Endocarditis Antigen Research Articles

Safety evaluation and bacteriocinogenic potential of Pediococcus acidilactici strains isolated from artisanal cheeses

Pediococcus acidilactici ST1607V, ST2104V and ST3105V, isolated from a Brazilian artisanal cheese expressed bacteriocins with high inhibitory activity against several strains of Listeria monocytogenes and Enterococcus spp., but not against most of the other tested lactic acid bacteria (LAB). Bacteriocinogenic activity against L. monocytogenes ATCC7644 was expressed with a high titre of 51200 AU/mL (ST1607V and ST2104V) and 25600 AU/mL (ST3105V), when cultured in MRS. Bacteriocins produced by strains ST1607V, ST2104V and ST3105V presented a bactericidal mode of action against L. monocytogenes ATCC7644 and Enterococcus faecium ATCC19434. DNA of the studied LAB strains was obtained and subjected to PCR assays targeting more than 50 genes related to biogenic amines production (histidine, tyrosine and ornithine decarboxylases), virulence (sex pheromones, gelatinase, cytolysin, hyaluronidase, aggregation substance, enterococcal surface protein, endocarditis antigen, adhesion of collagen, integration factors) and antibiotic resistance (vancomycin, tetracycline, erythromycin, gentamicin, chloramphenicol, bacitracin). A few safety-related genes were detected in some strains, indicating their safety for application in fermented food products. Special attention should be given to the presence of possible virulence factors, in addition to the production of biogenic amines and antibiotic resistance. Additional in vitro and in vivo experiments could be designed for future evaluation of possible expression of detected virulence genes.

Not Everything is a Question of Reputation: Safety of Bacteriocinogenic Lab Isolated from Smoked Salmon

Total DNA extracted from bacteriocinogenic and potential probiotic lactic acid bacteria (LAB) (Lactobacillus curvatus ET06, ET30 and ET31, Lactobacillus fermentum ET35, Lactobacillus delbrueckii ET32, Pediococcus acidilactici ET34 and Enterococcus faecium ET05, ET12 and ET88), have been screened for presence of more than 50 genes related to production of biogenic amines (histidine decarboxylase, tyrosine decarboxylase and ornithine decarboxylase), virulence factors (sex pheromones, gelatinase, cytolisin, hyaluronidase, aggregation substance, enterococcal surface protein, endocarditis antigen, adhesion of collagen, integration factors) and antibiotic resistance (vancomycin, tetracycline, erythromycin, gentamicin, chloramphenicol, bacitracin). Tested strains presented low frequencies of presence for virulence genes. Only few genes were detected in some strains, indicating their safety for application in fermented food products. Besides all beneficial properties studied for various LAB, most considered as GRAS, a special attention need to be addressed on the possible presence of virulence factors, production of biogenic amines and antibiotic resistance. Results from appropriate biochemical tests and detected main genes associated to virulence factors and antibiotic resistance in LAB strains, could be considered as potential hazard of application of this organisms in food products. Moreover, additional in vitro and in vivo experiments must be designed in the system simulating GIT conditions in order to investigate deeper the possible expression of the present virulence genes.

Comparison of antibiotic resistance and virulence in vancomycin-susceptible and vancomycin-resistant Enterococcus faecium strains

Aim. Today, infections caused by vancomycin-resistant Enterococcus faecium (VRE) are a major problem in the healthcare system. The aim of this study was to compare the antibiotic resistance and virulence traits between vancomycin-susceptible E. faecium (VSE) and VRE clinical isolates.Material and Methods. Studies were performed on 66 E. faecium (32 VRE and 34 VSE) strains. Susceptibility testing and identification were performed, and strains were examined for ß-lactamase, hemolysin and biofilm production. Isolates were tested for the presence of 5 van genes, 8 virulence genes and 6 aminoglycoside-modifying enzyme (AME) genes. Obtained amplicons were subjected to electrophoretical separation and DNA sequencing.Results. Among 32 VRE isolates, 28 were found to have the VanA phenotype, and 4 the VanB. The most frequent resistance and virulence profile among VRE strains was resistance to ampicillin, imipenem, gentamicin, streptomycin, teicoplanin, and vancomycin with enterococcal surface protein (esp), endocarditis antigen (efaA), collagen adhesin (acm), and hialuronidase (hyl) genes; among VSE: resistance to ampicillin, imipenem, gentamicin, streptomycin with esp, efaA, acm, and hyl genes. Conclusions. Our findings prove that both VRE and VSE strains were well equipped with virulence and resistance genes, although VRE strains were characterized by a greater variety and a higher number of these genes. However, statistical analysis revealed no significant differences between VSE and VRE strains (p > 0.05). Nevertheless, our results suggest that VRE strains may slowly acquire and incorporate resistance and virulence genes, due to their ability to survive in a hospital environment for a long time.

Safety of Lactobacillus plantarum ST8Sh and Its Bacteriocin.

Total DNA extracted from Lb. plantarum ST8Sh was screened for the presence of more than 50 genes related to production of biogenic amines (histidine decarboxylase, tyrosine decarboxylase, and ornithine decarboxylase), virulence factors (sex pheromones, gelatinase, cytolysin, hyaluronidase, aggregation substance, enterococcal surface protein, endocarditis antigen, adhesion of collagen, integration factors), and antibiotic resistance (vancomycin, tetracycline, erythromycin, gentamicin, chloramphenicol, bacitracin). Lb. plantarum ST8Sh showed a low presence of virulence genes. Only 13 genes were detected (related to sex pheromones, aggregation substance, adhesion of collagen, tetracycline, gentamicin, chloramphenicol, erythromycin, but not to vancomycin, and bacitracin) and may be considered as indication of safety for application in fermented food products. In addition, interaction between Lb. plantarum ST8Sh and drugs from different groups were determined in order to establish possible application of the strain in combination with commercial drugs. Cytotoxicity of the semi-purified bacteriocins produced by Lb. plantarum ST8Sh was depended on applied concentration-highly cytotoxic when applied at 25μg/mL and no cytotoxicity at 5μg/mL.

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.

Assessment of the safety and applications of bacteriocinogenic Enterococcus faecium Y31 as an adjunct culture in North-eastern Chinese traditional fermentation paocai

Enterococcus faecium Y31 is a class IIa bacteriocin producer found in traditional Chinese fermented foods. The bacteriocin produced by Y31 (bacteriocin Y31) has a broad inhibitory spectrum. Here, we investigated the safety and behaviours of E. faecium Y31 in North-eastern Chinese traditional fermentation paocai (NCTFP) to assess its suitability as an adjunct culture. E. faecium Y31 showed interesting features, such as the absence of virulence determinants (aggregation substance [agg], enterococcal surface protein [esp], endocarditis antigen [efaAfm], gelatinase [gelE], hemolysin [cylA, cylB, cylM], sex pheromone [cpd, cob, ccf], adhesion of collagen [ace], and hyaluronidase [hyl] genes), sensitivity to various antibiotics, lack of haemolytic compounds, and lack of biogenic amine production. E. faecium Y31 could propagate in raw Chinese cabbage and produce bacteriocin Y31. To evaluate its performance as an adjunct culture for NCTFP, changes in the overall quality of control (noninoculated) and experimental NCTFP (inoculated with E. faecium Y31 under previously optimised fermentation conditions) were determined in duplicate fermentations of Chinese cabbage using chemical, microbiological, texture, and sensory evaluations. In the presence of E. faecium Y31, faster fermentation and higher bacteriocin Y31 production were observed than in the control. Additionally, the nitrite concentration of the fermentation solution decreased more rapidly, the total viability of the culture increased, and the lactic acid bacterium population was higher than those in NCTFP produced by spontaneous fermentation. No yeasts and moulds were detected in NCTFP produced by inoculation fermentation or spontaneous fermentation, and the textural properties of these two products were similar. Sensory evaluation results for NCTFP produced by inoculation fermentation were superior to those produced by spontaneous fermentation. Therefore, E. faecium Y31 was sufficient as an adjunct culture to enhance both the hygienic and sensory attributes of NCTFP and its use may help to address the issues inherent in commercial production of NCTFP.

Prevalence of Enterococcus Species and Their Virulence Genes in Fresh Water Prior to and after Storm Events

Enterococcus spp. isolates (n = 286) collected from six surface water bodies in subtropical Brisbane, Australia, prior to and after storm events, were identified to species level and tested for the presence of seven clinically important virulence genes (VGs). Enterococcus faecalis (48%), Enterococcus faecium (14%), Enterococcus mundtii (13%), and Enterococcus casseliflavus (13%) were frequently detected at all sites. The frequency of E. faecium occurrence increased from 6% in the dry period to 18% after the wet period. The endocarditis antigen (efaA), gelatinase (gelE), collagen-binding protein (ace), and aggregation substance (asa1) were detected in 61%, 43%, 43%, and 23% of Enterococcus isolates, respectively. The chances of occurrence of ace, gelE, efaA, and asa1 genes in E. faecalis were found to be much higher compared to the other Enterococcus spp. The observed odds ratio of occurrence of ace and gelE genes in E. faecalis was much higher at 7.96 and 6.40 times, respectively. The hyl gene was 3.84 times more likely to be detected in E. casseliflavus. The presence of multiple VGs in most of the E. faecalis isolates underscores the importance of E. faecalis as a reservoir of VGs in the fresh water aquatic environment. Consequently, if contaminated surface water is to be used for production of potable and nonpotable water some degree of treatment depending upon intended use such as detention in basins prior to use or chlorination is required.

Incidence of virulence factors in enterococci from raw and fermented meat and biofilm forming capacity at 25 °C and 37 °C

Twenty-nine Enterococcus strains from raw and fermented meat products were screened for the presence of virulence genes, including those for aggregation substances (asa1 and asa373), cytolysin activator (cylA), collagen binding protein (ace), endocarditis antigen (efaA), enterococcal surface protein (esp) and gelatinase (gelE). Virulence gene occurrence, expression of gelatinase and pheromone aggregation was greater in Enterococcus faecalis than in Enterococcus faecium strains. All E. faecalis and 54% of E. faecium were positive for at least one or more virulence gene. The only strain of Enterococcus gallinarum tested also contained virulence genes. The effect of different growth temperatures (25 and 37°C) on biofilm formation using polystyrene plates was also assessed. Strong biofilm formation occurred at lower than optimum temperature in all three species of enterococci. Neither esp nor gelE was necessary for biofilm formation and this relationship was species rather than strain specific. This study emphasizes the importance of enterococci as a reservoir of virulence genes and the potential for their genetic transfer to human strains following consumption of uncooked or undercooked contaminated meat.

Characterization of Enterococcus faecium isolates and first report of vanB phenotype–vanA genotype incongruence in the Middle East

We aimed to characterize the vancomycin genotype/phenotype, carriage of putative virulence genes, and genetic relatedness of Enterococcus faecium isolates in Saudi Arabia. E. faecium isolated from inpatients at our medical center were studied. Sensitivity to ampicillin, linezolid, teicoplanin, quinupristin/dalfopristin, tetracycline, and ciprofloxacin was determined. The presence of van genes and virulence genes for aggregation substance (Asa-1), enterococcal surface proteins (esp), cytolysin (cylA, cylL, cylM), gelatinase (gelE), E. faecium endocarditis antigen (EfaA( fm )), hyaluronidase (hyl), and collagen adhesion (Ace) was assessed. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE). Twenty-nine E. faecium isolates were obtained and the majority of isolates (n/N = 22/29) were from stool specimens. PFGE analysis identified eight pulsotypes (A-H) based on 80% similarities. Isolates were represented in five major pulsotypes: type A (n = 5), type B (n = 3), type D (n = 6), type E (n = 5), and type F (n = 7). All isolates were vanA gene-positive. Thirteen isolates had vanA(+)/vanB(+) genotype. Of these, ten exhibited a vanB phenotype and three had a vanA phenotype. Eight isolates with vanA(+)/vanB(-) genotype exhibited vanB phenotype. Six of these eight isolates belonged to the same pulsotype. All isolates were positive for gelE, esp, and EfaA( fm ) genes. Five were CylA-positive and 24 had the hyl genes. Of the eight isolates harboring a combination of gelE, esp, EfaA( fm ), and hyl genes, five showed vanB phenotype-vanA genotype incongruence. This is the first report of vanB phenotype-vanA genotype incongruent E. faecium in the Middle East region. Molecular typing indicates clonal spread and high occurrence of virulence genes, especially esp genes, associated with epidemic clones.

Molecular identification of an Enterococcus faecalis endocarditis antigen efaA in root canals of therapy-resistant endodontic infections

Introduction:Enterococcus faecalis has long been implicated in persistent root canal infections and therapy-resistant endodontic infections. It has also been associated with bacteremia, that is, infective endocarditis arising from certain invasive dental procedures. E. faecalis endocarditis antigen (efaA) has been identified as one of the principal virulence factors associated with infective endocarditis.Aim:To detect the presence of putative E. faecalis virulence factor, efaA in root canals of therapy-resistant endodontic infections using polymerase chain reaction (PCR) amplification.Materials and Methods:Samples were obtained from 32 patients (20–70 years) undergoing endodontic retreatment, which were incubated in prereduced thioglycollate broth and subcultured onto ethyl violet azide broth (EVA; selective medium for E. faecalis). Deoxyribonucleic acid (DNA) was extracted from the samples and analyzed for the endocarditis virulence factor efaA using PCR.Results:Among the positive E. faecalis samples, efaA gene was identified in 11 out of 15 samples.Conclusion:Our findings indicate that efaA, a potent E. faecalis virulence gene can be found in E. faecalis strains detected in root canals of therapy-resistant endodontic infections similar to reports for ‘medical’ strains.

Frequency of Virulence-Associated Genes in Enterococccus faecalis Isolated in Kuwait Hospitals

Objective: The objective of this study was to investigate the carriage of 6 virulence-associated genes in Enterococcus faecalis isolates obtained from patients in 8 hospitals in Kuwait. Materials and Methods: In total, 466 E. faecalis isolates were obtained from 313 urine samples, 68 wound swabs, 36 blood samples, 25 rectal swabs, 12 high vaginal swabs and 12 miscellaneous sources. Genes for gelatinase(gelE),aggregation substance (aggA), hemolysin activation factor (cylA), enhanced expression of pheromone (eep), enterococcal surface protein (esp), and E. faecalis endocarditis antigen A (efaA) were detected in PCR assays. Results: Of 466 isolates, 423 (90.8%) were positive for 1 and up to 5 genes. However, none of the genes was detected in all of the isolates. The prevalence of the individual genes was eep: 31.9%; esp: 31.5%; gelE: 28.5%; efaA: 27.9%; aggA: 23.4%, and cylA: 18.5%. Of the 423 positive isolates, 148 (34.9%) were positive for 2 genes and 52 (12.3%), 15 (3.5%) and 5 (0.9%) isolates were positive for 3, 4 and 5 virulence genes, respectively. The efaA and esp combination was detected in isolates from all clinical sources. Conclusion: The study showed a high prevalence of virulence genes in E. faecalis isolated in Kuwait hospitals. The absence of a dominant gene in all of the isolates suggests that infections by E. faecalis may require the involvement of multiple virulence factors.

Evaluation of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 as probiotics by using a gastro-intestinal model with infant milk formulations as substrate

Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 produce bacteriocins with activity against a number of Gram-positive and Gram-negative bacteria. Both strains survived intestinal conditions simulated in a gastro-intestinal model (GIM) with infant milk formulations as substrate and prevented the growth of Listeria monocytogenes ScottA. The strains are inhibited by the antibiotics amoxicillin, cefadroxil, roxithromycin and doxycycline, anti-inflammatory medicaments containing meloxicam, ibuprofen and sodium diklofenak, and analgesics containing paracetamol, codeine phosphate and promethazine. Strain 423 is sensitive to vancomycin and does not contain genes encoding gelatinase, cell aggregation substance (AS), adhesion to collagen (Ace), enterococcus surface protein (Esp), Enterococcus faecalis endocarditis antigen (EfaAfs), cytolysin and non-cytolysin (β-hemolysin III). Genes encoding AS, cytolysin and non-cytolysin (β-hemolysin III) were amplified from the genome of strain ST4SA. Survival of strains ST4SA and 423 improved when used as combined cultures in the GIM and compared well with the survival of commercially available probiotics subjected to the same conditions.

Virulence and antimicrobial resistance in clinical Enterococcus faecium

The aim of the present study was to determine the occurrence of seven virulence determinants in Enterococcus faecium clinical blood culture isolates over a 6-year period and to investigate possible correlations between virulence and antibiotic resistance. Two hundred and sixty-three isolates were screened for the presence of genes coding for aggregation substance ( asa1), cytolysin ( cylA), collagen-binding protein ( ace), Enterococcus faecalis endocarditis antigen ( efaA fs), enterococcal surface protein ( esp fm), gelatinase ( gelE) and hyaluronidase ( hyl fm) by polymerase chain reaction. The minimum inhibitory concentrations (MICs) of ampicillin, ciprofloxacin, gentamicin, imipenem, linezolid and vancomycin were determined by the agar dilution method and the MIC of daptomycin was determined by Etest. The esp fm gene was found in 56% of the isolates, hyl fm in 4%, whilst the other virulence genes were detected only sporadically (≤1%). The level of antibiotic resistance was 77% to ampicillin, 90% to ciprofloxacin and 83% to imipenem; 5% of the isolates were resistant to vancomycin and 2% were resistant to gentamicin (high-level resistance, MIC ≥500 mg/L). A significant correlation was found between the presence of esp fm and resistance to ampicillin, ciprofloxacin and imipenem ( P < 0.01). Twelve isolates were esp fm-positive and ampicillin-susceptible.

Prevalence of putative virulence factors and antimicrobial susceptibility of Enterococcus faecalis isolates from patients with dental Diseases

BackgroundThis study investigated the prevalence of Enterococcus faecalis, its putative virulence factors and antimicrobial susceptibility in individuals with and without dental diseases. A total of 159 oral rinse specimens were collected from patients (n = 109) suffering from dental diseases and healthy controls (n = 50).ResultsE. faecalis was detected using only culture in 8/109 (7.3%) of the patients with various types of dental diseases, whereas no E. faecalis was found in the healthy controls weather using both culture and PCR. Phenotype characterizations of the 8 E. faecalis isolates indicated that 25% of the isolates produced haemolysin and 37.5% produced gelatinase. Most important virulence genes; collagen binding protein (ace) and endocarditis antigen (efaA) were present in all 8 E. faecalis isolates, while haemolysin activator gene (cylA) was detected only in 25% of isolates, and all isolates were negative for esp gene. All E. faecalis isolates were 100% susceptible to ampicillin, chloramphenicol, ciprofloxacin, vancomycin, and teicoplanin, and to less extent to erythromycin (62.5%).ConclusionThis study shows that all E. faecalis isolates were recovered only from patients with dental diseases especially necrotic pulps, and all isolates carried both collagen binding protein and endocarditis antigen genes and highly susceptible to frequently used antimicrobial drugs in Jordan.

Risk factors in enterococci isolated from foods in Morocco: Determination of antimicrobial resistance and incidence of virulence traits

A collection of enterococci isolated from meat, dairy and vegetable foods from Morocco including 23 Enterococus faecalis and 15 Enterococcus faecium isolates was studied. All isolates were sensitive to ampicillin, penicillin, and gentamicin. Many E. faecalis isolates were resistant to tetracycline (86.95%), followed by rifampicin (78.26% ciprofloxacin (60.87%), quinupristin/dalfopristin (56.52%), nitrofurantoin (43.47%), levofloxacin (39.13%), erythromycin (21.73%), streptomycin (17.39%), chloramphenicol (8.69%), vancomycin (8.69%), and teicoplanin (4.34%). E. faecium isolates showed a different antibiotic resistance profile: a high percentage were resistant to nitrofurantoin (73.33%), followed by erythromycin (66.60%), ciprofloxacin (66.66%), levofloxacin (60.00%), and rifampicin (26.66%), and only a very low percentage were resistant to tetracycline (6.66%). One isolate was resistant to vancomycin and teicoplanin. The incidence of virulence factors was much higher among E. faecalis isolates, especially for genes encoding for sex pheromones, collagen adhesin, enterococcal endocarditis antigen, and enterococcal surface protein. Isolates with multiple factors (both antibiotic resistance and virulence traits) were also more frequent among E. faecalis isolates, in which one isolate cumulated up to 15 traits. By contrast, several isolates of E. faecium had only very few unwanted traits as compared to only two isolates in E. faecalis. The high abundance of isolates carrying virulence factors and antibiotic resistance traits suggests that the sanitary quality of foods should be improved in order to decrease the incidence of enterococci.

Virulence, phenotype and genotype characteristics of endodontic Enterococcus spp.

Enterococci have been implicated in persistent root canal infections but their role in the infection process remains unclear. This study investigated the virulence, phenotype and genotype of 33 endodontic enterococcal isolates. Phenotypic tests were conducted for antibiotic resistance, clumping response to pheromone, and production of gelatinase, hemolysin and bacteriocin. Genotype analysis involved polymerase chain reaction amplification of virulence determinants encoding aggregation substances asa and asa373, cytolysin activator cylA, gelatinase gelE, gelatinase-negative phenotype ef1841/fsrC, adherence factors esp and ace, and endocarditis antigen efaA. Physical DNA characterization involved pulsed-field gel electrophoresis of genomic DNA, and plasmid analysis. Potential virulence traits expressed included production of gelatinase by Enterococcus faecalis (n=23), and response to pheromones in E. faecalis culture filtrate (n=16). Fourteen strains produced bacteriocin. Five strains were resistant to tetracycline and one to gentamicin, whereas all were susceptible to ampicillin, benzylpenicillin, chloramphenicol, erythromycin, fusidic acid, kanamycin, rifampin, streptomycin and vancomycin. Polymerase chain reaction products encoding efaA, ace, and asa were detected in all isolates; esp was detected in 20 isolates, cylA in six isolates, but asa373 was never detected. The gelatinase gene (gelE) was detected in all isolates of E. faecalis (n=31) but not in Enterococcus faecium (n=2); a 23.9 kb deletion sequence corresponding to the gelatinase-negative phenotype was detected in six of the eight E. faecalis isolates that did not produce gelatinase. Pulsed-field gel electrophoresis and plasmid analyses revealed genetic polymorphism with clonal types evident. Plasmid DNA was detected in 25 strains, with up to four plasmids per strain and a similar (5.1 kb) plasmid occurring in 16 isolates. Phenotypic and genotypic evidence of potential virulence factors were identified in endodontic Enterococcus spp., specifically production of gelatinase and response to pheromones.

Survey for virulence determinants among Enterococcus faecalis isolated from different sources.

A collection of Enterococcus faecalis strains from clinical isolates, healthy individuals and the environment was screened for the presence of virulence factor genes, such as those for collagen-binding protein (ace), endocarditis antigen (efaA), haemolysin activator (cylA), gelatinase (gelE), aggregation substances (asa1 and asa373), a surface protein (esp) and two novel putative surface antigens (EF0591 and EF3314). Apart from some genes that were present in all strains (ace, efaA and EF3314), the gelE gene was the most common factor, although its presence did not correlate with its expression. The genes that encode Esp and CylA were never detected in endocarditis isolates, whereas an association was noted between the esp gene and isolates from urinary tract infection (UTI) and bacteraemia. An aggregation substance gene was always present in commensal strains. As for gelatinase, the presence of the cylA and asa genes did not correlate completely with their phenotypic expression. Generally, isolates from endocarditis, biliary stents and the environment were equipped with fewer virulence factors than isolates from other sources. UTI strains possessed the highest number of factors.

Comparison of the incidence of virulence determinants and antibiotic resistance between Enterococcus faecium strains of dairy, animal and clinical origin

Enterococci are part of the dominant microbiota of several dairy products. They are also present in the gut of humans and animals. Their presence in traditional raw milk cheeses is probably due to faecal contamination of milk during milking. Due to their importance as a cause of nosocomial infections, enterococci are acquiring increased significance. Such infections are becoming more and more difficult to treat as resistance to antibiotics increases. The aim of this investigation was to compare the potential virulence of Enterococcus faecium isolated from different ecological habitats and to establish if strains isolated from dairy products should really be considered as potential pathogens. In the present work, the antibiotic resistance pattern of 40 E. faecium strains isolated from dairy products, 26 E. faecium isolated from ewes' faeces and 28 clinical isolates of the same species was studied, and checks were made to see if known virulence determinants were present. Resistance to 12 different antibiotics commonly used in the treatment of human infections was tested using the broth microdilution method as described by the NCCLS. In addition, polymerase chain reaction (PCR) tests were carried out to see if genes for vancomycin resistance were present. The presence of the aggregation substance (AS) gene, the surface protein gene esp, the accessory colonisation factor ace, the Enterococcus faecalis endocarditis antigen efaA and the gelatinase gelE gene, which are involved in the virulence of enterococci, were also tested by PCR. The results of this study clearly indicate that E. faecium strains isolated from both cheese and sheep faeces are less pathogenic than those isolated from clinical samples. A similar pattern of resistance to antibiotics was observed in both dairy and animal strains. It was also found that there was difference in the kind of virulence determinants present in dairy and clinical isolates, while no virulence traits were found in sheep faeces strains. The results of this study suggest that E. faecium from traditional Sardinian raw milk cheeses should not be considered to be the main source of untreatable nosocomial enterococcal infections in humans in the island of Sardinia.

Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy).

Enterococci are widely distributed in the environment; within the human body, they are normal commensals of the oral cavity, gastrointestinal tract and vagina. In recent years, enterococci have become one of the most frequent causes of acquired nosocomial infections worldwide. The molecular mechanism of virulence of these bacteria is still not completely understood. The aims of this work were to characterize phenotypically 47 isolates of Enterococcus faecalis and Enterococcus faecium collected in Sardinia (Italy) by their abilities to adhere to different epithelial cell lines (Vero and Caco-2 cells) and to associate their phenotypes with the presence of known virulence genes detected within their genomes by PCR. The following genes were amplified: AS (aggregation substance), esp (surface protein gene), ace (accessory colonization factor), efaA (E. faecalis endocarditis antigen) and gelE (gelatinase). The virulence genes were detected in E. faecalis isolates only, with the exception of esp, which was found in both species. The phenotypic and genotypic results were also compared with the susceptibility of isolates to various antibiotics.

Molecular typing of selected Enterococcus faecalis isolates: pilot study using multilocus sequence typing and pulsed-field gel electrophoresis.

The present study compared the recently developed multilocus sequence typing (MLST) approach with a well-established molecular typing technique, pulsed-field gel electrophoresis (PFGE), for subspecies differentiation of Enterococcus faecalis isolates. We sequenced intragenic regions of three E. faecalis antigen-encoding genes (ace, encoding a collagen and laminin adhesin; efaA, encoding an endocarditis antigen; and salA, encoding a cell wall associated antigen) and one housekeeping gene (pyrC) of 22 E. faecalis isolates chosen largely for their temporal and geographical diversity, but also including some outbreak isolates. MLST analysis of polymorphic regions of these four genes identified 13 distinct sequence types (STs) with different allelic profiles; the composite sequences generated from the four sequenced gene fragments of individual isolates showed 98.3 to 100% identity among the 22 isolates. We also found that the allelic profiles from two sequences, ace and salA, were sufficient to distinguish all 13 STs of this study. The 13 STs corresponded to 12 different PFGE types, with one previously designated PFGE clone (a widespread U.S. clone of beta-lactamase-producing isolates) being classified into two highly related STs which differed at 2 of 2,894 bases, both in the same allele. MLST also confirmed the clonal relationships among the isolates of two other PFGE clonal groups, including vancomycin resistant isolates. Thus, this pilot study with representative E. faecalis isolates suggests that, similar to PFGE, the sequence-based typing method may be useful for differentiating isolates of E. faecalis to the subspecies level in addition to identifying outbreak isolates.