Presence of Virulence Genes in Enterococcus Species Isolated from Meat Turkeys in Germany Does Not Correlate with Chicken Embryo Lethality.
ABSTRACT: Virulence-associated traits have frequently been studied in enterococci and are considered to contribute towards the pathogenicity of infections. In the present study, Enterococcus isolates were collected during diagnostic investigations from meat turkeys in Germany. Twenty-eight isolates of three different Enterococcus species were analyzed for five selected putative virulence traits to understand their potential role in the pathogenicity using the chicken embryo lethality assay. Ten E. faecalis, ten E. faecium, and eight E. gallinarum isolates were examined for the presence of common virulence genes and their phenotypic expression, namely, the cytolysin operon, five individual cyl genes (cylL L , cylL S , cylM, cylB, and cylA), gelatinase (gelE), hyaluronidase (hyl Efm ), aggregation substance (asa1), and enterococcal surface protein (esp). The Enterococcus isolates showed significant species-dependent differences in the presence of genotypic traits (p < 0.001 by Fisher's exact test; Cramer's V?=?0.68). At least one gene and up to three virulence traits were found in E. faecalis, while six E. faecium isolates and one E. gallinarum isolate did not display any virulence-associated pheno- or genotype. More than half of the Enterococcus isolates (n?=?15) harbored the gelE gene, but only E. faecalis (n?=?10) expressed the gelatinase activity in vitro. The hyl Efm gene was found in five E. gallinarum isolates only, while seven isolates showed the hyaluronidase activity in the phenotypic assay. In Cramer's V statistic, a moderate association was indicated for species (V???0.35) or genotype (V?
Project description:Enterococcus spp. are opportunistic pathogens that cause lameness in broiler chickens, resulting in serious economic losses worldwide. Virulence of Enterococcus spp. is associated with several putative virulence genes including fsr, efm, esp, cylA, cad1, ace, gelE, and asa1. In this study, multiplex polymerase chain reaction (PCR) for the simultaneous detection of these virulence genes in Enterococcus spp. was developed, and detection limits for E. faecium, E. faecalis, and E. hirae were 64.0 pg/?L, 320.0 pg/?L, and 1.6 ng/?L DNA, respectively. Among 80 Enterococcus isolates tested, efm and cad1 were detected in all 26 E. faecium samples, and only cad1 was observed in E. hirae. Additionally, the presence of virulence genes in 25 E. faecalis isolates were 100% for cad1, 88.0% for gelE, 64.0% for fsr, 44.0% for asa1, 16.0% for cylA, and 4.0% for esp. No virulence genes were found in E. gallinarum isolates. A total of 49 isolates were resistant to tigecycline and to at least 2 different classes of antibiotics. The most prevalent resistance was to ciprofloxacin (73.5%), quinupristin/dalfopristin (55.1%), and tetracycline (49.0%). No strains were resistant to vancomycin or linezolid. This is the first multiplex PCR assay to simultaneously detect eight virulence genes in Enterococcus spp., and the method provides diagnostic value for accurate, rapid, and convenient detection of virulence genes. Additionally, we report the prevalence of virulence genes and antimicrobial resistance in Enterococcus isolates from commercial broiler chickens suffering lameness.
Project description:OBJECTIVE:Virulence factors (VFs) among the clinical strains of enterococci play a vital role in pathogenesis. This study was aimed to screen for cylA, asa1, gelE, esp and hyl among Enterococcus faecalis (n?=?89) and E. faecium (n?=?51) by multiplex PCR. The previously reported multiplex PCR was modified to 2 duplex (asa1 and gelE, cylA and esp) PCRs and 1 simplex (hyl) PCR. The idea of the modification of the multiplex PCR proposed here emerged in the course of the research study when majority of the isolates which phenotypically exhibited virulence traits were found to be negative for the respective gene. RESULTS:cylA, gelE and asa1 were significantly predominant in E. faecalis (59.55%, 85.39%, 86.51%) than E. faecium (1.96%, 60.78%, 9.80%) (p?<?0.0001, p?=?0.001967, p?<?0.0001). hyl was detected in E. faecium (5.9%) only. The number of VFs detected in each isolate was recorded as the VF score. E. faecalis isolates had a VF score pattern of score 4 (34.83%), score 3 (26.96%), score 2 (28.08%) and score 1 (8.98%) while E. faecium had score 4 (1.96%), score 3 (7.84%), score 2 (25.49%) and score 1 (41.18%). This modification of the PCR protocol could resolve the problem of decreased detection of virulence determinants in enterococci.
Project description:BACKGROUND:This study was aimed to investigate the intestinal microbiota in racing pigeons with regard to Enterococcus species distribution, virulence factors and antibiotic susceptibility. Three methods (API, Multiplex sodA-PCR, 16S rRNA sequencing) were compared for Enterococcus species identification. Cloacal samples from 179 apparently healthy pigeons of 13 different flocks were tested. RESULTS:Multiplex sodA-PCR and 16S rRNA gene sequencing showed almost perfect agreement in Enterococcus species identification. Isolates were identified as Enterococcus columbae (34.5%), Enterococcus hirae (20.7%), Enterococcus faecalis (11.7%), Enterococcus faecium (11.7%), Enterococcus gallinarum (9%), Enterococcus mundtii (4.8%), Enterococcus casseliflavus (3.4%), Enterococcus cecorum (2.1%), Enterococcus durans (2.1%). More Enterococcus species were found after the race season than before. The study showed differences between Enterococcus species in relation to 68.8% (22/32) biochemical parameters. Six out of seven virulence genes were detected: gelE (43.5%), asa1 (42.1%), efaA (30.3%), ace (30.3%), cylA (27.6%), and esp (9%). None of the isolates harboured hyl gene. Overall 15.2% of Enterococcus isolates produced gelatinase, but 66.7% gelE genes were silent. Enterococcus faecalis showed the most often efaA, ace and gelatinase activity than other enterococcal species. Nearly all isolates (93.1%) were resistant to at least one antibiotic. The most frequent resistance was to enrofloxacin (80%), doxycycline with teicoplanin (73.1%), erythromycin (49.7%). The study revealed significant differences between some enterococcal species in the antibiotic susceptibility to different antibiotics. Enterococcus columbae and E. cecorum showed significantly more frequent resistance to chloramphenicol than other enterococci. The presence of VRE (19.3%), HLGR (2.8%) and no LRE were found. Overall 30.3% of isolates were positive for vancomycin resistance genes, where vanC1 (E. gallinarum), vanC2-C3 (E. hirae, E. casseliflavus), vanB (E. columbae) predominated. CONCLUSIONS:We conclude, that intestinal microbiota in racing pigeons is composed by 9 different Enterococcus species. Given that racing pigeons are kept in close contact with humans and backyard animals, combined with their long-distance flight abilities, they can serve as potential source of virulent and antibiotic resistant Enterococcus spp. in the environment.
Project description:This study is designed to discuss the antimicrobial resistance, virulence determinants and biofilm formation capacity of Enterococcus spp. isolated from milk of sheep and goat with subclinical mastitis in Qena, Egypt. The obtained isolates were identified by the VITEK2 system and 16S rDNA sequencing as E. faecalis, E. faecium, E. casseliflavus and E. hirae. Overall, E. faecalis and E. faecium were the dominant species recovered from mastitic milk samples. The antimicrobial susceptibility test evidenced multidrug resistance of the isolates against the following antimicrobials: oxacillin (89.2.%), followed by vancomycin (75.7%) and linezolid (70.3%). Also, most of these isolates (73%) could form biofilms. For example, 18.9% of Enterococcus strains formed strong biofilm, whereas 32.4% of isolates formed moderate biofilm and 21.6% of isolates formed weak biofilm. The most prevalent resistance genes found in our isolates were blaZ (54%), vanA (40%), ermB (51.4%), tetM (13.5%) and optrA (10.8%). Moreover, asa1 (37.8%), cylA (42.3%), gelE (78.4%), esp (32.4%), EF3314(48.6%) and ace (75.5%) were the most common virulence genes. A significant correlation was found between biofilm formation, multidrug resistance and virulence genes of the isolates. This study highlights several aspects of virulence and harmfulness of Enterococcus strains isolated from subclinical mastitic milk, which necessitates continuous inspection and monitoring of dairy animals.
Project description:The misuse/abuse of antibiotics in intensive animal rearing and communities led to the emergence of resistant isolates such as vancomycin-resistant enterococci (VREs) worldwide. This has become a major source of concern for the public health sector. The aim of this study was to report the antibiotic resistance profiles and to highlight the presence of virulence genes in VREs isolated from feedlots cattle of the North-West Province of South Africa. 384 faecal samples, 24 drinking troughs water, and 24 soil samples were collected aseptically from 6 registered feedlots. Biochemical and molecular methods were used to identify and categorise the enterococci isolates. Their antibiotic resistance profiles were assessed and genotypic methods were used to determine their antibiotic resistance and their virulence profiles. 527 presumptive isolates were recovered, out of which 289 isolates were confirmed as Enterococcus sp. Specifically, E. faecalis (9%), E. faecium (10%), E. durans (69%), E. gallinarum (6%), E. casseliflavus (2%), E. mundtii (2%), and E. avium (2%) were screened after molecular assays. VanA (62%), vanB (17%), and vanC (21%) resistance genes were detected in 176 Enterococcus sp., respectively. Moreover, tetK (26), tetL (57), msrA/B (111), and mefA (9) efflux pump genes were detected in 138 VRE isolates. Multiple antibiotic resistances were confirmed in all the VRE isolates of this study; the most common antibiotic resistance phenotype was TETR-AMPR-AMXR-VANR-PENR-LINR-ERYR. CylA, hyl, esp, gelE, and asa1 virulence genes were detected in 86 VREs with the exception of vancomycin-resistant E. mundtii isolates that did not display any virulence factor. Most VRE isolates had more than one virulence genes but the most encountered virulence profile was gelE-hyl. Potentially pathogenic multidrug resistant VREs were detected in this study; this highlights the impact of extensive usage of antimicrobials in intensive animal rearing and its implications on public health cannot be undermined.
Project description:The emergence of antimicrobial-resistant and virulent enterococci is a major public health concern. While enterococci are commonly found in food of animal origin, the knowledge on their zoonotic potential is limited. The aim of this study was to determine and compare the antimicrobial susceptibility and virulence traits of Enterococcus faecalis and Enterococcus faecium isolates from human clinical specimens and retail red meat in Slovenia. A total of 242 isolates were investigated: 101 from humans (71 E. faecalis, 30 E. faecium) and 141 from fresh beef and pork (120 E. faecalis, 21 E. faecium). The susceptibility to 12 antimicrobials was tested using a broth microdilution method, and the presence of seven common virulence genes was investigated using PCR. In both species, the distribution of several resistance phenotypes and virulence genes was disparate for isolates of different origin. All isolates were susceptible to daptomycin, linezolid, teicoplanin, and vancomycin. In both species, the susceptibility to antimicrobials was strongly associated with a food origin and the multidrug resistance, observed in 29.6% of E. faecalis and 73.3% E. faecium clinical isolates, with a clinical origin (Fisher's exact test). Among meat isolates, in total 66.0% of E. faecalis and E. faecium isolates were susceptible to all antimicrobials tested and 32.6% were resistant to either one or two antimicrobials. In E. faecalis, several virulence genes were significantly associated with a clinical origin; the most common (31.0%) gene pattern included all the tested genes except hyl. In meat isolates, the virulence genes were detected in E. faecalis only and the most common pattern included ace, efaA, and gelE (32.5%), of which gelE showed a statistically significant association with a clinical origin. These results emphasize the importance of E. faecalis in red meat as a reservoir of virulence genes involved in its persistence and human infections with reported severe outcomes.
Project description:Enterococci are now frequent causative agents of nosocomial infections. In this study, we analyzed the frequency and distribution of antibiotic resistance and virulence genotypes of Enterococcus isolates from broiler chickens. Fecal and cecal samples from nine commercial poultry farms were collected to quantify total enterococci. Sixty-nine presumptive enterococci were isolated and identified by API 20 Strep, and their susceptibilities to antibiotics were determined. Genotypes were assessed through the use of a novel DNA microarray carrying 70 taxonomic, 17 virulence, and 174 antibiotic resistance gene probes. Total enterococcal counts were different from farm to farm and between sample sources (P < 0.01). Fifty-one (74%) of the isolates were identified as E. faecium, whereas nine (13%), seven (10%), and two (3%) isolates were identified as E. hirae, E. faecalis, and E. gallinarum, respectively. Multiple-antibiotic resistance was evident in E. faecium and E. faecalis isolates. The most common multiple-antibiotic resistance phenotype was Bac Ery Tyl Lin Str Gen Tet Cip. Genes conferring resistance to aminoglycoside (aac, aacA-aphD, aadB, aphA, sat4), macrolide (ermA, ermB, ermAM, msrC), tetracycline (tetL, tetM, tetO), streptogramin (satG_vatE8), bacitracin (bcrR), and lincosamide (linB) antibiotics were detected in corresponding phenotypes. A range of 9 to 12 different virulence genes was found in E. faecalis, including ace, agg, agrB(Efs) (agrB gene of E. faecalis), cad1, the cAM373 and cCF10 genes, cob, cpd1, cylAB, efaA(Efs), and gelE. All seven E. faecalis isolates were found to carry the gelE gene and to hydrolize gelatin and bile salts. Results from this study showed the presence of enterococci of public and environmental health concerns in broiler chicken farms and demonstrated the utility of a microarray to quickly and reliably analyze resistance and virulence genotypes of Enterococcus spp.
Project description:BACKGROUND:Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted. RESULTS:VRE isolates, including E. faecalis (n?=?24), E. faecium (n?=?11), E. casseliflavus (n?=?2) and E. gallinarum (n?=?2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p?<?0.001) and E. faecalis (p?<?0.001) and with the number of AMR genes in E. faecium (p?=?0.005). Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG (E. faecalis), and vanC (E. casseliflavus/E. gallinarum), were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers. CONCLUSIONS:There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs. E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp.
Project description:Enterococci have emerged as important opportunistic pathogens in intensive care units (ICUs). In this study, enterococcal population size and Enterococcus isolates colonizing the intestinal tract of ICU patients receiving Selective Digestive Decontamination (SDD) were investigated. All nine patients included in the study showed substantial shifts in the enterococcal 16S rRNA gene copy number in the gut microbiota during the hospitalization period. Furthermore, 41 Enterococcus spp. strains were isolated and characterized from these patients at different time points during and after ICU hospitalization, including E. faecalis (n = 13), E. faecium (n = 23), and five isolates that could not unequivocally assigned to a specific species (E. sp. n = 5) Multi locus sequence typing revealed a high prevalence of ST 6 in E. faecalis isolates (46%) and ST 117 in E. faecium (52%). Furthermore, antibiotic resistance phenotypes, including macrolide and vancomycin resistance, as well as virulence factor-encoding genes [asa1, esp-fm, esp-fs, hyl, and cyl (B)] were investigated in all isolates. Resistance to ampicillin and tetracycline was observed in 25 (61%) and 19 (46%) isolates, respectively. Furthermore, 30 out of 41 isolates harbored the erm (B) gene, mainly present in E. faecium isolates (78%). The most prevalent virulence genes were asa1 in E. faecalis (54%) and esp (esp-fm, 74%; esp-fs, 39%). Six out of nine patients developed nosocomial enterococcal infections, however, corresponding clinical isolates were unfortunately not available for further analysis. Our results show that multiple Enterococcus species, carrying several antibiotic resistance and virulence genes, occurred simultaneously in patients receiving SDD therapy, with varying prevalence dynamics over time. Furthermore, simultaneous presence and/or replacement of E. faecium STs was observed-, reinforcing the importance of screening multiple isolates to comprehensively characterize enterococcal diversity in ICU patients.
Project description:Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants. The genus Enterococcus is of increased significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. In this study, we investigated the incidence of known virulence determinants in starter, food, and medical strains of Enterococcus faecalis, E. faecium, and E. durans. PCR and gene probe strategies were used to screen enterococcal isolates from both food and medical sources. Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E. faecium strains. Medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than did starter strains. All of the E. faecalis strains tested possessed multiple determinants (between 6 and 11). E. faecium strains were generally free of virulence determinants, with notable exceptions. Significantly, esp and gelE determinants were identified in E. faecium medical strains. These virulence determinants have not previously been identified in E. faecium strains and may result from regional differences or the evolution of pathogenic E. faecium. Phenotypic testing revealed the existence of apparently silent gelE and cyl genes. In E. faecalis, the trend in these silent genes mirrors that of the expressed determinants. The potential for starter strains to acquire virulence determinants by natural conjugation mechanisms was investigated. Transconjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. In addition, multiple pheromone-encoding genes were identified in both food and starter strains, indicating their potential to acquire other sex pheromone plasmids. These results suggest that the use of Enterococcus spp. in foods requires careful safety evaluation.