Abstract
The emergence of antimicrobial-resistant, livestock-associated Enterococcus faecalis represents a public health concern. Here, we report the isolation, molecular detection of virulence and antimicrobial resistance determinants, in addition to the phylogenetic analyses of 20 Enterococcus species using whole genome sequencing analysis of 15 Enterococcus faecalis strains including six strains of three novel sequence types, three Enterococcus faecium and two Enterococcus durans. All strains were isolated from food chain animals in South Africa. Enterococcus strains were isolated on bile aesculin azide agar, followed by identification using MALDI-TOF MS analysis. Antibiotic susceptibility testing was performed using the Kirby–Bauer disk diffusion method. The genomic DNA of the isolates was extracted and sequencing was performed using the Illumina MiSeq platform. Sequence reads were trimmed and de novo assembled. The assembled contigs were analyzed for antimicrobial resistance genes and chromosomal mutations, extra-chromosomal plasmids, and multi-locus sequence type (MLST). Multidrug antimicrobial resistance genes conferring resistance to aminoglycosides (ant(6)-Ia, aph(3′)-IIIa, sat4, and spw), lincosamides (lnu(B), lsa(A), and lsa(E)), macrolides (erm(B)), trimethoprim (dfrG) and tetracyclines (tet(L) and tet(M)) were identified. Plasmid replicons were detected in seven E. faecalis and three E. faecium isolates. The sequence type (ST) of each isolate was determined using the Enterococcus PubMLST database. Ten STs were identified in the collection, three of which (ST1240, ST1241, and ST1242) have not been previously reported and are described in the present study for the first time. To compare the sequenced strains to other previously sequenced E. faecalis strains, assembled sequences of E. faecalis from livestock were downloaded from the PubMLST database. Core genome-based phylogenetic analysis was performed using ParSNP. The detection of multiple drug-resistance in Enterococcus including E. faecalis and E. faecium highlights the significance of genomic surveillance to monitor the spread of antimicrobial resistance in food chain animals. In addition, the genome sequences of Enterococcus strains reported in the present study will serve as a reference point for future molecular epidemiological studies of livestock-associated and antibiotic-resistant E. faecalis in Africa. In addition, this study enables the in-depth analysis of E. faecalis genomic structure, as well as provides valuable information on the phenotypic and genotypic antimicrobial resistance, and the pathogenesis of livestock-associated E. faecalis and E. faecium.
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Introduction
Enterococcus species are Gram-positive, non-spore forming cocci which naturaly occur in healthy humans and animals. Enterococcus remain one of the most important zoonotic enteric pathogens that are responsible for morbidity and mortality in humans and livestock worldwide. They also serve as indicator bacteria in the surveillance for antimicrobial resistance in food chain animals1. Enterococcus spp. are important commensal bacteria of the normal gut flora of humans and animals2, however, they have recently emerged as leading causes of zoonotic and nosocomial infections3,4,5,6. Vancomycin-resistant enterococci (VRE) are among the critical-priority resistant bacteria in the “global priority list of antibiotic-resistant bacteria for the research and development of new antibiotics”1,7. Enterococcus faecium and Enterococcus faecalis are the most common nosocomial opportunistic pathogens responsible for approximately 10–15% and 80–90% of infections, respectively8. E. faecalis is an important zoonotic enteric pathogen that may cause serious infections in humans and animals, including gastrointestinal tract infections and extra-intestinal infections including urinary tract infections, meningitis, bacteremia, peritonitis, endocarditis, and periodontitis9,10,11. E. faecalis infections primarily spread from person to person through poor hygiene. The bacteria can contaminate foods through inadequate hygiene and food manipulation. Foodstuffs, particularly ready-to-eat foods and foods of animal origins, may be the sources of bacterial transmission supporting their zoonotic potential12,13,14,15.
Studies investigating food animals as a zoonotic reservoir for common human bacterial pathogens have confirmed the zoonotic origin of human bacterial pathogens such as Salmonella spp.16, Campylobacter spp.17 and pathogenic Escherichia coli18,19. These studies also highlight the possible zoonotic origin for other major human bacterial infections involving Staphylococcus aureus, E. faecalis, and E. faecium9. Food animals meat production settings and livestock production systems are important sources of antimicrobial resistance genes selected by the use of antibiotics, carried from poultry and livestock to humans by zoonotic bacterial clones. These resistance factors can be transferred to other bacteria present in the gut microbiota of the consumer through horizontal gene transfer1,7,9.
Enterococcus is responsible for a significant proportion of multidrug-resistant intestinal and extra-intestinal infections in humans and food animals. Virulent and multidrug-resistant strains of Enterococcus have been detected and isolated from domesticated animals including bovine, poultry, pigs, and pets as well as food products from different regions12. The detection and worldwide spread of resistance genes including the colistin mcr-1 genes in livestock and humans have spurred efforts of genomic surveillance for antimicrobial resistance in food animals20.
Genome sequence analysis of Enterococcus is essential because of the potential impact of these bacteria on human and animal health. Whole genome analysis is an ideal approach to detect genomic variations, and provides valuable information enabling us to understand virulence, pathogenesis, antimicrobial resistance, host specificity and phylogenetic relationships21,22,23. However, currently there is very little data concerning the genome sequence of Enterococcus spp. isolated from livestock in Africa are available. In addition, data on genetic characterization of Enterococcus spp from the livestock production sector in South Africa are limited, and whole genome sequencing is not routinely employed to evaluate virulence and AMR associated genes24. Here, we report the use of whole genome sequencing in analysis of fifteen E. faecalis, three E. faecium, and two E. durans strains isolated from different livestock animals (cattle, chicken, goat, horse and pigs) in KwaZulu-Natal and Eastern Cape Provinces in South Africa.
Materials and methods
Ethical statement
All procedures and methods were carried out in accordance with relevant guidelines and regulations and were reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). No anesthesia or euthanasia methods were employed or involved in the present study. The study was approved by the Animal Research Ethics Committee of the University of Kwa-Zulu Natal (Reference numbers AREC/051/017 M, AREC 071/017 and AREC014/018). The field sampling protocols, samples collected from animals, and the research were conducted in full compliance with Section 20 of the Animal Diseases Act of 1984 (Act No 35 of 1984) and were approved by the South African Department of Agriculture, Forestry and Fisheries (DAFF) (Section 20 approval reference number 12/11/1/5).
Sample collection
One hundred and seventy eight samples were collected from different animals housed in livestock farms in South Africa. The livestock production farms were small-scale commercial farms located in Camperdown, Amandawe, and Scottburgh in KwaZulu-Natal (KZN) and Flagstaff in Eastern Cape (EC) Provinces as shown in Fig. 1. Samples were collected from chickens (n = 60), cows (n = 25), ducks (n = 3), goats (n = 29), horses (n = 2), sheep (n = 17), and pigs (n = 17). Additional environmental samples were obtained from water (n = 20), feedlot (n = 1), and soil (n = 4). Samples were collected from animals by rectal, oral and faecal swabbing. Samples were collected using sterile cotton swabs. After sampling, the swabs were placed in 10 ml of 0.1% (w/v) peptone water (Merck, South Africa). All samples were immediately transported to the laboratory maintaining a cold chain temperature during transport of the samples.
Geographic map showing the areas where the samples in the present study were collected from livestock farms in KwaZulu-Natal and Eastern Cape Provinces in South Africa. The map was generated using the software ArcGIS Pro (v3.1, Environmental Systems Research Institute (ESRI), Redlands, CA, USA).
Isolation and identification of Enterococcus species
A total of 1 ml peptone water sample was inoculated into 10 ml of brain heart infusion broth (Merck, South Africa) for enrichment and incubated for a duration of 18–24 h at 37 °C. The broth cultures were streaked onto bile aesculin azide agar (Merck, South Africa) and further incubated for a period of 18–24 h at 37 °C. Presumptive Enterococcus isolates with brownish-black to black dew drop phenotype colonies were purified and isolated using trypticase soy agar (Merck, South Africa). The isolates were cultured on sheep blood agar for 18–24 h at 37 °C in the presence of 5% CO2. Pure colonies were suspended in trypticase soy broth to give an inoculum density equivalent to that of a 0.5 McFarland standard. An aliquot of overnight cultures of Enterococcus isolates in brain heart infusion broth was used for matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis for species identification as previously described25.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed against 12 different antibiotics using Kirby-Bauer method and results were interpreted according to the standard breakpoints for inhibition zone provided by Clinical and Laboratory Standard Institute as previously described26.
Briefly, the bacterial isolates were grown overnight in Mueller Hinton broth (Fisher Scientific Inc., TX, USA) at 37˚C. The isolates from the overnight culture were then diluted to an optical density (OD600) of 0.05 (2.5 × 107 colony forming unit per ml) in Mueller–Hinton broth. Approximately, a volume of 100 µl of the diluted bacteria was spread evenly on Mueller–Hinton agar plates. Antimicrobial disks were purchased from Hardy Diagnostics (Santa Maria, CA, USA) and each isolate was tested against ampicillin (10 µg, AMP 10), chloramphenicol (30 µg, C 30), azithromycin (15 µg, AZM 15), gentamicin (10 µg, GM 10), tetracycline (30 µg, TE 30), ciprofloxacin (5 µg, CIP 5), tigecycline (15 µg, TGC 15), vancomycin (30 µg, V 30), oxacillin (1 µg, OX 1), trimethoprim/sulfamethoxazole (25 µg, SXT 25), imipenem-relebactam (IMR 10/25), and levofloxacin (5 µg, LVX 5). The bacterial growth inhibition zone diameter (in mm) was measured after incubation of the bacterial isolates with the antibiotic discs at 37˚C for 12 h as previously reported27.
Whole genome sequencing
Whole genome sequencing of the bacterial isolates was performed as previously reported25,27. Briefly, an aliquot of the bacterial culture in tryptic soy broth was diluted with 0.85% sterile saline solution to the desired inoculum density of 1 × 106 CFU per ml using Thermo Scientific™ Sensititre™ Nephelometer and the chilled culture tubes were submitted for DNA extraction and sequencing. DNA extraction was performed using the Qiagen DNeasy Blood & Tissue kit (Lucigen, WI, USA) according to the manufacturer’s protocol as was previously described20. Sequencing libraries were prepared using the Nextera XT library preparation kit (Illumina Inc., CA, USA). Sequencing was performed on the Illumina MiSeq platform (Illumina Inc., CA, USA) using the v2 reagent kit (2 × 250 nt paired-end chemistry), which yielded 250-bp paired-end reads.
Bioinformatics analyses
Quality control, assembly of the raw reads and virulence gene determination were performed using the ASA3P pipeline28. The raw sequence data and the assembled contigs were uploaded to the NCBI database under the accession numbers listed in Supplementary Table 1. Analysis of antibiotic resistance genes (ResFinder 2.2) and multilocus sequence type (MLST 1.6) were performed using the CGE pipeline29. The PlasmidFinder tool available from the Center for Genomic Epidemiology (https://cge.cbs.dtu.dk/services) was used to identify plasmid incompatibility groups. Type (Strain) Genome Server (TYGS, https://tygs.dsmz.de/, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany) pipeline was used to verify the species determined by MALDI-TOF30.
Core-genome phylogeny was determined using ParSNP31. The phylogenetic trees were annotated using iTOL v6.732,33. Single nucleotide polymorphisms (SNPs) were determined using the output generated by ParSNP aligned in MEGA X34.
Results and discussion
A total of 178 samples were collected from livestock production systems and were screened for Enterococcus using bile esculin azide agar selective media. Based on the phenotypic colony morphology of brown and a black halo shape, 24 isolates (13.5%) were presumptively identified as Enterococcus spp. The isolates were identified using MALDI-TOF analysis to be E. faecalis (n = 18), E. faecium (n = 3) and E. durans (n = 3). The majority of Enterococcus strains (n = 23) were isolated from samples collected from four different animal species including goats (n = 5), chicken (n = 8), pigs (n = 4), cow (n = 5), and one E. faecalis strain was isolated from an environment water sample collected from a cow farm (Table 1). The majority of Enterococcus strains were isolated from KwaZulu-Natal Province (n = 19, 79%) and 16 isolates were obtained from fecal material while seven isolates were obtained from oral swabs.
Antimicrobial susceptibility results showed that isolates were resistant to oxacillin (100%), tetracycline (45%, n = 9), sulfamethoxazole/trimethoprim (35%, n = 7), ampicillin (10%, n = 2), ciprofloxacin, vancomycin, and gentamicin (5%, n = 1). Isolates were intermediate resistant to ciprofloxacin (65%) and vancomycin (20%). Isolates were susceptible to chloramphenicol (100%) and levofloxacin (90% susceptible) as shown in Fig. 2. Multidrug resistance (resistance to three or more different classes of antibiotics) was observed in nine E. faecalis isolates and one E. faecium isolate. Among the tested isolates, 30% (n = 6) were resistant to two antibiotics and 50% (n = 10) isolates were resistant to more than two antibiotics, as shown in Table 2.
Antibiotic susceptibility patterns of Enterococcus spp. isolates obtained from livestock in the present study.
Enterococcus isolates in the present study displayed high level of resistance to tetracycline (n = 45%) (confirmed by both Kirby-Bauer method and genome sequencing analysis) and macrolides (confirmed by the detection of genes encoding macrolides resistance). The results of the present study were similar to a previous study conducted in KwaZulu-Natal Province, South Africa which reported high level of resistance to tetracycline and erythromycin (macrolide) in Enterococcus isolated form poultry samples35.
Similar results may be associated with the extensive use of antibiotics in animal production systems, similarity in the geographical region and antibiotic resistance patterns. As a result of the emergence of antibiotic resistance patterns linked to the excessive use of antibiotics in food animals, the European Union implemented a ban on the use of most of these antibiotics as animal feed additives and growth promoters36.
Epidemiological evidence suggests an association between the occurrence of antibiotic resistance in bacterial infections in humans and animal production systems. In many cases such as in Enterococcus, for example, high level of resistance to vancomycin in humans has been linked with the cross resistance from avoparcin, a vancomycin analogue used as feed additive in poultry, pig, and cattle farms37. It was reported that Enterococcus can harbor vancomycin resistance genes (vanA, vanB ,vanC) even after years of use of avoparcin as feed additive38.
In the present study, 20 out of 24 Enterococcus isolates were sequenced. Three Enterococcus faecalis isolates and one Enterococcus durans isolate were only identified using MALDI-TOF analysis. Using the whole genome sequencing and TYGS, the 20 Enterococcus isolates were verified to be of three different Enterococcus species: Enterococcus faecalis (n = 15), Enterococcus faecium (n = 3) and Enterococcus durans (n = 2). Phylogenetic analysis was performed and phylogenetic trees of the E. faecalis isolates from the present study were constructed using the neighbor-joining algorithm and 1000 bootstrap replicated genomes after alignment of the genomes (Fig. 3) using Roary v3.11.231.
Phylogeny of E. faecalis isolates in the present study showing the distribution of MLST profiles and the detected antimicrobial resistance genes.
The multilocus sequence type (ST) of each Enterococcus faecalis isolate was determined using the Enterococcus faecalis PubMLST database (https://pubmlst.org/organisms/enterococcus-faecalis). Ten STs were identified in this collection (Table 2), three of which have not previously been described (ST1240, ST1241, and ST1242). The MLST alleles and distribution of STs of each Enterococcus faecalis isolate are listed in Table 2.
The ST of Enterococcus faecium isolates (n = 3) was determined using the Enterococcus faecium scheme in PubMLST (https://pubmlst.org/organisms/enterococcus-faecium). Two different STs were detected, ST195 and ST2155 (Table 3). ST2155 represents a new ST type, never described before. For Enterococcus durans, no MLST determination could be performed due to the current lack of an MLST scheme.
The detection of antibiotic resistance genes in livestock-associated pathogens of zoonotic potential is an escalating public health concern. To determine the AMR genes, the contig harbouring each AMR gene was compared to sequences available in the GenBank/NCBI database. Table 2 shows the different AMR genes identified in each E. faecalis genome.
Antimicrobial resistance genes conferring resistance to aminoglycosides (ant(6)-Ia, aph(3′)-IIIa, , and spw), lincosamides (lnu(B), lsa(A), and lsa(E)), streptothricin (sat4), macrolides (erm(B)), trimethoprim (dfrG) and tetracyclines (tet(L) and tet(M)) were identified in E. faecalis using the ResFinder database. No vancomycin resistance (van cluster) genes were detected in the genome sequences reported in the present study.
E. durans strains genomes harbored tet(L) and tet(M)-like genes, while the genomes of E. faecium harbored macrolide, aminoglycoside and tetracycline resistance genes as shown in Table 3. Plasmid replicons rep9a (MEZEF128), rep9b (MEZEF181, MEZEF183), rep9c (MEZEF124), and repUS43 (MEZEF126, MEZEF128, MEZEF159, MEZEF162, MEZEf181, MEZEF183) were detected in E. faecalis. Plasmid replicon types rep2 (MEZEF138) and rep18a (MEZEF3, MEZEF24) were detected in E. faecium.
In isolates harbouring tet(L) and tet(M), the tet(L) gene encodes an efflux pump39 whilst the tet(M) gene encodes a ribosomal protection protein40. Analysis revealed that isolate MEZEF128 harbored a contig (contig # 20) that encoded dfrG, tet(L) and tet(M), isolate.
MEZEF159 harbored a contig (contig # 19) that encoded erm(B), tet(L) and tet(M), isolate MEZEF181 harbored a contig (contig # 61) that encoded ant(6)-Ia, aph(3′)-IIIa, lnu(B), sat4, and spw, isolate MEZEF183 harbored a contig (contig # 64) that encoded aph(3′)-IIIa, erm(B) and sat4, and isolate MEZEF183 harbored a contig (contig # 72) that encoded lnu(B), lsa(E) and spw. To investigate the polymorphisms in parC and gyrA associated with quinolone resistance, the amino acid sequences encoded by the parC and gyrA genes of each isolate were analysed for mutations conferring resistance to quinolones. No known mutations were identified in the E. faecalis genomes.
The results of virulence determinant identification are depicted in Fig. 4. The number of virulence determinants differed by Enterococcus species. E. durans isolates did not harbor any virulence operons. E. faecium isolates harbored between one and three and E. faecalis isolates between eight to twelve virulence determinants.
Depiction of the virulence determinants detected in Enterococcus spp. isolates in the present study. Only complete virulence operons are depicted.
The most common virulence gene types in E. faecalis and E. faecium were genes involved in adhesion. E. faecium harbored only virulence determinants involved in adhesion, namely acm (100%), scm (67%) and sgrA (34%).
E. faecalis genomes harbored between one to four different operons involved in adhesion, including efaA (100%), ebp pilli (87%), ace (73%), AS (13%) and esp (7%).
Three different exoenzyme genes were detected in the 15 sequenced E. faecalis genomes, namely gelatinase (93%), sprE (93%) and hyaluronidase (20%). E. faecalis uses these exoenzymes to degrade polymers from its host to acquire carbon and nitrogen sources for its growth.
Genes involved in capsule and biofilm production were only detected in E. faecalis isolates (47% and 100%, respectively). Two different genes responsible for biofilm production were detected in E. faecalis, bopD (15/15) and fsr (6/15).
The phylogenetic relationship of E. faecalis isolates was determined and a phylogenetic tree was constructed using the neighbor-joining algorithm after alignment of the genomes and visualization/annotation in iTOL (Fig. 3).
The determination of the closest relative of our E. faecium and E. faecalis isolates with those available in PubMLST was performed based on the STs. For E. faecium ST2155 and E. faecalis ST93, ST931, ST1240, ST1241 and ST1242, no genomes were available, thus phylogenetic relatedness could not be determined. For E. faecium ST195 (Supplementary Table 2) and E. faecalis ST4, ST16, ST32, ST245 and ST300 (Supplementary Table 3), contigs from publicly available E. faecium and E. faecalis isolates of the same ST were downloaded from the PubMLST database. Core-genome phylogeny and single nucleotide polymorphisms were determined to detect closely related isolates to those from the current study.
For E. faecium ST195 isolate MEZEF138, the closest relative was the isolate TV42 (Fig. 5), an isolate detected in New Zealand from unknown source. It showed 3618 SNP differences towards MEZEF138 (Supplementary Table 4), thus indicating a very distant genetic relatedness.
Core genome-based phylogenetic tree of MEZEF138 and all publicly available ST195 E. faecium genomes available in NCBI produced by the HarvestSuite package32, visualized using iTOL v. 6.5.833, and adjusted using Inkscape 0.91 (https://inkscape.org/release/inkscape-0.91/).
For E. faecalis ST4 isolate MEZEF110, the closest relative was the isolate 90 (Fig. 6) from a poultry faecal sample in Ghana with a very high number of SNPs (n = 6581, Supplementary Table 5), thus indicating a distant genetic relatedness to MEZEF110.
Core genome-based phylogenetic tree of MEZEF110 and all publicly available ST4 E. faecalis genomes available in NCBI produced by the HarvestSuite package32, visualized using iTOL v. 6.5.833, and adjusted using Inkscape 0.91 (https://inkscape.org/release/inkscape-0.91/).
The next relatives to MEZEF181 (ST16) were the two isolates VAR472 (pig) and H120S2 (unknown source) detected in Belgium and Canada, respectively (Fig. 7). Both isolates harbored a relatively low number of SNPs as compared to MEZEF181 (n = 56, Supplementary Table 6) thus indicating a close genetic relatedness.
Core genome-based phylogenetic tree of MEZEF181 and all publicly available ST16 E. faecalis genomes available in NCBI produced by the HarvestSuite package32, visualized using iTOL v. 6.5.833, and adjusted using Inkscape 0.91 (https://inkscape.org/release/inkscape-0.91/).
The isolate 2UIK3 from the hospital environment in South Africa was the next relative to MEZEF183 (ST32, Fig. 8), albeit the number of SNPs (n = 391, Supplementary Table 7) indicates that these two isolates are not closely related.
Core genome-based phylogenetic tree of MEZEF183 and all publicly available ST32 E. fecalis genomes available in NCBI produced by the HarvestSuite package32, visualized using iTOL v. 6.5.833, and adjusted using Inkscape 0.91 (https://inkscape.org/release/inkscape-0.91/).
MEZEF121 and MEZEF124 (Fig. 9) were only distantly related to the only ST245 isolate designated “75” present in PubMLST (number of SNPs: n = 1450 and 1447, respectively, Supplementary Table 8). It was detected in sheep feces in Ghana.
Core genome-based phylogenetic tree of MEZEF121 and MEZEF124 and all publicly available ST245 E. faecalis genomes available in NCBI produced by the HarvestSuite package32, visualized using iTOL v. 6.5.833, and adjusted using Inkscape 0.91 (https://inkscape.org/release/inkscape-0.91/).
The only ST300 isolate present in PubMLST (designated 89, Fig. 10) detected in poultry feces in Ghana, was only distantly related to MEZEF126 (number of SNPs: n = 981, Supplementary Table 9).
Core genome-based phylogenetic tree of MEZEF126 and all publicly available ST300 E. faecalis genomes available in NCBI produced by the HarvestSuite package32, visualized using iTOL v. 6.5.833, and adjusted using Inkscape 0.91 (https://inkscape.org/release/inkscape-0.91/).
Analysis showed that none of the E. faecium and E. faecalis from the present study belongs to a clonal complex according to the definition of clonal complex (PubMLST).
The application and implementation of whole genome sequencing in genomic surveillance efforts for antimicrobial resistance have been recently reported in clinical and animal settings22,23,41,42. There is a paucity of data on the role of genomic surveillance in understanding antibiotic resistance in livestock, particularly in South Africa. To the authors’ knowledge, the genomes of Enterococcus spp. reported in the present study are the first reported enterococcal genome sequences isolated from livestock in South Africa. Similar studies worldwide reported the detection of antimicrobial resistance in livestock-associated Enterococcus from diverse sources including humans, avian (chicken and turkey), swine, bovine, and ovine, their products, and the environment43. It was recently reported that there is an overall high prevalence of VRE (26.8%) circulating in Africa and analysis showed that a high prevalence of VRE isolated from South African region with the highest prevalence of VRE was in South Africa 74.8% (95% CI; 51–99%; I2 = 99.9%; p < 0.001) followed by, Egypt 37.2% (95% CI; − 17–92%; I2 = 99.7%; p < 0.001) and (2.8%) in Algeria and Nigeria44. Additionally, analysis showed a higher prevalence of VRE was isolated from environmental samples, followed by the animal source as compared to a human source44. In the present study, 25 samples were obtained from environmental non-animal source and analysis revealed that a single E. fecalis isolate (MEZEF166) of novel and previously not reported ST1240 was obtained from a water sample collected from cow’s water bucket. This finding strained the significance of the One Health, which is an important approach to combat antimicrobial resistance which threatens the world and causing escalated public health concerns. Vancomycin resistance determinants (van cluster genes) were not detected in the enterococcal genome sequences reported in the present study. The VRE phenotype of one isolate (MEZEF124) in the present study may be explained by the natural-intrinsic antibiotic resistance which are numerous in this bacterial genus45,46,47. Our results coincide with another study conducted with aquatic environmental samples in South Africa where none of the tested samples were positive for vancomycin resistance genes however the genes encoding aminoglycosides (aph (3)-IIIa), macrolides (ermB) and tetracycline resistance (tetM and tetL) were detected by PCR which coincides with results of the present study48. However, a previous study reported the detection of vancomycin resistance genes (vanB and vanC1/C2/C3) in Enterococcus spp. isolates using PCR in pig dung in the Eastern Cape Province in South Africa43. Similarly, another study also reported the detection of vancomycin resistance genes vanC2/C3, vanC1 and vanB using PCR in fecal samples obtained from dairy cattle in South Africa49. This may be explained due to differences in sample source, time of sample collection, or changes in resistance trends and antimicrobial use practices. The same study also reported the detection of macrolide resistance gene erm(B) which coincides with the current study. Understanding the prevalence of bacteria and their antibiotic resistance patterns in food-producing animals such as dairy cattle, poultry, and pigs is crucial for gaining a comprehensive understanding of the potential health risks associated with farm-to-fork49,50.
To the best of the authors’ knowldege, no study has been conducted to date reporting the whole genome sequencing and sequence type of Enterococcus isolated from livestock in South Africa. In conclusion, the present study highlights the significance of continued genomic surveillance for antimicrobial resistance of livestock associated Enterococcus spp., which continue to pose a serious global threat to human health and food security, primarily a leading cause of foodborne illnesses and invasive infections in humans. The study highlights the importance of food animals as principal reservoirs of multidrug resistant pathogenic Enterococcus spp. The use of subtherapeutic doses of antimicrobial agents in animal feeds for prophylactic and growth-promoting purposes are among the major causes of the multiple drug resistance observed in the isolates reproted in the present study. The shedding of multiple drug-resistant, animal-associated Enterococcus in the environment through faecal contamination is a serious concern. Enterococcus spp. and important food pathogens of zoonotic potential and public health risks should be regularly surveyed and monitored in livestock production systems.
Data availability
This Whole Genome Shotgun project has been deposited at GenBank/NCBI under BioProject PRJNA716986, BioSample accession numbers SAMN19185853-SAMN19185872, and DDBJ/ENA/GenBank accession numbers JAHHFJ000000000-JAHHEQ000000000. The version described in this paper is version JAHHFJ010000000-JAHHEQ010000000. The sequences have been submitted to the Sequence Read Archive (SRA) under the accession numbers SRR14598116-SRR14598105. In addition, the fifteen E. faecalis genomes were submitted to Enterococcus fecalis PubMLST database (PubMLST ID: 2325-2339) and E. faecium genomes were submitted to E. faecium PubMLST database (PubMLST ID: 4561, 4562, and 4563).
Change history
14 November 2023
A Correction to this paper has been published: https://doi.org/10.1038/s41598-023-46976-2
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Acknowledgements
The authors thank the South African livestock farm owners for their cooperation in the study. Authors thank Keith D. Perret, DVM from the Epidemiology Section, KwaZulu-Natal Veterinary Services and the Department of Agriculture and Rural Development, KwaZulu-Natal, South African Department of Agriculture, Forestry and Fisheries (DAFF) for their support and help. Authors would like to thanks the two anonymous reviewers for their insightful comments which significantly improved the manuscript. We thank staff members from the Veterinary Diagnostic Laboratory, College of Veterinary Medicine, and the Genomics Center at the University of Minnesota (St. Paul, MN, USA) for their cooperation and help throughout the project. We thank staff members from the Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University (Raleigh, NC, USA), the GenomeTrakr Network, and the WGS Program for Foodborne Pathogen Traceback and the Center for Food Safety and Applied Nutrition (CFSAN), U.S. FDA, for their cooperation and support. We thank the NCBI GenBank submission staff for their help with the genome submission, decontamination, and deposition process. Prof. Mohamed E. El Zowalaty and co-authors thank Sean G Young, Ph.D., from Peter O’Donnell Jr. School of Public Health, the University of Texas Southwestern Medical Center, Dallas, TX, USA for his help and collaboration.
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Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the organizations or agencies that provided support for the project. No other person had any role in the study concept, study design, data collection, experimental work, data analysis, data interpretation or the decision to publish. Any use of commercial names, commercial diagnostic products, or firm names is for descriptive purposes only and does not imply endorsement by the funding agencies, the National Institutes of Health, Food and Drug Administration, or the United States Government. Any opinions, findings and conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the view of the funding agencies, the National Institutes of Health, or the United States Government. The funders of the study had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The corresponding author had full access to all the data in the study project and had the final responsibility for the decision to submit for publication.
Funding
The project was supported in part by funding from the United States National Institutes of Health/Food and Drug Administration (USA/NIH/FDA) under award 1U18FD006780-01, the Hessian Ministry of Higher Education, Research and Arts within the project HuKKH (Hessisches Universitaeres Kompetenzzentrum Krankenhaus Hygiene), and the South African National Research Foundation for supporting this research through the Thuthuka Funding Instrument (grant number TTK170411226583). The project was also supported by discretionary funds from Prof. Dr Mohamed Ezzat El Zowalaty and from Dr Yosra A. Helmy through start-up fund from the Department of Veterinary Science, College of Agriculture, Food, and Environment, University of Kentucky, USA. The Open access publication charges of this article were made possible by support from the University of KwaZulu-Natal, South Africa.
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M.E.Z.: Conceptualization, Study Design, Methodology, Investigation, Data curation, Formal analysis, Investigation, Visualization, Validation, Writing—original draft, Writing—review & editing, critical manuscript revisions, Project administration, Supervision, Funding acquisition; B.L. Writing—original draft, Writing—review & editing, Methodology, Formal analysis; LF: Conceptualization, Writing—original draft, Writing—review & editing, Formal analysis, Methodology, Visualization, Data curation, Software, Validation; S.M.: Methodology, Data curation, Formal analysis, Writing—original draft, Writing—review & editing, Investigation; O.Z.: Methodology, Investigation,, Supervision, Funding acquisition; S.F.: Data curation, Investigation, Visualization, Validation, Writing—review & editing; Y.A.H.: Conceptualization, Methodology, Validation, Writing—original draft, Writing—review & editing, Funding acquisition, Supervision.
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The authors declare no competing interests. Professor Dr. Mohamed Ezzat El Zowalaty is listed as a member of the One Health Initiative (One World One Health; https://onehealthinitiative.com/) and is listed as affiliate member of Biosafety Now, NJ, USA.
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The original online version of this Article was revised: The original version of this Article contained an error in the spelling of the author Yosra A. Helmy which was incorrectly given as Yosra A. Hemly.
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El Zowalaty, M.E., Lamichhane, B., Falgenhauer, L. et al. Antimicrobial resistance and whole genome sequencing of novel sequence types of Enterococcus faecalis, Enterococcus faecium, and Enterococcus durans isolated from livestock. Sci Rep 13, 18609 (2023). https://doi.org/10.1038/s41598-023-42838-z
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DOI: https://doi.org/10.1038/s41598-023-42838-z
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