubated\u0026nbsp;aerobically, to allow further selection for lactic acid strains,\u0026nbsp;at 37C.\u0026nbsp;Colonies were counted every day for 5 days \u0026ndash; until no new colonies appeared on the plates. Three dilutions, consisting of the countable dilution (between 25 and 250 colonies) and the dilutions above and below it, were chosen\u0026nbsp;for sequencing. None of the plate dilutions utilized represented a situation where a lawn was formed or the plate would not be able to be counted. 2ml\u0026nbsp;1XPBS\u0026nbsp;where administered\u0026nbsp;onto\u0026nbsp;the plates and\u0026nbsp;a spatula was used to mix the colonies on the plates.\u0026nbsp;~1.2ml was collected in 1.5ml eppendorph tubes, and centrifuged 15min 12000XG. Supernatant was removed at pellets were frozen at\u0026nbsp;-20 untill\u0026nbsp;DNA extraction.\u0026nbsp;The pelleted samples were then extracted as individual dilutions\u0026nbsp;using\u0026nbsp;ZymoBiomics DNA Kit\u0026nbsp;(D4300- zymoresearch).\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eDNA preparation for and 16S rRNA MiSeq sequencing:\u003c/strong\u003e All DNA samples were prepared for sequencing using the following protocol: \u0026ldquo;16S Metagenomic Sequencing Library Preparation\u0026nbsp;(\u0026ldquo;16s-Metagenomic-Library-Prep-Guide-15044223-b.Pdf\u0026rdquo; n.d.)\u0026rdquo;. In short:\u0026nbsp;Extracted DNA was diluted to 5 ng/ml for all samples to keep quantities constant for the amplification step. PCR was performed using\u0026nbsp;KAPA\u0026nbsp;PCR Master Mix\u0026nbsp;(Roche)\u0026nbsp;and tagged Illumina primers (10 mM concentration) in\u0026nbsp;25\u0026nbsp;ml reactions targeting the hypervariable\u0026nbsp;V3-V4\u0026nbsp;(341F - 785R)\u0026nbsp;region (primer\u0026nbsp;are\u0026nbsp;listed in Supplementary\u0026nbsp;Table 3). Illumina overhangs (100 mM concentration) were attached in a second PCR reaction by combining barcoded samples in equal amounts as template for amplifying multiple\u0026nbsp;50\u0026nbsp;ml reactions. Thermocycling conditions for both PCR steps were as follows, except 25 cycles in the first PCR and 8 cycles in the second PCR: initial denaturation 95\u0026nbsp;\u0026deg;C for\u0026nbsp;3\u0026nbsp;min, followed by\u0026nbsp;25 or 8\u0026nbsp;cycles of:\u0026nbsp;95\u0026nbsp;\u0026deg;C for\u0026nbsp;30 s,\u0026nbsp;55 \u0026deg;C for 30 s and 72 \u0026deg;C for 30 s, and a final elongation at 72 \u0026deg;C for\u0026nbsp;10\u0026nbsp;min. PCR product sizes were confirmed at each step and the final PCR product was purified using AMPure XP bead\u0026nbsp;(Beckman coulter). The samples were\u0026nbsp;measured with qubit and normalized to 10nM/ul (diluted in Tris_HCL pH:8.5), then run on Agilent 2100 Bioanalyzer (Covaris) to verify the size of the fragments, and\u0026nbsp;sequenced on an Illumina MiSeq\u0026nbsp;system.\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eSequence quality control and processing:\u003c/strong\u003e Fastq files were downloaded from Basespace.illumina.com. Qiime2 was used to process the fastq files to count matrices, followed by downstream data analysis using R. The following tutorial was used to perform the analysis using Qiime2\u0026nbsp;(\u0026ldquo;Metadata in QIIME 2 \u0026mdash; QIIME 2 2022.8.3 Documentation\u0026rdquo; n.d.). In short: fastq files were imported into qiime2 as paired end with input phred33. Next\u0026nbsp;quality filtering, chimera checking, and paired- end read joining\u0026nbsp;of the sequence data was perform with DADA2\u0026nbsp;(Callahan et al. 2016)\u0026nbsp;through the q2-dada2 plugin.\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eFor the ex vivo study:\u003c/strong\u003e reads were truncated when the quality score became approximately below 25 (forward reads at 285bp, reverse reads at 240bp). Reads were filtered from each sample (between 55.49% and 89.85% with a mean of 85.32% - reads per sample were between 4621 and 633583 with a mean of 89248 reads).\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eFor the in vivo study:\u003c/strong\u003e reads were truncated when the quality score became approximately below 25 (forward reads at 260bp, reverse reads at 240bp). Reads were filtered from each sample with a mean of 57% - reads per sample were between 401 and 104.077 with a mean of 70.255 reads).\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eFor both the ex vivo and in vivo study:\u003c/strong\u003e A feature table and feature data were generated using the command \u0026ldquo;qiime feature-table\u0026rdquo; describing the ASVs that were observed in each sample and how many times it was observed. To assign taxonomic information to the ASV sequences a trained classifier for the V3-V4 region based on the \u0026ldquo;SILVA release_139 nr99\u0026rdquo; SSU database, which uses 99% similarity to assign species to an ASV. The classifier was downloaded from Github: https://github.com/anweshmaile/silva-138_classifiers. The command \u0026ldquo;qiime feature-classifier\u0026rdquo; was used for this analysis, outputting a count matrix used for further processing in R using the phyloseq package. In R the further data processing was handled. ASVs were removed if they had less than 2 counts in at least 10% of samples. This reduced ASVs from 4541 to 332 taxa in the \u003cem\u003eex vivo\u003c/em\u003e study and removing ASVs with less than 2 counts in 5% of samples reduced the number of taxa from 4600 to 2293 in the \u003cem\u003ein vivo\u003c/em\u003e study. Rarefaction curves were made for both studies with curves showing max species at ~2000 species in both cases, therefore rarefaction was performed using 2000 species per sample. This removed five samples from the \u003cem\u003eex vivo\u003c/em\u003e data, to leave in total 76 samples, and one sample in the \u003cem\u003ein vivo\u003c/em\u003e data to leave in total 71 samples. Beta-diversity was examined using Bray-Curtis method quantifying the difference between the overall taxonomic composition between samples.\u003c/p\u003e\n\u003ch2\u003eIn vivo study:\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid and strain construction for expression in \u003cem\u003eE. coli\u003c/em\u003e Nissle:\u003c/strong\u003e Primers, promoters and sequences are listed in the Supplementary Table 3. Primers and geneblocks were ordered from Integrated DNA Technologies (IDT). The probiotic strain used in this study, \u003cem\u003eEcN\u003c/em\u003e_GFP, StrepR: \u003cem\u003eE. coli\u003c/em\u003e Nissle 1917 is a modified version of the wild-type \u003cem\u003eE. coli\u003c/em\u003e Nissle 1917 (tradename Mutaflor, Ardeypharm, Germany) strain(Jacobi and Malfertheiner 2011). Plasmid pMUT\u0026nbsp;(Zainuddin, Bai, and Mansell 2019)-kanR-HokSok was used as the vector. \u003cem\u003eEcN\u003c/em\u003e_GFP was used as production host using a strong constitutive promotor (#1.7\u0026nbsp;from the\u0026nbsp;Schantzetta library\u0026nbsp;(Armetta et al. 2021)). The bacteriocins were secreted using the OmpA signal sequence\u0026nbsp;(Freudl, Klose, and Henning 1990). The ribosomal binding site (RBS) of the bacteriocin gene was measured with salislab.net\u0026nbsp;(Salis, Mirsky, and Voigt 2009). Strength was ~5000 compared to the RBS in the pMUT plasmid for \u003cem\u003ein vitro\u003c/em\u003e expression of bacteriocins which was ~10.000. The following plasmids were cloned and expressed in\u003cem\u003e\u0026nbsp;EcN\u003c/em\u003e_GFP: \u003cem\u003eEcN-\u003c/em\u003epMUT0-no-bacteriocin, \u003cem\u003eEcN-\u003c/em\u003epMUT12-ubericinA, \u003cem\u003eEcN-\u003c/em\u003epMUT13-actifencin, \u003cem\u003eEcN-\u003c/em\u003epMUT22-bacteroidetocinA, and \u003cem\u003eEcN-\u003c/em\u003epMUT52_Bacteriocin31. Cloning and transformation, plasmid evaluation and purification was performed similar to the plasmid construction for\u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e BL21-AI expression.\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eGeneration of competent \u003cem\u003eEcN\u003c/em\u003e:\u0026nbsp;\u003c/strong\u003e\u003cem\u003eEcN\u003c/em\u003e_GFP was made competent for electroporation in the following way: Culture was streaked from -80C cryo-stock on LB-streptomycin (50ug/ml) agar plates and incubated at 37C for ~20 hours. Then, 1 colony was used to inoculate 5ml 2YT-streptomycin (50ug/ml) and incubated at 37C for ~20hours with shake. Cultures were diluted 1:100 and incubated at 37C with shake until OD reached 0.3-0.5 (~2 hours). When desired OD was reached cultures were placed on ice for 15mins, then centrifuged for 10 mins at 4C and 4500xG. Supernatant was removed and pellet was resuspended in 1ml MQ water+10% glycerol (4C). Cultures were centrifuged for 3 mins at 6500RPM at 4C, this step was repeated 3 times. After the last wash cells were resuspended in 50ul MQ water+10% glycerol (4C). 1ul of the purified plasmid was used for electroporation of the 50ul competent \u003cem\u003eEcN\u003c/em\u003e_GFP cells. Cells were recovered in 1ml fortified SOC for 1 hour at 37C with shake, then plated on LB agar plates containing 50ug/ml kanamycin and incubated at 37C for 14-18 hours. Colony PCR and gel electrophoresis was used to identify clones. Plasmids were extracted and subjected to whole plasmid sequencing using plasmidsaurus.com. Growth rate of the clones were evaluated using a plate reader (Synergy H1 - Holm and Halby). Two clones of each of the four \u003cem\u003eEcN\u003c/em\u003e_GFP strains were inoculated 1:100 in 3 replicates into 100ul LB-kanamycin (50ug/ml) in a 96-well plate. Growth was followed for 24hours in a plate reader (continuous shake 37C) (Synergy H1 - Holm and Halby). Data was extracted and doubling time was calculated using R.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;Animal experiment: Ethics:\u003c/strong\u003e The animal experiment was conducted according to the Danish Animal Experiments Act on protection of animals used for scientific purpose (LBK 1107 from 02/07/2022) and Directive 2012/63/EU of the European Parliament. In addition, the protocol was licensed accordingly by the Animal Experimentation Committee under the Ministry of Food, Fishing, and Agriculture (license number 2020-15-0201-00405). The study was carried out in accordance with the ARRIVE guidelines\u0026nbsp;(Percie du Sert et al. 2020). Animal study design: Twenty-four male C57BL/6NTac (Taconic Biosciences, Lille Skensved, Danmark) mice aged 5 weeks went through 7 days of acclimatization before being divided into four groups (n=6) based on weight stratification. Hereafter, the mice received 1 daily oral dosing of 100 \u0026micro;l either containing\u0026nbsp;PBS, \u003cem\u003eE. Coli\u003c/em\u003e Nissle pMUT-empty (CFU 10\u003csup\u003e11\u003c/sup\u003e),\u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e Nissle pMUT13_actifencin\u0026nbsp;(CFU 10\u003csup\u003e11\u003c/sup\u003e) or\u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e Nissle pMUT22_bacteroidetocinA\u0026nbsp;(CFU 10\u003csup\u003e11\u003c/sup\u003e). CFU was measured based on OD600 measurements and standard curves created by spotting different dilutions of the gavage. After 7 days the mice went through a washout period of 7 more days. Colonization was investigated by fecal sampling\u0026nbsp;on days: 0, 1,2,3,4,5,6,7, 9, 11, and 14.\u0026nbsp;The mice were co-housed 3 per cage in individually ventilated cages (IVC).\u0026nbsp;All mice were housed at\u0026nbsp;22\u0026nbsp;\u0026deg;C\u0026nbsp;\u0026plusmn;\u0026nbsp;2\u0026nbsp;\u0026deg;C, light cycle was 6 am to 6 pm,\u0026nbsp;and\u0026nbsp;the mice were\u0026nbsp;given ad libitum access to water and chow\u0026nbsp;diet\u0026nbsp;(Safe Diets, A30).\u0026nbsp;At end- study the mice were euthanized by CO\u003csub\u003e2\u003c/sub\u003e sedation and cervical dislocation. Content from\u0026nbsp;small intestine, cecum and colon\u0026nbsp;collected in 1xPBS to be tested for colonization (CFU count) immediately after collection.\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eColonization:\u003c/strong\u003e The faeces were collected in pre-weighed 2.0 mL Eppendorf tubes including 1 mL of 1x PBS. After fecal samples had been added to the tubes these were weighed again to determine the faecal weight. All sample preparation for assessing CFU numbers was kept on ice and followed the same practice. The faecal samples were homogenised by vortexed at ~2400 rpm for 20 min. The samples were then spun down at 100xG for 30 seconds, followed by a dilution series, where 5 \u0026mu;L of each dilution was plated on LB supplemented with 50mg/ml kanamycin and 50mg/ml streptomycin. (Sigma Aldrich). After 24 hours CFU was determined by counting.\u0026nbsp;Samples were then spun down for 20mins at 11000xG, supernatant was removed, and pellet was stored at -20C until DNA samples were extracted using DNeasy powersoil HTP 96 kit (qiagen Cat. No. 12955-4)\u003c/p\u003e\n\u003cp\u003eDNA preparation for MiSeq 16S rRNA amplicon sequencing and sequence quality control was performed in the same manner as described for the\u0026nbsp;\u003cem\u003eex vivo\u003c/em\u003e samples.\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eVerification of \u003cem\u003eEcN\u003c/em\u003e strains from mouse feces:\u003c/strong\u003e 2-3 Fecal pellets from mice at study day 6 from the #13_actifencin and #22_bacteroidetocinA groups were resuspended in 200ml 1X PBS and serial diluted to 10^-3. 100ul of each dilution was plated on LB-kanamycin plates and incubated for 20 hours at 37C. \u003cem\u003eEcN\u003c/em\u003e_pMUT22_bacteroidetocinA plates were transferred to ANO boxes and placed at 5C for 24 hours before performing the spot assay. 4 colonies from 2 replicate mouse feces of \u003cem\u003eEcN\u003c/em\u003e_pMUT13_actifencin was re-streaked on LB plates containing no antibiotics and incubated for additional 20hours before performing the spot assay. Plates containing \u003cem\u003eEcN\u003c/em\u003e_pMUT22_bacteroidetocinA was tested using the indicator strain \u003cem\u003eBacteroides vulgatus\u003c/em\u003e DSM 1447. \u003cem\u003eEcN\u003c/em\u003e_pMUT13_actifencin was tested using the indicator strain: \u003cem\u003eLactobacillus mali\u003c/em\u003e DSM 20444. Indicator strains were cultured similar to what is described in the\u003cem\u003e\u0026nbsp;in vitro\u0026nbsp;\u003c/em\u003espot assay section. After pouring the top agar on the respective plates these were incubated ANO in the case for \u003cem\u003eEcN\u003c/em\u003e_pMUT22_bacteroidetocinA and AE in the case for \u003cem\u003eEcN\u003c/em\u003e_pMUT13_actifencin at 37C. After ~20 hours the plates were inspected for inhibition zones. 9 colonies of each of the \u003cem\u003eEcN\u003c/em\u003e strains were subjected to colony PCR to verify that no mutations had occurred in the promotor or gene region of the plasmid. PCR product was Sanger sequenced with eurofinsgenomics.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eMedia and antibiotic concentrations used and antibiotic references:\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eFortified SOC medium recipe: 100ml SOC + 0.5ml 1M Mg2Cl2 + 2ml 1M glucose, Tween80: Sigma-Aldrich 102578383, kanamycin sulfate: Roth Art-Nr: T832.3 (concentration used: 50ug/ml), streptomycin sulfate salt: Sigma-Aldrich - Merck Life Science 9137, Vancomycin: Sigma-Aldrich - Merck Life Science -94747-1G, arabinose: Sigma Aldrich - L-(+)-Arabinose W325501, MRS deMan, Rogosa, Sharpe media: Milipore # 69966, MRS deMan, Rogosa, Sharpe agar: Milipore # 69964, mGAM media and agar (modified Gifu Anaerobic Media \u0026ndash; Nissui pharmaceutical CO.,LTD), BHI (Brain Heart Infusion, Merck #53286)\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and Consent to participate\u003c/h2\u003e\n\u003cp\u003eThe animal experiment was conducted according to the Danish Animal Experiments Act on protection of animals used for scientific purpose (LBK 1107 from 02/07/2022) and Directive 2012/63/EU of the European Parliament. In addition, the protocol was licensed accordingly by the Animal Experimentation Committee under the Ministry of Food, Fishing, and Agriculture (license number 2020-15-0201-00405). The study was carried out in accordance with the ARRIVE guidelines (Percie du Sert et al. 2020)\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets\u0026nbsp;supporting the conclusions of this article are available in the\u0026nbsp;NCBI\u0026nbsp;repository. \u003cem\u003eEx vivo\u003c/em\u003e data:\u0026nbsp;\u0026nbsp;BioProject ID PRJNA1007568\u0026nbsp;(\u0026ldquo;ID 1007563 - BioProject - NCBI\u0026rdquo; n.d.). The\u0026nbsp;\u003cem\u003eIn vivo\u003c/em\u003e dataset supporting the conclusions of this article are available in the NCBI repository:\u0026nbsp;BioProject ID: PRJNA1007563\u0026nbsp;(\u0026ldquo;ID 1007568 - BioProject - NCBI\u0026rdquo; n.d.)\u003c/p\u003e\n\u003cp\u003eThe datasets\u0026nbsp;supporting the conclusions of this article are included within the article\u0026nbsp;\u0026nbsp;and its additional files:\u003c/p\u003e\n\u003ch3\u003eSupplementary files:\u003c/h3\u003e\n\u003cp\u003eSupplementary Table 1: List of the bacteriocins cloned and expressed in this study.\u003c/p\u003e\n\u003cp\u003eSupplementary Table 2: list of representative gut microbiota strains.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 1:\u0026nbsp;Growth rate assesment for the EcN strains and spot assay for the EcN 13 and 22\u003cbr\u003e\u0026nbsp;Supplementary Table 3: Primers, promoters, and sequences\u003c/p\u003e\n\u003cp\u003eSupplementary Table 4: List of screened bacteriocins against representative gut strains including halo-size of inhibition.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors\u0026nbsp;declares\u0026nbsp;that they have no competing interests\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work received funding from The Novo Nordisk Foundation under NNF grant number: NNF20CC0035580\u0026nbsp;and The Novo Nordisk Foundation, Challenge programme, CaMiT under grant agreement: NNF17CO0028232\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003eMS: provided the initial and overall idea of the project. CR: designed the concepts for the \u003cem\u003ein vitro\u003c/em\u003e experiment, CR \u0026amp; RV: designed the concepts for the \u003cem\u003eex vivo\u003c/em\u003e experiment, CR \u0026amp; DL: designed and conducted the \u003cem\u003ein vivo\u003c/em\u003e study, CR: analyzed the data, CR: wrote the manuscripts with input from MS, RV, \u0026amp; DL.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u0026ldquo;16s-Metagenomic-Library-Prep-Guide-15044223-b.Pdf.\u0026rdquo; n.d. 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Kudva, Jang Won Yoon, Carl W. Hunt, Christopher J. Williams, and Carolyn J. Hovde. 2002. \u0026ldquo;Gastrointestinal Tract Location of Escherichia Coli O157:H7 in Ruminants.\u0026rdquo; \u003cem\u003eApplied and Environmental Microbiology\u003c/em\u003e 68 (5): 2269\u0026ndash;77. https://doi.org/10.1128/AEM.68.5.2269-2277.2002.\u003c/li\u003e\n \u003cli\u003eHammami, Riadh, Abdelmajid Zouhir, Christophe Le Lay, Jeannette Ben Hamida, and Ismail Fliss. 2010. \u0026ldquo;BACTIBASE Second Release: A Database and Tool Platform for Bacteriocin Characterization.\u0026rdquo; \u003cem\u003eBMC Microbiology\u003c/em\u003e 10 (January): 22. https://doi.org/10.1186/1471-2180-10-22.\u003c/li\u003e\n \u003cli\u003eHeel, Auke J. van, Anne de Jong, Manuel Montalb\u0026aacute;n-L\u0026oacute;pez, Jan Kok, and Oscar P. 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Kuipers. 2011. \u0026ldquo;Evaluating the Feasibility of Lantibiotics as an Alternative Therapy against Bacterial Infections in Humans.\u0026rdquo; \u003cem\u003eExpert Opinion on Drug Metabolism \u0026amp; Toxicology\u003c/em\u003e 7 (6): 675\u0026ndash;80. https://doi.org/10.1517/17425255.2011.573478.\u003c/li\u003e\n \u003cli\u003eHeilbronner, Simon, Bernhard Krismer, Heike Br\u0026ouml;tz-Oesterhelt, and Andreas Peschel. 2021. \u0026ldquo;The Microbiome-Shaping Roles of Bacteriocins.\u0026rdquo; \u003cem\u003eNature Reviews Microbiology\u003c/em\u003e 19 (11): 726\u0026ndash;39. https://doi.org/10.1038/s41579-021-00569-w.\u003c/li\u003e\n \u003cli\u003eHern\u0026aacute;ndez-Gonz\u0026aacute;lez, Juan Carlos, Abigail Mart\u0026iacute;nez-Tapia, Gebim Lazcano-Hern\u0026aacute;ndez, Blanca Estela Garc\u0026iacute;a-P\u0026eacute;rez, and Nayeli Shantal Castrej\u0026oacute;n-Jim\u0026eacute;nez. 2021. \u0026ldquo;Bacteriocins from Lactic Acid Bacteria. 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Mansell. 2019. \u0026ldquo;CRISPR-Based Curing and Analysis of Metabolic Burden of Cryptic Plasmids in Escherichia Coli Nissle 1917.\u0026rdquo; \u003cem\u003eEngineering in Life Sciences\u003c/em\u003e 19 (6): 478\u0026ndash;85. https://doi.org/10.1002/elsc.201900003.\u003c/li\u003e\n \u003cli\u003eZhang, L H, M J Fath, H K Mahanty, P C Tai, and R Kolter. 1995. \u0026ldquo;Genetic Analysis of the Colicin V Secretion Pathway.\u0026rdquo; \u003cem\u003eGenetics\u003c/em\u003e 141 (1): 25\u0026ndash;32. https://doi.org/10.1093/genetics/141.1.25.\u003c/li\u003e\n \u003cli\u003eZhang, Na, Chen Li, Zhihua Niu, Hongyan Kang, Miaoshu Wang, Bo Zhang, and Hongtao Tian. 2020. \u0026ldquo;Colonization and Immunoregulation of Lactobacillus Plantarum BF_15, a Novel Probiotic Strain from the Feces of Breast-Fed Infants.\u0026rdquo; \u003cem\u003eFood \u0026amp; Function\u003c/em\u003e 11 (4): 3156\u0026ndash;66. https://doi.org/10.1039/C9FO02745A.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Class II bacteriocins, gut microbiota, novel antimicrobial peptides, gut modulation strategies","lastPublishedDoi":"10.21203/rs.3.rs-3282788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3282788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBacteriocins are antimicrobial peptides with properties making them applicable for food preservation and pathogen control. However, their impact on the gut microbiota remains understudied. In this study we sought to examine the inhibitory spectrum of 75 class II bacteriocins against 49 representative species of the human gut microbiota including 21 Biosafety level 2 organisms. The bacteriocins were cloned and expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e and evaluated \u003cem\u003ein vitro, ex vivo\u003c/em\u003e and \u003cem\u003ein vivo.\u003c/em\u003e\u003cbr\u003e\n \u003cstrong\u003eResults\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e spot assays using \u003cem\u003eEscherichia coli\u003c/em\u003e BL21-AI expressing these bacteriocins revealed that 22 bacteriocins inhibited at least one species, with greater efficacy against Gram-positive than Gram-negative species. Two bacteriocins, Actifencin and Bacteroidetocin A, were selected based on their broad spectrum of inhibition for further characterization and applied to murine feces \u003cem\u003eex vivo\u003c/em\u003e. Application of these bacteriocins led to substantial modifications in the composition of the microbial community of murine feces. Yet, these findings could not be replicated \u003cem\u003ein vivo\u003c/em\u003e when bacteriocin producing \u003cem\u003eEscherichia coli \u003c/em\u003eNissle strains were dosed to mice. \u003cbr\u003e\n\u003cstrong\u003eConclusions\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study evaluate the potential and expands our knowledge of the inhibitory spectrum of class II bacteriocins against a large and representative collection of bacterial isolates from the gut microbiota and underscores that further optimization is needed to use bacteriocins \u003cem\u003ein vivo\u003c/em\u003efor modulating microbial community composition.\u003c/p\u003e","manuscriptTitle":"Antimicrobial potential of class II bacteriocins on gut microbiota species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-28 19:44:20","doi":"10.21203/rs.3.rs-3282788/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ffb88e24-3f94-42ff-833c-af22ac5a6be3","owner":[],"postedDate":"August 28th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-09-27T22:59:17+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-28 19:44:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3282788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3282788","identity":"rs-3282788","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}