Genomic and Bioinformatic Insights into Enterococcus faecalis from Retail Meats in Nigeria

Enterococcus faecalis, antimicrobial resistance, whole-genome sequencing, virulence, 22 plasmids, retail meat, Nigeria 23 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 2

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Enterococcus faecalis (E. faecalis) is a commensal and opportunistic pathogen 25 increasingly recognized for its antimicrobial resistance (AMR) and zoonotic potential. This study 26 employs whole -genome sequencing (WGS) to characterize E. faecalis isolates from retail meat 27 samples, focusing on antimicrobial resistance genes (ARGs), virulence determinants, mobile genetic 28 elements, and phylogenomic relationships. Materials and Methods: Fifty raw meat samples, 29 including chicken (n=18), beef (n=17), and turkey (n=15), were collected from retail markets in 30 Akungba-Akoko, Nigeria. E. faecalis isolates were identified using standard microbiological 31

and subjected to antimicrobial susceptibility testing were further analysed using WGS. 32

Ten E. faecalis isolates were recovered, with the highest prevalence in chicken (n=6), 33 followed by beef (n=2) and turkey (n=2). All isolates were resistant to clindamycin, erythromycin, 34 and tetracycline. Frequent ARGs included aac(6’)-aph(2’’), ant(6)-Ia, lsa(A), erm(B), tet(M), 35 and tet(L). Plasmid replicons rep9c and repUS43 showed ST-specific associations with ST477 and 36 ST16, respectively. MGEs such as IS3, IS6, IS256, and IS1380 co-localized with ARGs and 37 virulence determinants. Phylogenomic analysis revealed two major lineages, with ST477 distributed 38 across meat types and ST16 restricted to chicken. Comparative genomic analysis with publicly 39 available African E. faecalis isolates revealed distinct clonal lineages and geographic clustering 40 across the continent. Conclusion: The co-occurrence of multidrug resistance, virulence factors, and 41 MGEs in foodborne E. faecalis poses a public health concern due to the risk of horizontal gene 42 transfer and zoonotic spread. These findings underscore the need for genomic surveillance and 43 antimicrobial stewardship in food systems, particularly in low- and middle-income countries. 44 45 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 3 1. Introduction 46 Enterococcus faecalis is a commensal bacterium of the gastrointestinal tracts of humans and 47 animals, yet it has emerged as a notable opportunistic pathogen, especially in healthcare settings 48 where multidrug -resistant (MDR) strains contribute to severe and difficult -to-treat infections 49 (Farman et al., 2019). Beyond clinical contexts, its presence in food system particularly in raw meats 50 raises significant concerns about its role in the dissemination of antimicrobial resistance genes 51 (ARGs) and virulence factors via the food chain (de Mesquita Souza Saraiva et al., 2022). The 52 species' adaptability is bolstered by its remarkable capacity to acquire and transfer mobile genetic 53 elements (MGEs), which facilitates horizontal gene transfer and complicates therapeutic strategies 54 (Hegstad et al., 2010). These characteristics collectively pose a dual threat to both food safety and 55 public health, necessitating a comprehensive understanding of its genomic architecture across 56 diverse ecological niches. 57 Globally, genomic studies have examined the resistance mechanisms and genetic diversity 58 of E. faecalis across clinical, livestock, and environmental settings (Daniel et al., 2017; Guan et al., 59 2024). However, substantial gaps remain in low -resource regions where genomic surveillance of 60 foodborne isolates is limited (Okeke et al., 2022). In Nigeria, retail meat is a dietary staple, yet little 61 is known about the genomic features of E. faecalis circulating in these products (Wada et al., 2020). 62 Existing research has largely focused on phenotypic antibiotic resistance (Ndahi et al., 2023), with 63 minimal exploration into the genetic determinants of resistance, virulence, and gene transfer that 64 contribute to its pathogenic potential (Okeke et al., 2022). This is particularly concerning in a country 65 where antibiotic use in agriculture remains poorly regulated and surveillance infrastructure is still 66 evolving (Schnirring, 2023), potentially accelerating the emergence of MDR lineages. 67 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 4 The public health implications of foodborne E. faecalis are further underscored by increasing 68 evidence of clonal transmission across animals, humans, and the environment (Monteiro Marques 69 et al., 2023; Poulsen et al., 2012). Hospital-adapted lineages of E. faecalis have been shown to carry 70 MGEs conferring resistance to clinically critical antibiotics such as vancomycin and β -lactams, as 71 well as virulence genes that promote biofilm formation, immune evasion, and tissue invasion (Raven 72 et al., 2016; Hourigan et al., 2024). If food -derived strains harbor similar genomic traits, this could 73 signal a critical interface between agricultural and clinical reservoirs, a hypothesis that remains 74 largely untested in key distribution hubs such as Akungba -Akoko, a prominent meat market in 75 southwestern Nigeria (Alimi, 2013). 76 This study addresses this knowledge gap by performing a comprehensive genomic 77 characterization of E. faecalis isolates recovered from retail meat in Akungba -Akoko. Utilizing 78 whole-genome sequencing (WGS) and bioinformatics approaches, we aim to (1) assess the 79 prevalence and diversity of ARGs, including those conferring resistance to critically important 80 antimicrobials; (2) characterize virulence determinants associated with adhesion, biofilm formation, 81 and immune evasion; and (3) investigate the mobile genetic elements (MGEs) facilitating gene 82 exchange. These findings will contribute to our understanding of the genomic plasticity of E. 83 faecalis in Nigeria’s food systems and inform mitigation strategies to reduce the public health risks 84 posed by this emerging foodborne pathogen. 85 86 2. Materials and methods 87 2.1. Sample collection and study site 88 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 5 The study was carried out over a three-month period between April and June 2022: 50 samples of 89 raw retail meat, including chicken (n=18), beef (n=17), and turkey (n=15), from the Ibaka market 90 (7.473500° N, 5.736250° E) in Akungba Akoko, Nigeria. The Ibaka market is a rural periodic day 91 market located in Akoko, which is the host community of Adekunle Ajasin University. 92 2.2. Isolation and identification of Enterococcus faecalis isolates 93 In the laboratory, each meat sample was aseptically homogenized. Smears of the homogenates were 94 prepared and subjected to Gram staining to identify gram -positive cocci arranged in pairs or short 95 chains, which are characteristic of Enterococcus species. For bacterial isolation, aliquots of the 96 homogenized samples were inoculated onto blood agar plates and incubated aerobically at 37°C for 97 24–48 hours. Colonies exhibiting typical Enterococcus morphology were selected for further 98 testing. Presumptive Enterococcus isolates were identified on the basis of their Gram staining 99 characteristics and ability to grow on blood agar. Biochemical tests were performed to confirm that 100 the isolates were Enterococcus species. These tests included the Voges‒Proskauer (VP) test for 101 detecting acetoin production and the potassium tellurite (PT) test to assess the ability to reduce 102 tellurite. Additionally, fermentation tests for glucose, lactose, and sucrose were conducted to 103 evaluate the carbohydrate utilization profiles of the isolates. Staphylococcus aureus ATCC 29213 104 and E. faecalis ATCC 29212 served as negative and positive controls, respectively. Confirmed E. 105 faecalis isolates were preserved by storing them in brain heart infusion broth (Difco) supplemented 106 with 20% glycerol at −70°C for long-term storage. 107 2.3. Antibiotic susceptibility testing of Enterococcus faecalis strains 108 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 6 The antibiotic resistance profiles of E. faecalis isolates were determined via the disk diffusion 109

on Mueller‒Hinton agar (MHA), following the guidelines of the Clinical and Laboratory 110 Standards Institute (CLSI, 2023). Overnight cultures of the isolates were used to prepare bacterial 111 suspensions adjusted to a turbidity equivalent to a 0.5 McFarland standard. A sterile cotton swab 112 was dipped into each suspension and evenly streaked across the entire surface of the MHA plates to 113 ensure a uniform bacterial lawn. Commercial antibiotic disks (Hi -Media, India) were placed onto 114 the inoculated plates via sterile forceps. The antibiotics used and their corresponding disk 115 concentrations were as follows: tetracycline (30 μg), chloramphenicol (30 μg), streptomycin (10 μg), 116 kanamycin (30 μg), erythromycin (15 μg), vancomycin (30 μg), clindamycin (2 μg), and tobramycin 117 (10 μg). The plates were incubated aerobically at 37 °C for 18 –24 hours. After incubation, the 118 diameters of the inhibition zones around each antibiotic disk were measured in millimeters. 119 Staphylococcus aureus ATCC 25923 was used as the control. The results were interpreted according 120 to CLSI guidelines (CLSI, 2023), categorizing the isolates as susceptible, intermediate, or resistant 121 to each antibiotic tested. 122 2.4. DNA Extraction, Whole -Genome Sequencing, and Assembly of Enterococcus 123 faecium strains 124 Genomic DNA was extracted from E. faecalis isolates via the MasterPure™ Gram Positive DNA 125 Purification Kit (Lucigen, Middleton, WI, USA) according to the manufacturer's instructions. The 126 quality and concentration of the extracted DNA were assessed via a NanoDrop 1000 127 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The genomic DNA libraries 128 were prepared via the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) 129 following the manufacturer's protocol. Sequencing was performed on an Illumina NovaSeq 6000 130 system (Illumina, San Diego, CA, USA) to generate paired-end reads. The raw sequence reads were 131 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 7 assembled into contigs via the Shovill pipeline version 1.0.4, which incorporates Trimmomatic 132 version 0.38 for read trimming and quality control. Genome annotation was conducted via Prokka 133 version 1.13.3. 134 2.5. Identification of resistance genes, virulence genes, plasmids and multi -locus 135 sequence typing 136 Antimicrobial resistance genes were identified via ResFinder version 4. 6.0 137 (http://genepi.food.dtu.dk/resfinder). Virulence genes were detected via VirulenceFinder version 2.0 138 (https://cge.food.dtu.dk/services/VirulenceFinder/). Plasmid types were determined by analysing the 139 assembled genome sequences with PlasmidFinder version 2.1 140 (https://cge.food.dtu.dk/services/PlasmidFinder/). Multilocus sequence typing (MLST) was 141 performed via the MLST tool version 2.0 (https://cge.food.dtu.dk/services/MLST/) to assign 142 sequence types to the E. faecalis isolates. 143 2.6. Phylogenomic analysis and metadata insights 144 The de novo assembled contigs of the E. faecalis isolates were submitted to CSI Phylogeny version 145 1.4 ( https://cge.cbs.dtu.dk/services/CSIPhylogeny-1.4), an online tool that identifies single 146 nucleotide polymorphisms (SNPs) from whole-genome sequencing (WGS) data, filters and validates 147 SNP positions, and infers phylogeny on the basis of concatenated SNP profiles. The Enterococcus 148 faecalis ATCC BAA-2128 strain (Accession number: NAQY00000000.1) was used as an outgroup 149 to root the tree, facilitating the assessment of phylogenetic relationships among the E. 150 faecalis strains. The phylogenetic tree was visualized and annotated with isolate metadata, including 151 demographic information, sequence types, resistome profiles, and mobile genetic elements (MGE), 152 via ITOL version 7 (https://itol.embl.de). This approach provided a comprehensive analysis of the 153 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 8 phylogenomic relationships among the isolates. Additionally, whole -genome sequences of E. 154 faecalis isolates from Africa, curated from public databases such as PATRIC 155 (https://www.patricbrc.org/) and NCBI between 201 3 and 2023, were downloaded and included in 156 the phylogenomic analysis to provide epidemiological and evolutionary context (Table S1). The 157 trees were edited and visualized via FigTree version 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). 158 Isolates belonging to the same STs are highlighted with the same color, and isolates from the same 159 geographical regions are labelled with the same text color to facilitate visual interpretation. 160 2.7. Nucleotide sequence 161 The sequences of the E. faecalis strains analysed in this study were deposited in the National Center 162 for Biotechnology Information GenBank database under BioProject number PRJNA928459. 163 164 3. Results 165 3.1. Prevalence and Antibiotic Resistance Patterns of E. faecalis in Retail Meats 166 From the 50 retail meat samples analyzed, a total of 10 Enterococcus faecalis isolates were 167 recovered, corresponding to an overall prevalence of 20%. The isolates were unevenly distributed 168 among the meat types: chicken accounted for the highest number of isolates (n=6, 60%), followed 169 by beef (n=2, 20%) and turkey (n=2, 20%). Antimicrobial susceptibility testing revealed consistent 170 resistance profiles across the isolates. All strains exhibited complete resistance (100%) to 171 clindamycin, erythromycin, and tetracycline, antibiotics commonly used in veterinary and clinical 172 settings. Moderate resistance was observed against streptomycin (80%), and tobramycin (80%). In 173 contrast, resistance to chloramphenicol was comparatively lower (20%), and no resistance to 174 vancomycin was detected among any of the isolates. The distribution and co-occurrence of resistance 175 phenotypes are illustrated in Figure 1 using an Upset plot. 176 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 9 177 Figure 1. The Upset plot illustrates the distribution of antimicrobial resistance patterns across the 178 isolates. The top bar chart quantifies unique combinations of antibiotic resistance among the isolates, 179 while the horizontal bar chart on the left shows the number of isolates resistant to individual 180 antibiotics. Co -resistance to clindamycin, erythromycin, tetracycline, and aminoglycosides 181 (streptomycin and tobramycin) was common. Vancomycin was excluded from the visualization due 182 to the absence of resistance. The arrangement highlights both dominant and infrequent co-resistance 183 profiles across the dataset. 184 3.2. ARG profiles and mobile genetic elements 185 Genomic analysis identified a diverse repertoire of antimicrobial resistance genes (ARGs) among 186 the E. faecalis isolates, many of which confer resistance to critically important antimicrobials. The 187 most frequently detected ARGs included aac(6’)-aph(2’’) and ant(6)-Ia (aminoglycoside 188 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 10 resistance), lsa(A) (lincosamide resistance), erm(B) (macrolide resistance), 189 and tet(M) and tet(L) (tetracycline resistance). Notably, dfrG (trimethoprim resistance) and catA8 190 (chloramphenicol resistance) were detected exclusively in two isolates recovered from retail 191 chicken, suggesting lineage-specific acquisition or exposure to unique selective pressures. 192 Across meat sources, the core resistome was largely conserved; however, isolates NigeriaC1 193 and NigeriaC11 (from chicken) harbored a broader range of ARGs, reflecting potential differential 194 antibiotic exposure in poultry production systems (Table 1). Analysis of mobile genetic elements 195 (MGEs) revealed lineage - and source-specific patterns. The plasmid replicon rep9cwas the most 196 prevalent, detected in all isolates regardless of meat source and consistently associated with sequence 197 type ST477. Conversely, repUS43 was uniquely found in ST16 isolates from retail chicken, 198 indicating a possible plasmid-lineage specificity. 199 Insertion sequences (ISs) played a prominent role in the resistome architecture. The IS6 200 family was frequently identified in ST477 isolates from all meat types. A unique combination of IS 201 elements; IS3, IS6, IS110, IS256, and IS1380 was observed only in ST16 isolates, co -localized 202 with dfrG and catA8. Further analysis showed identified a resistance gene cassette 203 comprising aac(6’)-aph(2’’), ant(6)-Ia, and tet(M) embedded within a genomic region enriched with 204 mobile genetic elements, including IS1380 and IS6 family transposases, as well as plasmid 205 recombinase family proteins (Figure 2). 206 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 11 207 Figure 2. Circular genomic annotation of the genetic cassette carrying resistance genes 208 in Enterococcus faecalis isolate NigeriaC2. This figure presents a visualization of the genomic 209 region containing resistance determinants aac(6’)-aph(2’’), ant(6)-Ia, and tet(M) in isolate 210 NigeriaC2 (Accession number: JAQOOR010000014). The annotation highlights the relative 211 positioning and orientation of these resistance genes alongside associated mobile genetic elements, 212 including IS1380 transposase, IS6 family transposase, and plasmid recombinase family proteins. 213 Genes are color -coded, with green representing resistance genes and regulatory elements, while 214 yellow indicates protein-coding sequences (CDS). Arrows denote the transcriptional orientation of 215 individual genes, providing insights into their synteny and potential functional interactions within 216 the genomic context. 217 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 12 Table 1: Genomic and Phenotypic Characteristics of E. faecalis Isolates 218 219 220 Isolate ID Isolation Source Antibiogram Resistance Genes Insertion Sequences Plasmid replicons MLST Virulence Factors NigeriaB142 Beef STP-TOB-TET- ERY-CLI aac(6’)-aph(2’’), ant(6)-Ia, lsa(A), erm(B), tet(M), tet(L) IS6 rep9c ST477 ElrA, SrtA, ace, cCF10, cOB1, cad, camE, ebpA, ebpB, ebpC, efaAfs, fsrB, gelE, hylB, tpx NigeriaB242 Beef STP-TOB-TET- ERY-CLI aac(6’)-aph(2’’), ant(6)-Ia, lsa(A), erm(B), tet(M), tet(L) IS6 rep9c ST477 ElrA, SrtA, ace, cCF10, cOB1, cad, came, ebpA, ebpB, ebpC, efaAfs, fsrB, gelE, hylB, tpx NigeriaC1 Retailed Chicken TET-ERY-CLI-CHL lsa(A), erm(B), catA8, tet(M), dfrG IS3, IS6, IS110, IS256, IS1380 repUS43 ST16 ElrA, SrtA, ace, agg, cCF10, cOB1, cad, came, cylA, cylB, cylL, cylM, ebpA, ebpB, ebpC, efaAfs, hylA, tpx NigeriaC11 Retailed Chicken TET-ERY-CLI-CHL lsa(A), erm(B), catA8, tet(M), dfrG IS3, IS6, IS110, IS256, IS1380 repUS43 ST16 ElrA, SrtA, ace, agg, cCF10, cOB1, cad, came, cylA, cylB, cylL, cylM, ebpA, ebpB, ebpC, efaAfs, hylA, tpx NigeriaC2 Retailed Chicken STP-TOB-TET- ERY-CLI aac(6’)-aph(2’’), ant(6)-Ia, lsa(A), erm(B), tet(M), tet(L) IS6 rep9c ST477 ElrA, SrtA, ace, cCF10, cOB1, cad, camE, ebpA, ebpB, ebpC, efaAfs, fsrB, gelE, hylB, tpx NigeriaC24 Retailed Chicken STP-TOB-TET- ERY-CLI aac(6’)-aph(2’’), ant(6)-Ia, lsa(A), erm(B), tet(M), tet(L) IS6 rep9c ST477 ElrA, SrtA, ace, cCF10, cOB1, cad, came, ebpA, ebpB, ebpC, efaAfs, fsrB, gelE, hylB, tpx NigeriaC242 Retailed Chicken STP-TOB-TET- ERY-CLI aac(6’)-aph(2’’), ant(6)-Ia, lsa(A), erm(B), tet(M), tet(L) IS6 rep9c ST477 ElrA, SrtA, ace, cCF10, cOB1, cad, came, ebpA, ebpB, ebpC, efaAfs, fsrB, gelE, hylB, tpx NigeriaC243 Retailed Chicken STP-TOB-TET- ERY-CLI aac(6’)-aph(2’’), ant(6)-Ia, lsa(A), erm(B), tet(M), tet(L) IS6 rep9c ST477 ElrA, SrtA, ace, cCF10, cOB1, cad, camE, ebpA, ebpB, ebpC, efaAfs, fsrB, gelE, hylB, tpx NigeriaT23 Retailed Turkey STP-TOB-TET- ERY-CLI aac(6’)-aph(2’’), ant(6)-Ia, lsa(A), erm(B), tet(M), tet(L) IS6 rep9c ST477 ElrA, SrtA, ace, cCF10, cOB1, cad, camE, ebpA, ebpB, ebpC, efaAfs, fsrB, gelE, hylB, tpx NigeriaT44 Retailed Turkey STP-TOB-TET- ERY-CLI aac(6’)-aph(2’’), ant(6)-Ia, lsa(A), erm(B), tet(M), tet(L) IS6 rep9c ST477 ElrA, SrtA, ace, cCF10, cOB1, cad, camE, ebpA, ebpB, ebpC, efaAfs, fsrB, gelE, hylB, tpx .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 13 3.3. Virulence factors, sequence types and phylogenetic insights 221 Genome analysis revealed that E. faecalis isolates harbored an extensive repertoire of virulence 222 factors associated with colonization, tissue invasion, and immune evasion. Conserved genes across 223 all isolates included ace (collagen adhesion), efaAfs (endocarditis 224 antigen), ElrA (adhesion), SrtA (anchoring protein), and the biofilm -associated pili 225 genes ebpA, ebpB, and ebpC. Additionally, all isolates carried cCF10 and cOB1, encoding 226 aggregation substances that enhance horizontal gene transfer and biofilm development. 227 The distribution of virulence genes displayed clear sequence type (ST) -specific patterns. 228 ST477, the most prevalent lineage, was recovered from beef, chicken, and turkey, and consistently 229 carried fsrB, gelE (gelatinase), and hylB (hyaluronidase), key factors implicated in biofilm 230 formation, extracellular matrix degradation, and immune modulation. In contrast, ST16 isolates 231 (NigeriaC1 and NigeriaC11), found exclusively in chicken, exhibited a distinct virulence profile. 232 These isolates harbored agg, cylA, cylB, cylL, and cylM genes encoding the cytolysin toxin complex, 233 which contributes to tissue damage and enhanced pathogenicity. ST16 also possessed hylA, an 234 alternative hyaluronidase variant, and camE (calcium-binding protein). 235 Plasmid replicons showed strong lineage associations. rep9c was universally detected in 236 ST477 isolates and co -occurred with the fsrB–gelE–hylB virulence gene set. 237 Conversely, repUS43 was exclusive to ST16 and linked to the presence of cytolysin genes and hylA. 238 Insertion sequences (ISs) were also associated with virulence profiles. ST477 isolates commonly 239 carried IS6, while ST16 harbored a broader array of MGEs, including IS3, IS110, IS256, and IS1380, 240 possibly facilitating mobilization of cytolysin and adhesion genes. Phylogenetic reconstruction using 241 SNP-based analysis revealed two well -defined clades corresponding to ST16 and ST477 lineages 242 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 14 (Figure 3). ST16 isolates clustered together and were uniquely associated with poultry, distinct 243 ARGs, and virulence factors. ST477 formed a separate, more diverse clade encompassing isolates 244 from all meat types and displaying a conserved resistome-virulome profile. 245 246 Figure 3: SNP-based maximum likelihood tree showing phylogenetic relationships among E. 247 faecium isolates recovered from retail meat. The tree illustrates two main clades, each linked to 248 specific sequence types (STs) and genomic characteristics. Annotations highlight key genomic 249 features, including STs, isolation sources, resistance genes, plasmid replicons, and insertion 250 sequences. Isolates are visually distinguished by color -coded boxes, indicating their distribution 251 across different meat sources. 252 3.4. Comparative phylogenomic analysis and metadata insights of E. faecalis isolates 253 from Africa 254 To contextualize the Nigerian E. faecalis isolates within broader regional dynamics, we conducted 255 a comparative phylogenomic analysis of 149 publicly available African genomes collected between 256 2013 and 2023. South Africa contributed the highest number of isolates (n=60), followed by 257 Tanzania (n=33) and Ghana (n=20), reflecting the uneven distribution of genomic surveillance 258 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 15 efforts across the continent. Nigeria, the focus of this study, accounted for 10 isolates, while 259 Cameroon, Zimbabwe, Tunisia, and Egypt contributed fewer (Figures 4 and 5a). 260 Temporally, most isolates were obtained between 2017 and 2021, with peaks in 2017 (n=42) 261 and 2021 (n=57). Earlier years (2013–2016) were underrepresented, limiting historical comparisons 262 but indicating a growing interest in enterococcal genomics in recent years (Figure 5b). Sequence 263 type analysis identified 47 distinct STs across the dataset, with both shared and country -specific 264 lineages. ST16 was the most widely distributed, detected in South Africa, Tanzania, Cameroon, and 265 Ghana (Figure 5c). In contrast, ST477 found exclusively in the current Nigerian isolates appeared 266 geographically restricted. Other notable country -specific lineages included ST21 (Tunisia and 267 Egypt) and ST646 (South Africa). A network analysis of the top 10 STs and their country 268 associations (Figure 5d) further illustrated these patterns. South Africa and Tanzania exhibited the 269 highest ST diversity, with multiple connections to ST6, ST16, and ST646 (Figure 5d). 270 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 16 271 Figure 4. Maximum likelihood phylogenetic tree of Enterococcus faecalis isolates from African 272 countries (2013 –2023). A core -genome phylogenetic tree was constructed from 149 E. 273 faecalis genomes retrieved from BV -BRC and annotated using iTOL. The tree is rooted using E. 274 faecalis ATCC BAA-2128 (Accession: NAQY00000000.1) as the reference genome. The outer ring 275 represents the sequence type (ST), the middle ring denotes the year of isolation, and the inner ring 276 displays the country of origin . The phylogeny illustrates both temporal and geographic diversity 277 of E. faecalis across the African continent. 278 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 17 279 Figure 5. (a) Geographic distribution of E. faecalis isolates across Africa. A bar chart illustrating 280 the number of E. faecalis isolates obtained from different African countries. (b) Temporal trends 281 in E. faecalis isolations across Africa. A bar chart depicting the number of E. faecalis isolates 282 collected per year from 2013 to 2023. (c) Distribution of major sequence types ( STs) among 283 African E. faecalis Isolates. A stacked bar chart showing the distribution of the most prevalent STs 284 across different African countries. (d) A network graph visualizing relationships between African 285 countries and their associated E. faecalis STs. Nodes represent countries and STs, with edges 286 indicating connections based on isolate presence. 287 288 4. Discussion 289 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 18 The presence of E. faecalis in retail meats highlights the potential role of foodborne transmission in 290 the dissemination of AMR (Conceição et al, 2023). Consistent with previous reports, chicken meat 291 yielded the highest recovery rate of E. faecalis , reinforcing its role as a major reservoir for 292 enterococci in food systems (Hayes et al., 2003; Aslam et al., 2012). Although not a classical 293 foodborne pathogen, the species’ capacity to horizontally transfer resistance genes within the human 294 gut microbiota elevates its public health significance (Krawczyk et al., 2021). These findings support 295 calls for strengthened hygiene protocols and antimicrobial stewardship in poultry production, a 296 sector characterized by intensive antimicrobial use (Conceição et al., 2023). 297 All isolates exhibited resistance to clindamycin, erythromycin, and tetracycline mirroring 298 global resistance trends and suggesting sustained selection pressure from widespread use of these 299 agents in animal husbandry (Arias & Murray, 2012; Lebreton et al., 2014; Landers et al., 2012). The 300 absence of vancomycin resistance is encouraging; however, the species’ genomic flexibility raises 301 concerns about future acquisition through horizontal gene transfer (van Hal et al., 2016). The 302 detection of catA8 among chicken isolates, despite the relatively low phenotypic resistance to 303 chloramphenicol, signals the emergence of latent resistance and highlights the risk of resurgence 304 (Bae et al., 2021). These findings reinforce the critical need for integrated AMR surveillance across 305 food and clinical sectors, especially in high-burden regions (WHO, 2019). 306 Genomic analysis revealed a consistent resistome dominated by aac(6')-aph(2''), ant(6)-307 Ia, lsa(A), erm(B), tet(M), and tet(L) conferring resistance to aminoglycosides, macrolides, 308 lincosamides, and tetracyclines. These genes, conserved across meat types and lineages, mirror 309 globally recognized resistance profiles and likely reflect co -selection pressures exerted by 310 agricultural antibiotic use (Hegstad et al., 2010). Of particular note, dfrG and catA8 were confined 311 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 19 to ST16 isolates from chicken, suggesting lineage-specific resistance acquisition possibly driven by 312 poultry-associated selective environments (Kristich et al., 2014). These resistance determinants were 313 not randomly distributed but instead embedded within complex mobile genetic architectures. A 314 multidrug resistance gene cassette co-localizing aac(6’)-aph(2’'), ant(6)-Ia, and tet(M) was detected 315 within a genomic region enriched with IS6 and IS1380 transposases and plasmid recombinase genes, 316 forming a transferable module capable of en bloc dissemination of resistance traits. Such modularity 317 enhances the potential for inter -species gene flow, especially within gut microbiota of exposed 318 consumers (Hegstad et al., 2010). 319 Plasmid replicons further delineated lineage -specific resistance pathways. The 320 widespread rep9c replicon, present in all ST477 isolates, was consistently co-located with the fsrB–321 gelE–hylB virulence cluster, suggesting clonal expansion and vertical maintenance of resistance –322 virulence hybrids (Willems et al., 2012). In contrast, repUS43 was exclusive to ST16 isolates, co -323 occurring with catA8 and dfrG, reinforcing its role in ST -specific resistance dissemination (van 324 Schaik et al., 2010). The structural linkage between plasmid types, insertion sequences, and ARGs 325 underscores the dynamic interplay of vertical inheritance and horizontal transfer in shaping the 326 resistome of E. faecalis. Insertion sequences (IS3, IS6, IS110, IS256, and IS1380) were found at 327 multiple resistance loci and appear to drive genomic fluidity by facilitating recombination and gene 328 mobilization. Their lineage -specific distribution highlights ongoing adaptive evolution under 329 antimicrobial pressure. These findings illustrate a highly structured yet flexible resistance landscape 330 in foodborne E. faecalis , propelled by mobile elements and reinforced by selective agricultural 331 practices. 332 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 20 The widespread detection of virulence genes among E. faecalis isolates from retail meats 333 reveals a pathogenic potential far beyond commensal behavior. Conserved adhesion factors 334 including ace, efaAfs, ElrA, SrtA, and the ebp operon (ebpA, ebpB, ebpC) were uniformly present, 335 underscoring a baseline capacity for epithelial colonization, biofilm formation, and immune 336 modulation (Nallapareddy et al., 2006; Șchiopu et al., 2023). The concurrent presence of conjugative 337 factors ( cCF10, cOB1) suggests an additional role in promoting gene exchange within host or 338 environmental niches, facilitating co-selection and persistence of resistance–virulence traits. 339 Lineage-specific virulence signatures were particularly striking. ST477 isolates recovered 340 from beef, chicken, and turkey harbored the fsrB–gelE–hylB cluster, a constellation of genes 341 associated with quorum sensing, extracellular matrix degradation, and immune evasion, previously 342 linked to device- and bloodstream-associated infections (Van Tyne et al., 2013; Johnson et al., 2024). 343 This combination of biofilm -promoting and immunomodulatory functions positions ST477 as a 344 high-risk foodborne lineage with potential for clinical crossover. In contrast, ST16 isolates 345 exclusively from chicken exhibited an enhanced virulence profile characterized by cytolysin operon 346 genes ( cylA, cylB, cylL, cylM) and hylA, features associated with epithelial disruption and pro -347 inflammatory host responses (Zheng et al., 2017). Notably, these isolates also carried agg, a plasmid-348 borne aggregation substance linked to increased virulence and conjugation efficiency. The co -349 occurrence of repUS43 with this virulence suite suggests a plasmid-mediated mechanism facilitating 350 the emergence of hypervirulent clones. 351 The convergence of resistance and virulence within specific plasmid backgrounds and 352 sequence types especially ST16 and ST477 raises substantial public health concerns. Mobile 353 elements, including IS3, IS110, IS256, and IS1380, were frequently associated with virulence loci, 354 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 21 further implicating horizontal gene transfer in the amplification of pathogenic potential. These 355 findings parallel global reports of virulence -enriched E. faecalis STs and affirm their relevance in 356 zoonotic transmission and foodborne disease (McBride et al., 2007; Fiore et al., 2019). In this 357 context, foodborne E. faecalis strains function not only as reservoirs of resistance but also as 358 potential vectors for invasive disease, particularly in immunocompromised hosts. Their emergence 359 in the food chain paired with genomic signatures linked to hospital-adapted strains underscores the 360 urgency of a One Health surveillance framework integrating food safety, environmental monitoring, 361 and clinical microbiology. 362 Comparative phylogenomics of E. faecalis isolates across Africa uncovered a geographically 363 structured but genetically diverse population. South Africa contributed the highest number of 364 isolates an observation likely shaped by differences in surveillance capacity, sequencing 365 infrastructure, and public health prioritization. The scarcity of isolates from earlier years (2013 –366 2016) and the sharp rise in submissions post-2017, interrupted briefly by the COVID-19 pandemic, 367 reflect both historical data gaps and the growing momentum of genomics -based AMR monitoring 368 on the continent (Baker et al., 2023; Kajumbula et al., 2024; Tegally et al., 2022). 369 Temporal and geographic analyses revealed that sequence type ST16 was the most broadly 370 distributed, identified in multiple countries and across diverse ecological contexts, suggesting a well-371 adapted and potentially mobile lineage (Zaheer et al., 2020; Monteiro Marques et al., 2023). Its 372 widespread detection aligns with prior observations of ST16’s capacity for environmental 373 persistence and inter -host transmission. In contrast, ST477 was restricted to Nigeria, while ST21 374 was confined to Tunisia and Egypt, implying localized evolutionary trajectories shaped by selective 375 pressures such as antimicrobial usage patterns, ecological boundaries, and food production systems 376 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 22 (Baquero et al., 2021; Bottery et al., 2021). Network-based analysis reinforced these findings by 377 revealing distinct ST -country associations, with South Africa and Tanzania exhibiting the highest 378 sequence type diversity. These clusters suggest transboundary transmission possibly facilitated by 379 trade, food import/export routes, or shared agricultural practices (Tatem et al., 2006; Salem et al., 380 2023). Conversely, the geographic confinement of ST477 to Nigeria raises concerns about the 381 emergence of a potentially endemic, foodborne high-risk clone with a stable resistome and virulome 382 signature. 383 These patterns support a dual model of E. faecalis evolution in Africa: one shaped by clonal 384 expansion of regionally successful lineages, and another driven by horizontal gene transfer across 385 environmental and host reservoirs. This genomic duality complicates control efforts and underscores 386 the need for longitudinal, cross-sectoral surveillance. The convergence of clinically relevant traits in 387 isolates recovered from food reinforces the risk of zoonotic spill -over, particularly in settings with 388 limited food safety regulation and AMR control. In light of these findings, a coordinated genomic 389 surveillance strategy that integrates human, animal, and environmental health guided by the One 390 Health framework is essential for tracking the emergence, evolution, and dissemination of high -391 risk E. faecalis clones in Africa. This study, while limited by its modest sample size and geographic 392 scope, contributes a critical dataset to the continental AMR landscape and provides a foundation for 393 future longitudinal studies on E. faecalis as a foodborne pathogen of increasing public health 394 concern. 395 396

397 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 23 This study provides critical genomic insights into the antimicrobial resistance and virulence 398 landscape of Enterococcus faecalis isolates recovered from retail meats in Nigeria, underscoring the 399 role of food systems as reservoirs and potential amplifiers of clinically relevant pathogens. The 400 identification of high -risk lineages such as ST477 and ST16 harboring multidrug resistance 401 determinants, virulence genes, and mobile genetic elements highlights the convergence of resistance 402 and pathogenicity within the food chain. The presence of plasmid -encoded resistance –virulence 403 modules and the widespread occurrence of insertion sequences suggest an active mobilome 404 facilitating gene exchange and adaptation across ecological boundaries. Comparative phylogenomic 405 analysis across Africa revealed geographically structured transmission dynamics, marked by the 406 emergence of regionally dominant clones and country -specific evolutionary trajectories. These 407 findings reflect broader challenges in AMR control, particularly in low - and middle -income 408 countries where food safety infrastructure and genomic surveillance remain limited. 409 To mitigate the growing threat of foodborne antimicrobial resistance, we advocate for 410 enhanced genomic monitoring of foodborne pathogens, stringent regulation of antimicrobial use in 411 agriculture, and integration of One Health strategies across the human –animal–environment 412 interface. The insights presented here serve as a foundation for future regional surveillance initiatives 413 and emphasize the need for proactive, genomics-informed interventions to safeguard public health. 414 415 Declarations 416 Funding: This research received no specific grant from any funding agency in the public, 417 commercial, or not-for-profit sectors. 418 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 16, 2025. ; https://doi.org/10.1101/2025.04.15.648955doi: bioRxiv preprint 24 Conflicts of Interest: The authors declare no competing interests. 419 Ethics Approval: Institutional approval for the study protocol and sampling approach was granted 420 by the Department of Microbiology, Adekunle Ajasin University (Approval Reference: 421 DM:AAU/2021). All meat samples were obtained from open retail markets in Akungba -Akoko, 422 Nigeria, and were purchased anonymously as part of routine food supply, without involving live 423 animals or interventions. The objectives of the study were clearly explained to meat vendors to 424 ensure transparency and voluntary participation in the sampling process. 425 Clinical Trial: Not applicable 426 427 Data Availability 428 The s equence data that support the findings of this study has been deposited in GenBank and 429 assigned accession numbers under BioProject PRJNA928459. All other data supporting this study 430 findings are available within the manuscript and supplementary material. 431 432

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