Esp Gene Research Articles

Assessment of food safety using a new real-time PCR assay for detection and quantification of virulence factors of enterococci in food samples.

Development of Taqman MGB real-time PCR (q-PCR) assays for the quantitative detection of virulence factor genes in pure culture and food samples with regard to food safety assessment. New Taqman primers and probes were designed for the ace, esp and gelE genes based on the determinants of virulence profiles of enterococcal strains from GenBank. The high specificity and accuracy of the Taqman probe assay was confirmed. The limit of detection for the different virulence genes was 102 CFUml-1 or CFUg-1 for pure culture and meat samples, and 103 CFUg-1 for cheese samples. This method provides the specific and rapid detection and quantification of ace, esp and gelE genes compared to conventional PCR assays, thus allowing the rapid and direct safety assessment of Enterococcus genus in food samples. This study presents efficient methods that can be used directly on food products for the rapid quantification and tracing of virulence genes, regarding food safety assessment. Moreover, this is the first study to quantify these virulence factors using a specific Taqman q-PCR assay in food samples.

Detection of Virulence Genes in Enterococci Isolated From the Human Normal Flora by Multiplex-Polymerase Chain Reaction

Background Enterococcus species recently evolved from a common avirulent normal flora into a multidrug–resistant, health care–associated pathogen causing difficult-to-treat diseases. The aim of the current study was the detection of aggregation substance (asa), hyaluronidase (hyl), cytolysin (cyl), enterococcal surface protein (esp), collagen-binding protein (acm), gelatinase (gelE), and Enterococcus faecalis endocarditis antigen genes in Enterococcus faecium isolated from healthy volunteers by the Multiplex-polymerase chain reaction (PCR) method. Methods Stool samples were obtained from 24 healthy volunteers. After biochemical and microbiological tests, all isolates were tested for the presence of the virulence genes by Multiplex-PCR. Results In total, 91 E. faecium strains were collected from 54 different REP-PCR patterns. The distribution of the virulence genes showed that 33 (36%), 14 (15%), 3 (3%), 1 (1.1%), and 0 isolates were positive for acm, asa, esp, cylA, hyl, and gelE genes, respectively. Conclusions Despite the fact that E. faecium isolated from normal human intestines usually shows low incidence of virulence genes, they may act as harbors of virulence factors, allowing the distribution of these genes to the human normal flora through the food chain.

Antibiotic Susceptibility Pattern and Virulence Genes in Enterococcus spp. Isolated From Clinical Samples of Milad Hospital of Tehran, Iran

Background: Enterococcus spp. are part of the normal flora of humans and animals. The nosocomial pathogenicity of Enterococcus spp. has emerged in recent years and has caused great concern due to developing of resistance to many antimicrobial agents. Objectives: The current study aimed to determine the resistance pattern and the type of virulence genes in Enterococcus spp. isolated from Milad hospital of Tehran, Iran. Materials and Methods: The current observational study was conducted from Apr 2014 to Feb 2015 on a total of 149 Enterococcus species isolated from Milad hospital in Tehran, Iran. The antibiotic susceptibility pattern of the bacteria was determined by the disc diffusion method for eight antibiotics. Minimum inhibitory concentration (MIC) of vancomycin was also done using agar-dilution assay by clinical and laboratory standards institute (CLSI) recommendations. The sodA, esp, cyl, ace and gelE genes were detected by polymerase chain reaction (PCR) assay. Results: About 37.5%, 73%, 86.6%, 35.8%, 69%, 60.8%, 45% and 79% of the isolates were resistant to vancomycin, tetracycline, gentamicin, chloramphenicol, ciprofloxacin, penicillin, ampicillin and erythromycin, respectively. MIC on 38% of the isolates was 256 g/mL. Although, the prevalence of vancomycin-resistant Enterococcus (VRE) strains belonged to two species, E. faecium showed high resistance to a broad range of antibiotics. In total, 94 isolates were positive for esp, and 59, 48 and 3 isolates were positive for ace, cylA and gelE, Respectively. Conclusions: The results of the current study designate the important role of medical samples as reservoirs of resistance inducing elements. Early detection of VRE with their virulence trait will help to prevent the spread of vancomycin resistant Enterococcus species. Supervision for antibiotic usage in hospitals, especially for last option antibiotics, can prevent the spread of resistant isolates and losing all treatment options in the future.

Characterization and rapid control of a vancomycin-resistant Enterococcus faecium (VREF) outbreak in a renal transplant unit in Spain: The environment matters

ObjectiveTo describe a clonal outbreak due to vancomycin-resistant Enterococcus faecium (VREF) in the nephrology and renal transplant unit of a tertiary teaching hospital in Barcelona, Spain, and to highlight how active patient and environment surveillance cultures, as well as prompt and directed intervention strategies, mainly environmental, helped to successfully bring it under control. Patients and methodsA study was conducted on patients admitted to the nephrology ward with any culture positive for VREF over a 6-month period (August 2012–January 2013). Based on the identification of a clonal link between the isolates, weekly rectal screening using swabs was implemented for all patients, as well as environmental cultures and cleaning of medical equipment and the ward. VREF isolates were identified by MicroScan and confirmed by Etest. Bacterial identification was confirmed by MALDI-TOF MS. The presence of van genes, and esp and hyl virulence genes was determined using PCR. The clonal relationship between the isolates was studied first with DiversiLab (bioMérieux), and then by PFGE-Smal and MLST. A two-tier sequence of infection control measures was implemented. ResultsDuring the study period, VREF was isolated from 13 patients. All cases were colonized with no criteria for infection. VREF isolates were also extensively recovered from the environment and medical equipment. Isolates carried the vanA gene, and were multidrug-resistant, including high-level resistance (MIC >16mg/L) to vancomycin and teicoplanin. Molecular analysis showed that all VREF isolates belonged to sequence type 17 (ST17) carrying hyl virulence genes. After implementing infection control measures in a two-tier sequence, and reinforcing particularly environmental and medical equipment cleaning, no further cases were detected in the follow-up year. ConclusionA clonal outbreak of VREF-ST17 involving only colonization is reported. The prompt implementation of aggressive infection control measures in patients and the environment was effective in controlling the outbreak and avoided the potential emergence of infection among patients.

MOLECULAR DETECTION AND ANTIMICROBIAL ACTIVITY OF ENTEROCOCCUS FAECALIS AND ENTEROCOCCUS FAECIUM ISOLATED FROM URINARY TRACT INFECTION (UTI) PATIENTS, ANIMALS AND POULTRY

Microbiological and polymerase chain reaction (PCR) methods was used to detect the occurrence ofEnterococcus faecalis and Enterococcus faecium among 95 urine samples collected from patients suffering from urinary tract infection (UTI) recurrence admitted to outpatient clinics of private and governmental hospitals in Sohag city and 102 fecal and cloacal swabs collected from reared animals and / or poultry in some patient´s households. The results revealed that E. faecalis and E. faecium was detected in 13 (13.7%) patients, among them 11 patients were reared animals and / or poultry in their households, also isolated from 15 (14.7%) out of 102 fecal and cloacal samples of animals and poultry reared in 11 patient´s households. Referring to antimicrobial resistance and presence of esp gene among all enterococcal strains, E. faecalis and E. faecium isolates of both human and animals possess resistance to some antimicrobials with clinically importance for human therapy and esp gene was detected in 11 (84.65) out of 13 E. faecalis and E. faecium strains isolated from UTI patients and in 9 (60%) out of 15 E. faecalis and E. faecium strains isolated from the reared animals and poultry. This study suggests that reared animals and poultry, miss use of antimicrobials and presence of esp gene considered a risk factors for UTI recurrence caused by enterococci in human.

Detection of virulence genes in resistant enterococci isolated from pediatric patients at high risk for nosocomial infections

BackgroundThe study of virulence genes carried by enterococci has become of greater relevance as nosocomial enterococcal infections have become more prevalent and possibly more severe. MethodsSurveillance swabs were performed on children less than 18 months of age in an intensive care unit in Iran in 2012–2013. Multiplex PCR and sequencing methods were used to detect gelE, esp and asa1 genes in enterococci with intermediate or full resistance to vancomycin. ResultsThe rate of carriage of the genes was gelE (91%), esp (79%) and asa1 (87%). ConclusionThe majority of enterococcal strains with resistance to vancomycin carry genes for all three potential virulence factors that were analyzed in this study. This might explain why enterococcal infections appear to be more virulent than in the past.

Emergence of antimicrobial resistance and virulence factors among the unusual species of enterococci, from North India.

Several enterococcal species are increasingly being reported from clinical infections, besides the major species. This study was undertaken to determine the prevalence of unusual enterococcal species and their antimicrobial susceptibility patterns, virulence factors, and molecular characterization. The study was conducted in Department of Microbiology and associated Tertiary Care University Hospital in North India. Enterococcal isolates were collected for a period of 2 years from clinical specimens. Identification and elaborate phenotypic characterization was done biochemically. All the isolates were tested by Kirby-Bauer disc diffusion method and breakpoint minimum inhibitory concentration for susceptibility against standard antibiotics. Screening for vancomycin-resistant enterococci (VRE), high-level aminoglycoside resistance was done on brain heart infusion agar incorporated with 6 μg/ml vancomycin, 500 μg/ml gentamicin, and 2000 μg/ml streptomycin, respectively. VRE isolates were tested for the presence of vanA, vanB, and vanC genes and high-level gentamicin resistant (HLGR) isolates for aac-6'- aph-2' gene by polymerase chain reaction (PCR). Hemolysin and gelatinase production, hemagglutination and biofilm formation were detected along with asa1, gelE, esp, hyl, and cylA genes by multiplex PCR. Of 403 enterococci, 93 (23.07%) isolates were identified as unusual species and atypical variants. Resistance of 52.68%, 46.23%, 44.08%, and 6.45% for ampicillin, ciprofloxacin, high strength gentamicin, and vancomycin, respectively were noted. Presence of vanC gene in Enterococcus gallinarum and Enterococcus casseliflavus isolates and vanA gene in Enterococcus durans and Enterococcus hirae and aac-6'- aph-2'' gene was found in 33.14% (14/41) of the HLGR isolates. The most frequent virulence factor was biofilm production. Only a few isolates harbored asa1 (2), gelE (9), and hyl (3) genes. Considerable prevalence of pathogenic unusual species of enterococci was seen along with their emerging drug resistance and virulence. Complete identification and routine speciation is essential to limit their emergence as major species in near future.

Molecular Characterization of Virulence Genes in Vancomycin-Resistant and Vancomycin-Sensitive Enterococci.

Background:The aim of this study was to find out the correlation between presence of virulence (gelatinase [gel E], enterococcal surface protein [esp], cytolysin A [cyl A], hyaluronidase [hyl], and aggregation substance [asa1]) and vancomycin-resistant genes (van A and van B) in enterococci, with their phenotypic expression.Materials and Methods:A total of 500 isolates (250 each clinical and fecal) were processed. Enterococci were isolated from various clinical samples and from fecal specimens of colonized patients. Various virulence determinants namely asa1, esp, hyl, gel E, and cyl were detected by phenotypic methods. Minimum inhibitory concentration (MIC) of vancomycin was determined by agar dilution method. Multiplex polymerase chain reaction (PCR) was used to detect the presence of virulence and van genes.Results:Out of all the samples processed, 12.0% (60/500) isolates carried van A or van B genes as confirmed by MIC test and PCR methods. Genes responsible for virulence were detected by multiplex PCR and at least one of the five was detected in all the clinical vancomycin-resistant enterococci (VRE) and vancomycin-sensitive enterococci (VSE). gel E, esp, and hyl genes were found to be significantly higher in clinical VRE. Of the fecal isolates, presence of gel E, esp, and asa1 was significantly higher in VRE as compared to VSE. The presence of hyl gene in the clinical VRE was found to be statistically significant (P = 0.043) as against the fecal VRE. Correlation between the presence of virulence genes and their expression as detected by phenotypic tests showed that while biofilm production was seen in 61.1% (22/36) of clinical VRE, the corresponding genes, i.e., asa1 and esp were detected in 30.5% (11/36) and 27.8% (10/36) of strains only.Conclusion:Enterococcus faecium isolates were found to carry esp gene, a phenomenon that has been described previously only for Enterococcus faecalis, but we were unable to correlate the presence of esp with their capacity to form biofilms.

Gentamicin induces efaA expression and biofilm formation in Enterococcus faecalis

Enterococci have been ranked among the leading causes of nosocomial bacteremia and urinary tract infection. This study aimed to investigate the effect of ampicillin, vancomycin, gentamicin and ceftizoxime on biofilm formation and gene expression of colonization factors on Enterococcus faecalis. Twelve clinical isolates of E. faecalis were used to investigate the effect of antibiotics on biofilm formation and gene expression of efaA, asa1, ebpA, esp and ace. Flow system assay and Microtiter plates were used for biofilm assay. Two hundred clinical isolates were used for confirming the effect of antibiotics on biofilm formation. Ampicillin, vancomycin and ceftizoxime did not have any significant effect on biofilm formation, but gentamicin induced biofilm formation in 89% of isolates. In twelve selected isolate gentamicin increased expression of esp (+50.9%) and efaA (+33.9%) genes and reduced or maintained expression of others (asa1:−47.4%, ebpA: 0, ace:−19.2%). Vancomycin increased expression of esp (+89.1%) but reduced the others (asa1: −34.9%, ebpA:−11%, ace:−30%, efaA:−60%). Ceftizoxime increased slightly ebpA (+19.7%) and reduced others (asa1:−66.2%, esp:−35%, ace:−28.1%, efaA:−38.4%). and ampicillin strongly increased expression of ace (+231%), esp (+131%) and ebpA (+83%) but reduced others (asa1:−85.5%, efaA:−47.4%). The findings of the present study showed that antibiotics may have a role in biofilm formation and sustainability of enterococci, especially in case of gentamicin. efaA gene may have an important role, especially in antibiotic induced biofilm formation by gentamicin. Experiments with efaA mutants are needed to investigate the exact effect of efaA on biofilm formation with antibiotic induced cells.

Virulence and antimicrobial resistance of Enterococcus faecium isolated from water samples.

The aim of this study was to determine the incidence of Enterococcus species and six virulence factors of Enterococcus faecium which were isolated from surface water and wells. Fifteen different water samples, which were used for drinking as well as agricultural irrigation, were collected from nine private wells and surface water from six rivers located at the east of Tehran. The Ent.faecium isolates were tested for their resistance to 10 antibiotics and their virulence factors were detected using multiplex PCR for esp, acm, gelE, asa1, cylA and hyl genes. The most predominant species in 315 isolates were Ent.faecium (n=118) followed by Enterococcus galinarom (n=110), Enterococcus mundeti (n=18), Enterococcus hirea (n=37) and Enterococcus casselifelavus (n=32). The resistance rates were observed in 41·5, 27·1, 12·7, 6·8 and 1·7% isolates for tetracycline, erythromycin, ampicillin, ciprofloxacin and chloramphenicol respectively. None of the Ent.faecium isolates were resistant to vancomycin, teicoplanin, linezolid, gentamicin and quinuspristin-dalfopristin. Virulence determinant was found in 84·7, 33·9, 16·1 and 2·5% of isolates for acm, asa1, esp, cylA respectively. None of the isolates carried hyl and gelE gene. The presence of virulence factors and antibiotic resistance indicated that water might be an important source of dissemination of virulent enterococci. Contamination of drinking or recreational water by human or animal faecal waste is a major public health threat. In this study, we determine the incidence of Enterococcus species and six virulence factors of Enterococcus faecium which were isolated from surface water and wells. Results from this study suggest that the presence of Ent.faecium in natural and well waters was found to be significant in rural areas of Tehran. Resistant to erythromycin among Ent.faecium was relatively high and the incidence of acm and asa1 among our isolates was common overall.

The rise of ampicillin-resistant Enterococcus faecium high-risk clones as a frequent intestinal colonizer in oncohaematological neutropenic patients on levofloxacin prophylaxis: a risk for bacteraemia?

Levofloxacin extended prophylaxis (LEP), recommended in oncohaematological neutropenic patients to reduce infections, might select resistant bacteria in the intestine acting as a source of endogenous infection. In a prospective observational study we evaluated intestinal emergence and persistence of ampicillin-resistant Enterococcus faecium (AREfm), a marker of hospital adapted high-risk clones. AREfm was recovered from the faeces of 52 patients with prolonged neutropenia after chemotherapy, at admission (Basal), during LEP, and twice weekly until discharge (Pos-LEP). Antibiotic susceptibility, virulence traits and population structure (pulsed-field gel electrophoresis and multilocus sequence typing) were determined and compared with bacteraemic isolates. Gut enterococcal population was monitored using a quantitative PCR quantification approach. AREfm colonized 61.4% of patients (194/482 faecal samples). Sequential AREfm acquisition (25% Basal, 36.5% LEP, 50% Pos-LEP) and high persistent colonization rates (76.9–89.5%) associated with a decrease in clonal diversity were demonstrated. Isolates were clustered into 24 PFGE-patterns within 13 sequence types, 95.8% of them belonging to hospital-associated Bayesian analysis of population structure subgroups 2.1a and 3.3a. Levofloxacin resistance and high-level streptomycin resistance were a common trait of these high-risk clones. AREfm-ST117, the most persistent clone, was dominant (60.0% isolates, 32.6% patients). It presented esp gene and caused 18.2% of all bacteraemia episodes in 21% of patients previously colonized by this clone. In AREfm-colonized patients, intestinal enrichment in the E. faecium population with a decline in total bacterial load was observed. AREfm intestinal colonization increases during hospital stay and coincides with enterococci population enrichment in the gut. Dominance and intestinal persistence of the ST117 clone might increase the risk of bacteraemia.

Environmental waters as a source of antibiotic-resistant Enterococcus species in Belgrade, Serbia.

Despite the number of studies on antibiotic-resistant enterococci from Serbian clinical settings, there are no data about environmental contamination with these bacteria. Thus, this study investigated the prevalence of antibiotic-resistant enterococci in Belgrade, Serbia. Enterococcus species collected from ten surface water sites, including a lake, two major river systems, and springs, were tested. Among enterococci, we found single (21.7 %), double (17.4 %), and multiple antibiotic resistance patterns (56.3 %). Vancomycin-resistant strains were not found, indicating that their abundance in Belgrade is tightly linked to clinical settings. The multiple drug-resistant strains Enterococcus faecalis, Enterococcus faecium, and Enterococcus mundtii were frequently detected in the lake during the swimming season and in the rivers near industrial zones. We confirmed the presence of ermB, ermC, ant(6)-Ia, tetM, and tetL and mutations in gyrA genes. The phylogenetic analysis of 16S rRNA gene of E. faecium isolates that harbor esp gene classified them into two groups based on high-bootstraps scores in the tree analysis. Pulsed-field gel electrophoresis analysis of antibiotic-resistant enterococci revealed genomic similarity ranging from 75 to 100 %. This study indicates the importance of anthropogenic impact to the spread of antibiotic-resistant enterococci in environmental waters of Belgrade, Serbia.

Biofilm synthesis and presence of virulence factors among enterococci isolated from patients and water samples.

The goal of this study was to compare biofilm synthesis among enterococci recovered from clinical samples (infection or colonization) of patients as well as environmental samples in order to determine possible virulence factors and clonal relationship. During a two-year period, clinical samples (blood, catheter tips, bronchial secretions, wounds, peritoneal fluid, urine) and rectal swabs collected from hospitalized patients as well as environmental water samples were tested for the presence of Enterococcus faecalis and Enterococcus faecium. Antibiotic susceptibility testing was performed by the disc diffusion method and Etest. Strains were tested for the presence of vanA, vanB, esp, ace and asp genes by PCR. Clones were identified by PFGE (SmaI). From infected patients, 48 strains were identified: 24 Enterococcus faecium (10 vanA-positive, 14 vancomycin-susceptible) and 24 Enterococcus faecalis (one vanA-positive, 23 vancomycin-susceptible). Among 143 colonizing isolates, 134 were Enterococcus faecium (58 vanA-positive, 11 vanB-positive, 65 vancomycin-susceptible) and nine Enterococcus faecalis (three vanA-positive, two vanB-positive, four vancomycin-susceptible). Among 167 environmental water samples, 51 Enterococcus faecalis and 19 Enterococcus faecium isolates, all glycopeptide-susceptible, were recovered. In total, 64 strains produced biofilm, whereas 34 were esp-positive, 64 asp-positive and 54 ace-positive. Biofilm production was associated with the presence of esp (P < 0.001) and ace genes (P = 0.021), being higher in infecting (P < 0.001) and water (P 0.005) isolates as compared with colonizing ones. Clones of environmental water-strains were different than the patients' clones. The differences found in the incidence of antibiotic resistance, virulence factors and clones suggest that hospital and water enterococci are of different origin.

Epidemiology and molecular typing of VRE bloodstream isolates in an Irish tertiary care hospital.

Ireland has the highest rate of vancomycin-resistant Enterococcus faecium (VREfm) isolated from blood of nosocomial patients in Europe, which rose from 33% (110/330) in 2007 to 45% (178/392) in 2012. No other European country had a VREfm rate from blood cultures of >25%. Our aim was to elucidate the reasons for this significantly higher rate in Ireland. The epidemiology and molecular typing of VRE from bloodstream infections (BSIs) was examined in a tertiary care referral hospital and isolates were compared with those from other tertiary care referral centres in the region. The most common source of VRE BSIs was intra-abdominal sepsis, followed by line-related infection and febrile neutropenia. Most of the isolates were positive for vanA; 52% (43/83) possessed the esp gene and 12% (10/83) possessed the hyl gene. Genotyping by SmaI macrorestriction analysis (PFGE) of isolates revealed clonal relatedness between bloodstream isolates and environmental isolates. VRE BSI isolates from two other tertiary care hospitals in the Dublin region showed relatedness by PFGE analysis. MLST revealed four STs (ST17, ST18, ST78 and ST203), all belonging to the clonal complex of hospital-associated strains. Irish VRE BSI isolates have virulence factor profiles as previously reported from Europe. Typing analysis shows the spread of individual clones within the hospital and between regional tertiary care hospitals. Apart from transmission of VRE within the hospital and transfer of colonized patients between Irish hospitals, no other explanation for the persistently high VREfm BSI rate in Ireland has been found.

Spread of Enterococcal Surface Protein in Antibiotic Resistant Entero-coccus faecium and Enterococcus faecalis isolates from Urinary Tract Infections.

Enterococci rank among leading cause of nosocomial bacteremia and urinary tract infection in hospital and community acquired infections. Several traits that may contribute to enhanced virulence have been identified in Enterococci. Extracellular surface protein (Esp) is a virulence factor that contributes in biofilm formation and resistance to environmental stresses. In this study we aimed to determine occurrence of esp in E. faecium and E. faecalis isolates isolated from urinary tract infections and to investigate whether there is any correlation between presence of esp and antibiotic resistance. One hundred and sixty six isolates were collected from patients with UTI and after identification by biochemical and PCR, antibiotic resistances were examined. The presence of esp was investigated by primer-specific PCR. 43.3% of isolates identified as E. faecium and 56.7% as E. faecalis. The esp gene was found in 76.1% of E. faecium isolates and 77.9% of E. faecalis isolate. There were significant correlation between esp positive E. faecium and resistance to Vancomycin (p<0.01), also in E.faecalis we found correlation between esp positive and resistance to Ampicillin, Chloramphenicol and Tetracycline (p<0.01, p<0.01, p<0.01 respectively). Occurrence of esp in our isolates from urinary tract infection was high that indicates importance of this gene in urinary tract infections and shows importance of ability to forming biofilm and hydrophobicity of surface of Enterococci for causing urinary infection by Enterococci. Also, our finding showed significant correlation between resistance to antibiotics and presence of esp in Enterococci.

Molecular typing, pathogenicity factor genes and antimicrobial susceptibility of vancomycin resistant enterococci in Belgrade, Serbia.

In this study the distribution of species and antimicrobial resistance among vancomycin resistant enterococci (VRE) recovered from clinical specimens obtained from five hospitals in Belgrade was analyzed. Strains were further characterized by pulsed-field gel electrophoresis (PFGE). Polymerase chain reaction (PCR) was used to investigate the presence of vanA and vanB genes and pathogenicity factor genes. Identification of 194 VRE isolates revealed 154 Enterococcus faecium, 21 Enterococcus faecalis, 10 Enterococcus raffinosus and 9 Enterococcus gallinarum. This study revealed existence of 8 major clones of VRE. PCR determined vanA gene to be present in all of the VRE studied. Esp and hyl genes were present in 29.22% and 27.92% of E. faecium, respectively, and in 76.19% and 0 of E. faecalis, respectively. Esp and hyl genes were not found more frequently in members of predominant clones of E. faecium than in single isolates; nor was their presence connected to invasiveness.

Exploration of the mechanisms of biofilm formation by Enterococcus faecalis in glucose starvation environments.

Enterococcus faecalis is the most common single species present in teeth after failed root canal therapy. This is mainly due to its ability to maintain viability for a long time in filled root canals where nutrients are normally sparse. The aim of this study was to explore the mechanism of E. faecalis survival and biofilm formation in glucose-starved environments. Enterococcus faecalis ATCC 33186 was inoculated in energy starvation media for biofilm formation. Confocal laser scanning microscopy and fluorescent DNA-binding agents were employed to assess biofilm-forming ability. The physiochemical properties of the biofilm cell wall were investigated by measuring the hydrophobicity, extracellular polysaccharide and ATPase activity. The expression of stress and virulence genes was quantified by real-time quantitative polymerase chain reaction. The ability for biofilm formation decreased with a decreasing concentration of glucose. The cell surface hydrophobicity increased dramatically with decreasing glucose concentration. Water-soluble exopolysaccharide (WSE) synthesis decreased in glucose starvation media, whereas water-insoluble exopolysaccharide (WIE) synthesis increased. Amarked decrease in ATPase activity was observed only in a no glucose medium. In addition, transcription of ace, fsrB and gelE genes increased under glucose starvation stress while atpE, salB and esp genes were down-regulated. Enterococcus faecalis survival and biofilm formation under glucose starvation stress may be attributed to an increase in cell-surface hydrophobicity and additionally to the up-regulation of some genes involved in stress response and biofilm formation. These characteristics may explain why E. faecalis can maintain viability for a long time in an energy-starved environment and why it is frequently isolated from persistently infected root canals.

Molecular characterization of vancomycin-resistant Enterococcus faecium strains isolated from carriage and clinical samples in a tertiary hospital, Turkey

This study aimed to determine the presence of vancomycin resistance (vanA and vanB) and virulence genes (esp, asa1, gelE, ace, hyl, cylA, cpd and ebpA) in vancomycin-resistant Enterococcus faecium (VREfm) strains and to analyse the clonal relationships among the strains. E. faecium strains were identified from rectal and clinical specimens by biochemical tests and the API-20 Strep kit. Susceptibility testing was performed using disc-diffusion and broth-dilution methods. PFGE was used for molecular typing of the VREfm strains. The vancomycin resistance and virulence genes were amplified by two-step multiplex PCR. All 55 VREfm isolates were resistant to penicillin G, ampicillin and high-level gentamicin but were susceptible to quinupristin/dalfopristin and linezolid. Multiplex PCR analysis indicated that all isolates harboured vanA and that 41 (75 %) were positive for virulence genes. The esp gene was the most common virulence factor and was detected in nine (41 %) invasive and 32 (96.7 %) non-invasive isolates. Multiple virulence genes were observed only in two non-invasive isolates; one harboured esp and ebpA and the other harboured esp, ebpA, asa1, gelE and cpd. PFGE typing yielded 16 different types, seven of which were clusters with two to 14 strains each. The clustering rates of the rectal swab, blood and urine isolates were 72.7 %, 61.5 % and 87.5 %, respectively. The genetic similarity observed among the VREfm isolates indicated cross-transmission in the hospital. Further studies on the virulence factors present in the strains might provide insight into the acquisition of these traits and their contribution to increased prevalence of VREfm.

Detection of vancomycin-resistant Enterococcus faecalis ST6-vanB2 and E. faecium ST915-vanA in faecal samples of wild Rattus rattus in Spain

The detection of vancomycin-resistant-enterococci (VRE) among wild animals represents a worrisome public health concern. The objectives of the study were to determine the possible presence of VRE in faecal samples of wild small mammals in Spain, to characterize the vancomycin resistance mechanisms and genetic lineages of recovered isolates and to know the diversity of enterococcal species in these animals. A total of 155 faecal samples from small mammals were inoculated in Slanetz–Bartley agar supplemented or not with vancomycin (Van-SB/SB plates). The antimicrobial susceptibility profile to 12 antimicrobials and the presence of 20 antimicrobial resistance genes was analyzed. The structure of Tn1546 and the presence of gelE, cylA, asa, esp and hyl genes was studied. Multilocus-sequence-typing (MLST) technique was also performed. VRE isolates were recovered in Van-SB plates in 11 samples. Two samples contained vanB2-positive E. faecalis isolates of lineage ST6, which showed a multiresistance phenotype and harboured the virulence genes gelE and asa. One sample contained a vancomycin-resistant E. faecium isolate of the new lineage ST915, with the vanA gene included into Tn1546 (truncated with IS1542 and IS1216 elements). The vanB2 and vanA isolates were obtained from Rattus rattus. The remaining eight VRE-positive samples contained species with intrinsic vancomycin-resistance mechanisms: E. casseliflavus (n=5) and E. gallinarum (n=3). One hundred and forty-seven vancomycin-susceptible-enterococcal isolates were obtained in SB plates, and E. faecalis and E. faecium were the most frequent detected species. This is the first report of vanB2-containing enterococci in wild animals.

Assessment of biofilm formation by enterococci isolates from urinary tract infections with different virulence profiles

This study aimed to investigate possible associations between virulence profiles and biofilm formation in Clinical UTI isolates. Isolates were collected from five university hospitals and identified and characterized for the presence of virulence factors by PCR. Biofilm assays were conducted in 96 well microtiter plates by reading the OD570 after crystal violet staining. 75% of isolates had esp gene, 38.77% had asa1, 84.18% had ace, 81.63% had efaA, 93.36% had ebpR, 34.18% had cylA, 81.63% had gelE and 17.35% had hyl. Biofilm experiences were done and isolates having asa1 or efaA genes produced more biofilms than negative ones (P=0.011, P=0.008), but the presence of esp, ace, cylA or gelE genes in isolates had no effect on biofilm formation. Isolates possessing hyl had much lower biofilm formation (P=0.000). Present study showed that the esp, ace, gelE and cylA genes do not seem to be necessary nor sufficient for the production of biofilm in enterococci but the presence of efaA and asa1 correlates with increased biofilm formation of urinary tract isolates. Also the low prevalence of hyl among enterococci isolated from UTIS and its association with poor biofilm production indicate that the absence of this gene can be an advantage in the pathogenesis of UTIs.