Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.     

Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates

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.     

Incidence of Virulence Factors and Antibiotic Resistance among Enterococci Isolated from Food

The incidence of virulence factors among 48 Enterococcus faecium and 47 Enterococcus faecalis strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of E. faecium strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of E. faecalis strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.     

Antimicrobial Resistance of Enterococcus Species Isolated from Produce

The purpose of this study was to characterize the antibiotic resistance profiles of Enterococcus species isolated from fresh produce harvested in the southwestern United States. Among the 185 Enterococcus isolates obtained, 97 (52%) were Enterococcus faecium, 38 (21%) were Enterococcus faecalis, and 50 (27%) were other Enterococcus species. Of human clinical importance, E. faecium strains had a much higher prevalence of resistance to ciprofloxacin, tetracycline, and nitrofurantoin than E. faecalis. E. faecalis strains had a low prevalence of resistance to antibiotics used to treat E. faecalis infections of both clinical and of agricultural relevance, excluding its intrinsic resistance patterns. Thirty-four percent of the isolates had multiple-drug-resistance patterns, excluding intrinsic resistance. Data on the prevalence and types of antibiotic resistance in Enterococcus species isolated from fresh produce may be used to describe baseline antibiotic susceptibility profiles associated with Enterococcus spp. isolated from the environment. The data collected may also help elucidate the role of foods in the transmission of antibiotic-resistant strains to human populations.     

Nonclinical and Clinical Enterococcus faecium Strains,but Not Enterococcus faecalis Strains,Have Distinct Structural and Functional Genomic Features

Certain strains of Enterococcus faecium and Enterococcus faecalis contribute beneficially to animal health and food production, while others are associated with nosocomial infections. To determine whether there are structural and functional genomic features that are distinct between nonclinical (NC) and clinical (CL) strains of those species, we analyzed the genomes of 31 E. faecium and 38 E. faecalis strains. Hierarchical clustering of 7,017 orthologs found in the E. faecium pangenome revealed that NC strains clustered into two clades and are distinct from CL strains. NC E. faecium genomes are significantly smaller than CL genomes, and this difference was partly explained by significantly fewer mobile genetic elements (ME), virulence factors (VF), and antibiotic resistance (AR) genes. E. faecium ortholog comparisons identified 68 and 153 genes that are enriched for NC and CL strains, respectively. Proximity analysis showed that CL-enriched loci, and not NC-enriched loci, are more frequently colocalized on the genome with ME. In CL genomes, AR genes are also colocalized with ME, and VF are more frequently associated with CL-enriched loci. Genes in 23 functional groups are also differentially enriched between NC and CL E. faecium genomes. In contrast, differences were not observed between NC and CL E. faecalis genomes despite their having larger genomes than E. faecium. Our findings show that unlike E. faecalis, NC and CL E. faecium strains are equipped with distinct structural and functional genomic features indicative of adaptation to different environments.     

Phenotypic and Genotypic Characterization of Bacteriocinogenic Enterococci Against <Emphasis Type="Italic">Clostridium botulinum</Emphasis>

The present study aimed to characterize Enterococcus faecalis (n = ?6) and Enterococcus faecium (n = 1) isolated from healthy chickens to find a novel perspective probiotic candidate that antagonize Clostridium botulinum types A, B, D, and E. The isolated enterococci were characterized based on phenotypic properties, PCR, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). The virulence determinants including hemolytic activity on blood agar, gelatinase activity, sensitivity to vancomycin, and presence of gelatinase (gelE) and enterococcal surface protein (esp) virulence genes were investigated. Also, the presence of enterocin structural genes enterocin A, enterocin B, enterocin P, enterocin L50A/B, bacteriocin 31, enterocin AS48, enterocin 1071A/1071B, and enterocin 96 were assessed using PCR. Lastly, the antagonistic effect of the selected Enterococcus spp. on the growth of C. botulinum types A, B, D, and E was studied. The obtained results showed that four out of six E. faecalis and one E. faecium proved to be free from the tested virulence markers. All tested enterococci strains exhibited more than one of the tested enterocin. Interestingly, E. faecalis and E. faecium significantly restrained the growth of C. botulinum types A, B, D, and E. In conclusion, although, the data presented showed that bacteriocinogenic Enterococcus strains lacking of virulence determinants could be potentially used as a probiotic candidate against C. botulinum in vitro; however, further investigations are still urgently required to verify the beneficial effects of the tested Enterococcus spp. in vivo.     

Streptogramin resistance patterns and virulence determinants in vancomycin-susceptible enterococci isolated from multi-component deli salads

Aims: This study examined vancomycin-susceptible Enterococcus (VSE) from deli salads for streptogramin resistance and presence of cpd, agg and gelE genes. Methods and Results: Fifteen VSE from retail salads were isolated for identification and antimicrobial susceptibility testing by MicroScan, Etest and agar diffusion. Clinical vancomycin-resistant Enterococcus (n = 32) and animal VSE (n = 17) were included for comparative purposes. Multiplex PCR was used to detect the following genes: agg, gelE, cpd, vatD, vatE and sodA. Results showed fewer streptogramin-susceptible Enterococcus faecium isolated from salad (1/6, 17%) and animals (6/10, 60%) than from clinical (26/29, 90%) sources. A low level of erythromycin susceptibility was detected among salad (2/6, 33%) and animal (3/10, 30%) Ent. faecium isolates. Food and animal VSE demonstrated similarities in antimicrobial resistance profiles. All Enterococcus faecalis carried one or more of the selected genes cpd (40%), gelE (33%) and agg (27%). The vatD or vatE genes were not detected in any of the isolates. Conclusions: Experiments demonstrated that streptogramin resistance and virulence genes agg, cpd and gelE are present in enterococci isolated from deli salads. Significance and Impact of the Study: This study provides useful information regarding streptogramin resistance and virulence determinants in enterococci from foods associated with multi-component ingredients.     

Virulence genes,antibiotic resistance and plasmid profiles of Enterococcus faecalis and Enterococcus faecium from naturally fermented Turkish foods

Aim: To determine the virulence genes, antibiotic resistance and plasmid profiles of 16 Enterococcus faecium and 68 Enterococcus faecalis strains isolated from various naturally fermented foods. Methods and Results: The presence of virulence genes (agg₂, gelE, cylM, cylB, cylA, espfs, espfm, efaAfs, efaAfm, cpd, cop, ccf, cad) and also the genes vanA and vanB were investigated by polymerase chain reaction (PCR). Antibiotic resistance of the isolates was determined by disc diffusion method. Most of the tested isolates were positive for virulence genes and resistant to some antibiotics. One of the Ent. faecalis strains isolated from a cheese sample carried the vanA gene and was intermediately resistant to vancomycin. The strains usually contained large plasmids, which might harbour acquired antibiotic resistance. Conclusion: The study showed that Ent. faecium and Ent. faecalis strains isolated from naturally fermented Turkish foods may be potential risk factors for consumer health in terms of virulence genes and acquired antibiotic resistance. Significance and Impact of the Study: The results indicate the importance of enterococcal contamination in terms of the safety of some fermented Turkish foods.     

Efficient Transfer of the Pheromone-Independent Enterococcus faecium Plasmid pMG1 (Gmr) (65.1 Kilobases) to Enterococcus Strains during Broth Mating

Plasmid pMG1 (65.1 kb) was isolated from a gentamicin-resistant Enterococcus faecium clinical isolate and was found to encode gentamicin resistance. EcoRI restriction of pMG1 produced five fragments, A through E, with molecular sizes of 50.2, 11.5, 2.0, 0.7, and 0.7 kb, respectively. The clockwise order of the fragments was ACDEB. pMG1 transferred at high frequency to Enterococcus strains in broth mating. pMG1 transferred between Enterococcus faecalis strains, between E. faecium strains, and between E. faecium and E. faecalis strains at a frequency of approximately 10⁻⁴ per donor cell after 3 h of mating. The pMG1 transfers were not induced by the exposure of the donor cell to culture filtrates of plasmid-free E. faecalis FA2-2 or an E. faecium strain. Mating aggregates were not observed by the naked eye during broth mating. Small mating aggregates of several cells in the broth matings were observed by microscopy, while no aggregates of donor cells which had been exposed to a culture filtrate of E. faecalis FA2-2 or an E. faecium strain were observed, even by microscopy. pMG1 DNA did not show any homology in Southern hybridization with that of the pheromone-responsive plasmids and broad-host-range plasmids pAMβ1 and pIP501. These results indicate that there is another efficient transfer system in the conjugative plasmids of Enterococcus and that this system is different from the pheromone-induced transfer system of E. faecalis plasmids.     

Genomic Features and Niche-Adaptation of Enterococcus faecium Strains from Korean Soybean-Fermented Foods

Certain strains of Enterococcus faecium contribute beneficially to human health and food fermentation. However, other E. faecium strains are opportunistic pathogens due to the acquisition of virulence factors and antibiotic resistance determinants. To characterize E. faecium from soybean fermentation, we sequenced the genomes of 10 E. faecium strains from Korean soybean-fermented foods and analyzed their genomes by comparing them with 51 clinical and 52 non-clinical strains of different origins. Hierarchical clustering based on 13,820 orthologous genes from all E. faecium genomes showed that the 10 strains are distinguished from most of the clinical strains. Like non-clinical strains, their genomes are significantly smaller than clinical strains due to fewer accessory genes associated with antibiotic resistance, virulence, and mobile genetic elements. Moreover, we identified niche-associated gene gain and loss from the soybean strains. Thus, we conclude that soybean E. faecium strains might have evolved to have distinctive genomic features that may contribute to its ability to thrive during soybean fermentation.     

Prevalence of antimicrobial resistance and molecular characterization of tetracycline resistance mediated by tet(M) and tet(L) genes in Enterococcus spp. isolated from food in Southern Brazil

Enterococcus spp. are opportunistic pathogens that are widely distributed in the natural environment. Two remarkable characteristics of enterococci is their intrinsic resistance against several of the antimicrobial agents routinely prescribed in the treatment of Gram-positive cocci, and their enormous capacity to acquire different genetic markers by conjugation. The aim of this study was to evaluate the prevalence of antimicrobial resistance and the frequency of tet(M) and tet(L) genes in 112 Enterococcus spp. strains isolated from food. Fifty-two strains (64%) of Enterococcus faecalis, 10 (55%) of Enterococcus faecium, 2 (66%) of Enterococcus casseliflavus and 3 (42%) of Enterococcus gallinarum showed multidrug resistance. Tet(M) gene associated with or without the tet(L) gene was the most prevalent genotype found in food. Nine erythromycin-resistant and tetracycline-susceptible enterococci strains harbor silencing tet(M) or tet(L) genes were present in our investigation. In conclusion, antibiotic-resistant enterococci current in food may act as a reservoir of resistant strains creating a potential route of genes transference by horizontal gene transfer.     

Occurrence of multidrug-resistant Enterococcus faecium isolated from environmental samples

Enterococcus species are present in the microbiota of humans and animals and have also been described in the environment. Among the species, Enterococcus faecium is one of the main pathogens associated with nosocomial infections worldwide. Enterococcus faecium isolates resistant to different classes of antimicrobials have been increasingly reported, including multidrug-resistant (MDR) isolates in environmental sources, which is worrying. Therefore, this study aimed to characterize E. faecium isolates obtained from soil and water samples regarding antimicrobial resistance and virulence determinants. A total 40 E. faecium isolates were recovered from 171 environmental samples. All isolates were classified as MDR, highlighting the resistance to the fluoroquinolones class, linezolid and vancomycin. Furthermore, high-level aminoglycoside resistance and high-level ciprofloxacin resistance were detected in some isolates. Several clinically relevant antimicrobial resistance genes were found, including vanC1, ermB, ermC, mefAE, tetM, tetL, ant(6′)-Ia, ant(4′)-Ia, aph(3′)-IIIa and aac(6′)-Ie-aph(2″)-Ia. Three virulence genes were detected among the MDR E. faecium isolates, such as esp, gelE and ace. The results of this study contribute to a better understanding of MDR E. faecium isolates carrying antimicrobial resistance and virulence genes in environmental sources and report for the first time in the world the presence of vanC1-producing E. faecium isolated from soil.     

Safety of the Surrogate Microorganism Enterococcus faecium NRRL B-2354 for Use in Thermal Process Validation

Enterococcus faecium NRRL B-2354 is a surrogate microorganism used in place of pathogens for validation of thermal processing technologies and systems. We evaluated the safety of strain NRRL B-2354 based on its genomic and functional characteristics. The genome of E. faecium NRRL B-2354 was sequenced and found to comprise a 2,635,572-bp chromosome and a 214,319-bp megaplasmid. A total of 2,639 coding sequences were identified, including 45 genes unique to this strain. Hierarchical clustering of the NRRL B-2354 genome with 126 other E. faecium genomes as well as pbp5 locus comparisons and multilocus sequence typing (MLST) showed that the genotype of this strain is most similar to commensal, or community-associated, strains of this species. E. faecium NRRL B-2354 lacks antibiotic resistance genes, and both NRRL B-2354 and its clonal relative ATCC 8459 are sensitive to clinically relevant antibiotics. This organism also lacks, or contains nonfunctional copies of, enterococcal virulence genes including acm, cyl, the ebp operon, esp, gelE, hyl, IS16, and associated phenotypes. It does contain scm, sagA, efaA, and pilA, although either these genes were not expressed or their roles in enterococcal virulence are not well understood. Compared with the clinical strains TX0082 and 1,231,502, E. faecium NRRL B-2354 was more resistant to acidic conditions (pH 2.4) and high temperatures (60°C) and was able to grow in 8% ethanol. These findings support the continued use of E. faecium NRRL B-2354 in thermal process validation of food products.     

Detection of bacteriocin production and virulence traits in vancomycin-resistant enterococci of different sources

Aim: Three hundred and two enterococci were isolated from food, animal and clinical samples in order to evaluate the incidence of vancomycin‐resistant enterococci (VRE) and bacteriocin, cytolysin, haemolysin, gelatinase production. Methods and Results: Among the isolates, 27 (8·9%) were VRE, and 17 (63%) of these showed, by the deferred antagonism method, bacteriocin production against Gram‐positive and some Gram‐negative indicators. Eight bacteriocin producers displayed by polymerase chain reaction an enterocin structural gene: six Enterococcus faecium the Enterocin A, two Enterococcus faecalis the Enterocin P genes. The enterocins AS‐48, 31, L50 and 1071A/B genes were not found. Regarding the virulence factors, two VRE produced gelatinase and seven were haemolytic. Gelatinase gelE gene was found in 19 strains and cytolysin cylL_L gene in eight. Among the strains showing the cylL_L gene, only two E. faecalis expressed a β‐haemolysis. Conclusions: Our results showed the persistence of VRE in food, animal and clinical samples. Many of these strains displayed antibacterial activity and sometimes different components of virulence, which could emphasize their pathogenicity. Significance and Impact of the Study: This work indicates the need of a constant monitoring of enterococci in order to assess their possible pathogenic properties. The strains of interest in the food industry or used as probiotics should be tested for antibiotic resistance and virulence traits.     

Influx of Enterococci and Associated Antibiotic Resistance and Virulence Genes from Ready-To-Eat Food to the Human Digestive Tract

The influx of enterococcal antibiotic resistance (AR) and virulence genes from ready-to-eat food (RTEF) to the human digestive tract was assessed. Three RTEFs (chicken salad, chicken burger, and carrot cake) were sampled from five fast-food restaurants five times in summer (SU) and winter (WI). The prevalence of enterococci was significantly higher in SU (92.0% of salad samples and 64.0% of burger samples) than in WI (64.0% of salad samples and 24.0% of burger samples). The overall concentrations of enterococci during the two seasons were similar (~10³ CFU/g); the most prevalent were Enterococcus casseliflavus (41.5% of isolates) and Enterococcus hirae (41.5%) in WI and Enterococcus faecium (36.8%), E. casseliflavus (27.6%), and Enterococcus faecalis (22.4%) in SU. Resistance in WI was detected primarily to tetracycline (50.8%), ciprofloxacin (13.8%), and erythromycin (4.6%). SU isolates were resistant mainly to tetracycline (22.8%), erythromycin (22.1%), and kanamycin (13.0%). The most common tet gene was tet(M) (35.4% of WI isolates and 11.9% of SU isolates). The prevalence of virulence genes (gelE, asa1, cylA, and esp) and marker genes for clinical isolates (EF_0573, EF_0592, EF_0605, EF_1420, EF_2144, and pathogenicity island EF_0050) was low (≤12.3%). Genotyping of E. faecalis and E. faecium using pulsed-field gel electrophoresis revealed that the food contamination likely originated from various sources and that it was not clonal. Our conservative estimate (single AR gene copy per cell) for the influx of tet genes alone to the human digestive tract is 3.8 × 10⁵ per meal (chicken salad). This AR gene influx is frequent because RTEFs are commonly consumed and that may play a role in the acquisition of AR determinants in the human digestive tract.     

VanA-Type Enterococci from Humans, Animals, and Food: Species Distribution, Population Structure, Tn1546 Typing and Location, and Virulence Determinants

VanA-type human (n = 69), animal (n = 49), and food (n = 36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n = 4) and M49 (n = 13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P > 0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P < 0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.     

Prevalence, distribution and antibiotic resistance pattern among enterococci species in two traditional fermented dairy foods

The prevalence, distributions and antibiotic resistance pattern among enterococci species were determined. A total of 30 samples of Nigerian traditional fermented dairy food were positive to presence of enterococci, with viable counts of 4.17 log CFU/g in nunu, lower than 4.55 log CFU/g observed in wara samples. Twenty-five representative strains were characterized by a combination of phenotypic and genomic typing based on 16S rRNA gene and multi-locus sequencing analysis (MLSA) of RNA polymerase A (rpoA) and phenylanaline synthase (pheS) genes sequencing; these strains were identified as Enterococcus faecium (84 %) and Enterococcus faecalis (16 %). All the 95 enterococci isolated from wara and nunu samples were alpha haemolytic with multi-drug resistance to 10 antimicrobials regardless of class. Four strains were sensitive to chloramphenicol (30 μg) while 33.7 % of the total isolates were resistant to vancomycin from 5 μg. This information will enhance understanding of Enterococcus drug resistance and distribution in traditional fermented foods to support safety and guarantee quality of traditional foods in West Africa.     

Identification of fitness determinants in Enterococcus faecalis by differential proteomics

Enterococcus (E.) faecalis is found as commensal in healthy humans, in a variety of fermented foods. It can serve as probiotic but also as pathogen causing endocarditis, bacteremia and urinary tract infections. We have employed a proteomic study with E. faecalis strain OG1RF under different growth conditions and in contact to mouse intestinal cells to identify novel latent and adaptive fitness determinants. These relate to changes in catabolic pathways (BudA), protein biosynthesis (AsnS), cellular surface biosynthesis (RmlA) and regulatory mechanisms (OmpR). This knowledge can be used to derive novel evidence-based targets, which can be used to further elucidate gene expression changes enhancing pathogenicity or fitness in a commensal strain and possibly delineate this species into groups of higher and lower risk for applications in a food or a medical context versus improved treatment strategies of the so far hard to cure diseases.     

Genome sequence of Desulfitobacterium hafniense DCB-2, a Gram-positive anaerobe capable of dehalogenation and metal reduction

Background

Enterococci are among the leading causes of hospital-acquired infections in the United States and Europe, with Enterococcus faecalis and Enterococcus faecium being the two most common species isolated from enterococcal infections. In the last decade, the proportion of enterococcal infections caused by E. faecium has steadily increased compared to other Enterococcus species. Although the underlying mechanism for the gradual replacement of E. faecalis by E. faecium in the hospital environment is not yet understood, many studies using genotyping and phylogenetic analysis have shown the emergence of a globally dispersed polyclonal subcluster of E. faecium strains in clinical environments. Systematic study of the molecular epidemiology and pathogenesis of E. faecium has been hindered by the lack of closed, complete E. faecium genomes that can be used as references.

Results

In this study, we report the complete genome sequence of the E. faecium strain TX16, also known as DO, which belongs to multilocus sequence type (ST) 18, and was the first E. faecium strain ever sequenced. Whole genome comparison of the TX16 genome with 21 E. faecium draft genomes confirmed that most clinical, outbreak, and hospital-associated (HA) strains (including STs 16, 17, 18, and 78), in addition to strains of non-hospital origin, group in the same clade (referred to as the HA clade) and are evolutionally considerably more closely related to each other by phylogenetic and gene content similarity analyses than to isolates in the community-associated (CA) clade with approximately a 3?C4% average nucleotide sequence difference between the two clades at the core genome level. Our study also revealed that many genomic loci in the TX16 genome are unique to the HA clade. 380 ORFs in TX16 are HA-clade specific and antibiotic resistance genes are enriched in HA-clade strains. Mobile elements such as IS16 and transposons were also found almost exclusively in HA strains, as previously reported.

Conclusions

Our findings along with other studies show that HA clonal lineages harbor specific genetic elements as well as sequence differences in the core genome which may confer selection advantages over the more heterogeneous CA E. faecium isolates. Which of these differences are important for the success of specific E. faecium lineages in the hospital environment remain(s) to be determined.     

Ecology of Antibiotic Resistance Genes: Characterization of Enterococci from Houseflies Collected in Food Settings

In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 × 10³ CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria.