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PubMed2006

Detection of antimicrobial activities and bacteriocin structural genes in faecal enterococci of wild animals.

The production of antimicrobial activities as well as the presence of bacteriocin structural genes (entA, entB, entP, entQ, cylL, entAS-48, bac31, and entL50A/B) were studied in 140 non-selected faecal enterococcal isolates recovered from wild animals. Eight different indicator strains (including Listeria monocytogenes, Pediococcus pentosaceus, and different enterococcal species) were used for antimicrobial activity detection. Twenty-five of the 140 enterococci (18%) showed antimicrobial activity against L. monocytogenes and 33 additional isolates (24%) showed antimicrobial activity against other indicator strains, but Listeria. At least one bacteriocin structural gene was detected in 17 of the 25 enterococci with antimicrobial activity against L. monocytogenes and different combinations of entA, entB, entP, entQ, entL50A/B, and cylL genes were detected; entA and entB were the most prevalent detected genes, and they were generally associated. Bacteriocin structural genes were detected in 10 of 33 isolates with antimicrobial activity against indicator strains other than Listeria, and the cylL gene was the most prevalent one, especially in E. faecalis isolates.

Animals↗
PubMed2005

[VCA agar (bioMérieux) for selective isolation of vancomycin-resistant Enterococci (VRE) from fecal specimens].

Screening for Vancomycin Resistant Enterococci (VRE) is recommended for preventing nosocomial infections with VRE. The aim of this study was to assess the performance of VCA3 agar (bioMérieux) in VRE isolation from fecal specimens. 220 specimens were cultured on VCA3 agar, which contains vancomycin and in parallel, on CAP agar (Oxoid), which is vancomycin-free. 36 vancomycin resistant enterococci were isolated: 24 isolates of Enterococcus faecium expressed a high-level resistance to vancomycin and 12 isolates of E. gallinarum/casseliflavus exhibited resistance at low-level. The sensitivity of VCA3 appeared greater than that of CAP for VRE isolation: 92% (22/24) vs 79% (19/24) for E. faecium (NS, P>0.05) ; 83% (10/12) vs 50% (6/12) for E. gallinarum/casselliflavus (NS, P>0.05). As expected, initial cultures of multiple gram positive organisms were far more frequent on CAP agar than on VCA3 agar. The isolation rate of vancomycin susceptible gram positive strains was impressively lower on VCA3 medium than on CAP medium. VCA3 agar avoided therefore additional subcultures, useless identification and susceptibility tests. In conclusion, VCA3 medium could be useful for the direct, rapid and selective isolation of VRE from fecal specimens.

Cross Infection↗
PubMed2006

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food.

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.

Drug Resistance, Bacterial↗
PubMed2004

Selection of enterococci for potential canine probiotic additives.

Enterococci are important inhabitants of animal intestine and are widely used in probiotic products. A potentially successful probiotic strain is expected to have several desirable properties in order to be able to exert its beneficial effects. Forty enterococcal isolates from dog faeces were tested for characters believed to be important for probiotic strains; bacteriocin production, resistance or tolerance to antibiotics, low pH, bile tolerance and adhesive activity. The total count of enterococci was found to be 3.3-7.3log(10)CFU/g of faeces. Most identified strains were Enterococcus faecium. All strains were sensitive to vancomycin, ampicillin, penicillin and chloramphenicol. Thirty-three percentage of strains were resistant to erythromycin and 28% to tetracycline. Among 40 isolates, 75% showed a broad inhibitory spectrum only against Gram-positive indicator bacteria. Seven strains with broad bacteriocin activity were selected for further assays. In the presence of 1% bile, the survival rate of selected strains ranged between 72 and 98%. Survival of strains at pH 3.0 was found in the range between 76 and 87% after 3h. The adhesion of the tested strains to intestinal mucus ranged from 4 to 11% for canine mucus and from 5 to 8% for human mucus. E. faecalis EE4 and E. faecium EF01 showed the best probiotic properties. It indicates that they could be used as new candidate probiotic strains after in vivo testing.

Animals↗
PubMed1997

Controlling the spread of vancomycin-resistant enterococci with a rehabilitation cohort unit.

Enterococci are common to the human gastrointestinal tract. Recently there has been an emergence of vancomycin-resistant enterococci (VRE); infection requires strict contact isolation. Patients with VRE infections are at higher risk for morbidity and mortality. As a result of the high prevalence of VRE, it was recommended that a cohort unit be established to control its spread within our metropolitan community hospital. We report the development of a rehabilitation VRE cohort unit. We present case studies of five patients who developed nosocomial colonization and one with an infection with VRE; all were treated on the rehabilitation cohort unit. Protocols for VRE isolation and procedures for decontamination in the cohort unit were developed. If a cohort unit is necessary, it is feasible to conduct a rehabilitation program in a cohort unit with strict adherence to contact isolation.

Adult↗
PubMed2002

Characterization of sewage waters by biochemical fingerprinting of Enterococci.

Three sewage waters with different sources of faecal pollution (human, pig and mixed origin) were analysed. Enumeration of Enterococci was performed and representative strains for each sample were isolated. Biochemical fingerprints of these strains were obtained by using a simple automated microplate system. The structure and composition of the Enterococci populations were studied and the most representative biochemical profiles were determined by cluster analysis. The diversity index was high for all the sewage waters sampled. The enterococcal populations detected in the three kinds of sewage waters were similar. Although many of the representative strains were found in the three kinds of samples, some strains were detected only in human sewage and others only in pig slurry.

Animals↗
PubMed1999

Reverse transcription polymerase chain reaction method for the detection of glycopeptide resistance in enterococci.

In this work we have developed reverse transcription polymerase chain reaction (RT-PCR) methods for detecting specific mRNA from enterococci, particularly vanA and vanB genes, responsible for glycopeptide resistance in this genus. mRNA from the two genes was detected immediately after RNA extraction of a midlog phase culture, determined by growth rate analysis. Because of the short half-life associated with many bacterial RNA species (1.5-2 min), time is an important factor in obtaining RNA of good yield and high purity. Our results showed that: (i) the transcription of mRNA related to vanA ligase in enterococci showing Van A phenotype happens only after induction with both vancomycin and teicoplanin; (ii) the transcription of mRNA related to vanB ligase happens only in the presence of vancomycin and (iii) there was no transcription of mRNA in the two strains positive to vanA gene after PCR experiments. RT-PCR methodology can have numerous applications in microbiology for studying gene expression in isolated bacteria and also in nonculturable cells in environmental samples, for studies of mechanisms and/or as an indicator of viability in bacterial communities.

Anti-Bacterial Agents↗
PubMed2002

Vancomycin-resistant enterococci in shellfish, unchlorinated waters, and chicken.

Vancomycin-resistant enterococci (VRE) have been a cause of increasing concern chiefly regarding the infection of hospital patients. There is suspicion, but limited evidence, that food and environmental spread may be important. Biomonitoring by examination of bivalve shellfish was used to assess the occurrence of VRE entering the environment. Using pre-enrichment and Lewisham and Slanetz and Bartley agars, 2/125 (1.6%) of shellfish were found to contain enterococci resistant to high levels of vancomycin. Lewisham agar allows relatively rapid identification of VRE. In a second phase of the work using pre-enrichment and Slanetz and Bartley agar, 4/151 (2.7%) shellfish and 5/27 (18.5%) raw chickens contained VRE. Using filtration and pre-enrichment, no VRE were found in 54 unchlorinated water samples. The study shows that environmental prevalence of VRE is low, and that raw chickens are frequently contaminated.

Agar↗
PubMed2003

Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins.

The inhibitory activity of 122 out of 426 Enterococcus strains of geographically widespread origin and from different sources (food and feed, animal isolates, clinical and nonclinical human isolates) was tested against a wide range of indicator bacteria. Seventy-two strains, mainly belonging to the species Enterococcus faecium and Enterococcus faecalis were bacteriocinogenic. A remarkable variation of inhibitory spectra occurred among the strains tested, including inhibition of, for instance, only closely related enterococci, other lactic acid bacteria (LAB), food spoilage and pathogenic bacteria. No correlation could be found between the origin of the strains and the type of inhibitory spectrum, although a clustering of human isolates from both fecal and clinical origin was observed in the group of strains inhibiting lactic acid bacteria, Listeria, and either Staphylococcus or Clostridium. No relationship could be established between the presence of enterocin structural genes and the origin of the strain either, and hence no correlation seemed to exist between the presence of known enterocin genes and the activity spectra of these enterococci. The structural gene of enterocin A was widely distributed among E. faecium strains, whereas that of enterocin B only occurred in the presence of enterocin A. The vancomycin resistance phenotype as well as the presence of vancomycin resistance genes was also investigated. The vanA gene only occurred among E. faecium strains. The incidence of beta-hemolysis was not restricted to E. faecalis strains, but among the E. faecium strains the structural genes of cytolysin were not detected. beta-Hemolysis occurred in strains both from food and nonfood origin. It has been concluded that bacteriocin-producing E. faecium strains lacking hemolytic activity and not carrying cytolysin nor vancomycin resistance genes may be useful as starter cultures, cocultures, or probiotics.

Animals↗
PubMed2003

The potential of vancomycin-resistant enterococci to persist in fermented and pasteurised meat products.

Experiments with 148 isolates of vancomycin-resistant enterococci (VRE) were performed to assess their potential to persist and grow in fermented sausages and pasteurised meat products. All strains were meat isolates and Van-type A, except a single VanC1 strain. In total, 143 strains of Enterococcus faecium were involved. Eight selected strains were examined for their potential to grow at high salt and nitrite levels and at reduced pH. The same isolates were used in experiments with fermented sausages. All available strains were subjected to heating tests in meat suspensions with added curing ingredients. All but one of the eight tested isolates grew at pH 4.0 in tryptone soya broth (TSB). With the combination of 8% w/w NaCl, 400 ppm NaNO2 and 0.5% w/w glucose in the meat suspension, all isolates grew at 37 degrees C, whereas none grew at 7 degrees C even after 56 days. With the addition of 10% w/w NaCl, 200 ppm NaNO2 and 0.5% w/w glucose, still one E. faecium isolate grew at 37 degrees C, although very slowly. Overall, the strains tolerated high salt and nitrite concentrations and reduced pH very well, even beyond levels applied in the regular production of fermented and/or pasteurised meat products. The tested strains could be isolated after the fermentation and further ripening of "boerenmetworst" and "snijworst". Overall, their colony counts decreased on average about 1 log-unit over a period of 60 days after batter manufacture. All 148 isolates demonstrated a relatively weak thermal resistance compared to results for selected vancomycin-sensitive enterococci strains reported in the literature and to results collected under identical experimental conditions in this laboratory. None of the strains (log inoculation level about 5-6 ml(-1) for each isolate) could be cultured after heating at 70 degrees C for 10 min.

Animals↗
PubMed2000

Vancomycin-resistant enterococci isolated from animals and food.

One hundred and one chicken products, boiled ham and turkey cold meat were acquired from 18 different supermarkets in Spain during October 1997 to June 1998 and were analyzed for vancomycin-resistant enterococci (VRE). In the same way, 50 intestinal chicken samples from a slaughterhouse were also studied. VRE were detected in 25 of 92 samples of food of chicken origin (27.2%), but no VRE were found in cooked pork or turkey products. VRE were also detected in 8 of 50 intestinal chicken samples from the slaughterhouse (16%). VRE were identified as Enterococcus durans (n = 11), Enterococcus faecalis (n = 10), Enterococcus faecium (n = 10) and Enterococcus hirae (n = 2). All these strains were characterized as belonging to the vanA genotype by polymerase chain reaction. Ampicillin, quinupristin/dalfopristin and high level aminoglycoside resistance were frequently found among these strains. Heterogeneity was observed in susceptibility patterns among VRE strains, even in those of the same species. The high rate of colonization of chicken products by vanA containing enterococci detected 6 months to 1 year after the banning of avoparcin as a growth promoter, supports other studies suggesting that the food chain could be a source of VRE colonization in humans and thus a source of VRE infections.

Animals↗
PubMed1995

Nosocomial enterococci: resistance to heat and sodium hypochlorite.

Six strains each of Enterococcus faecium and E. faecalis were investigated with respect to their resistance to heat and sodium hypochlorite. All enterococci survived the temperatures and holding times specified by the Department of Health (DoH) for the disinfection of 'foul and used' or 'infected' linen (65 degrees C for 10 min or 71 degrees C for 3 min). In addition, three strains (one E. faecium and two E. faecalis) could withstand 150 ppm available chlorine for 5 min, the treatment suggested by the DoH for the disinfection of heat labile materials. Further, our results showed that four strains of E. faecium were able to survive the British Standard for heat disinfection of bedpans (80 degrees C for 1 min). The significance of these findings with particular reference to the potential for enterococci to survive and disseminate in the hospital environment is discussed.

Cross Infection↗
PubMed1996

Heat and chemical resistance of enterococci.

Recent reports have highlighted the tolerance of vancomycin-resistant strains of enterococci to heat. This study examined the tolerance of vancomycin-resistant and sensitive strains of enterococci and an NCTC type strain to 65, 71 and 80 degrees C, and also to low concentrations of a chlorine-releasing agent, alcohol and glutaraldehyde. Variation in the tolerance to chemicals was observed but there was no correlation between vancomycin resistance and tolerance to chemical disinfectants. The NCTC type strain was killed within the time/temperature parameters set by the Department of Health for thermal washer/disinfectors, i.e. 65 degrees C for 10 min, 71 degrees C for 3 min and 80 degrees C for 1 min. However, the clinical strains showed varying resistance to heat, irrespective of their vancomycin susceptibility. One strain survived 80 degrees C for 3 min. These results showed that clinical isolates can be resistant to commonly used disinfection processes, although the practical significance of these results is debatable.

Alcohols↗
PubMed1997

Epidemiology of vancomycin-resistant enterococci in the community and the relevance of farm animals to human infection.

Several reports have documented the presence of vancomycin-resistant enterococci (VRE) in the stools of asymptomatic individuals from the community who have neither recently been in hospital nor received antibiotics. Such findings were contrary to the then existing perception of VRE as a strictly hospital-acquired infection of debilitated and immunocompromised patients on specialized units. Community-acquired infections with VRE are extremely rare but those that do occur may be conspicuous because of their serious nature, for example, endocarditis. If asymptomatic faecal carriage of VRE is present in the community, individuals admitted to hospital and subjected to the selective pressures of antibiotics on the normal gut flora, may act as the source of hospital outbreaks. VRE have also been found in sewage, from stools of healthy farm animals and animal products. Avoparcin, a glycopeptide showing cross-resistance to medically important glycopeptides, has been used in the European Community as a growth promoter in animal feeds. A possible link between the use of avoparcin, the selection of VRE, and humans becoming colonized via the food chain exists. To prove such a link is beset with many difficulties: it is necessary to explain the presence of VRE in the United States where avoparcin is not used, and the predominance of the VanA gene over the VanB gene. It is also proving difficult to show that animal and human strains are identical by means of molecular typing. To date, molecular typing of strains is only suggestive of a link, but epidemiological studies of farms that use avoparcin have shown a significant association with the presence of VRE in animal stools. As long ago as 1969, the Swann report declared that an antibiotic of medical importance should not be used as a growth promoter in animal feeds. The vasy array of antibiotics now being used in animal husbandry and fish-farming, and the cross-resistance of some antibiotics to their medically important counterparts is a real cause for concern. The emergence of multi-resistant enterococci causing human infections and the possibility of the transfer of the VanA gene from VRE to methicillin-resistant Staphylococcus aureus (MRSA) emphasizes the importance of this problem.

Animal Feed↗
PubMed1998

Comparison of three methods for the molecular typing of Singapore isolates of enterococci with high-level aminoglycoside resistances.

Enterococci are frequently isolated as nosocomial pathogens and have often acquired intrinsic drug resistances. Molecular typing techniques have been developed to assist in epidemological and infection control measures. This study investigates enterococci with high-level aminoglycoside resistance (HLAR) from the National University Hospital (NUH) of Singapore, and evaluates and compares three methods for typing: restriction enzyme analysis by conventional gel electrophoresis [restriction fragment length polymorphism (RFLP)], pulsed-field gel electrophoresis (PFGE), and polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD). Fifty-two isolates of Enterococcus faecalis and 13 isolates of Enterococcus faecium were used for the study. The numbers of patterns obtained for E. faecalis and E. faecium were 26 and 4, respectively by the RFLP method, and very similar discrimination was obtained by PFGE. RAPD PCR results were not reliably reproducible. A single pattern type by RFLP accounted for 16 of the E. faecalis isolates, suggesting hospital spread.

Aminoglycosides↗
PubMed1999

Molecular epidemiology of vancomycin-resistant enterococci from 6 hospitals in New York State.

BACKGROUND: Vancomycin resistance among enterococci is an emerging nosocomial problem. Consequently, it is important to understand the distribution of vancomycin-resistant enterococci (VRE) within and between hospitals to implement appropriate infection control measures. METHODS: In this study, 116 VRE isolates obtained from patients in 6 New York State hospitals were analyzed by antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE) fingerprinting, plasmid profile analysis, vanA and vanB polymerase chain reaction, and DNA:DNA hybridization with vanA and vanB probes. RESULTS: PFGE and plasmid typing generally agreed, but plasmid profiles were more variable. These analyses revealed that genetic heterogeneity among isolates from within each of the 6 hospitals varied considerably. Among 23 Enterococcus faecium isolates from one hospital, there were only 3 PFGE types, and 20 isolates had the same type. However, in another hospital, each isolate was genetically distinct. Closely related strains were not found in separate hospitals. VRE strains with vanA genes and strains with vanB genes were found in 3 hospitals. Both plasmid and chromosomal carriage of these genes was detected. CONCLUSIONS: PFGE typing showed that nosocomial VRE transmission had occurred in some hospitals. However, there was no evidence for it in others. Neither was there evidence for intrahospital transmission or for emergence of an endemic strain. These observations demonstrate that it is important to evaluate genetic heterogeneity among VRE before implementation of infection control measures. PFGE is the method of choice for epidemiologic typing, but polymerase chain reaction, plasmid, and hybridization studies can provide important information concerning the presence and potential for transfer of vancomycin resistance genes.

DNA Probes↗
PubMed1998

Application of 23S rDNA-targeted oligonucleotide probes specific for enterococci to water hygiene control.

Identification of enterococci species by hybridization with recently designed species-specific and group-specific 23S rDNA-targeted oligonucleotide probes was superior to results obtained with a common biochemical test panel. Considering these findings, a molecular biological procedure for the detection of enterococci in water samples was developed. A short enrichment is followed by an amplification step and a hybridization reaction in microtiter plate format. The detection limit is about 1 CFU/ml, and results are available within 26 h.

Bacterial Typing Techniques↗
PubMed1999

Determination of the nucleotide sequence of the 23S ribosomal RNA and flanking spacers of an Enterococcus faecium strain, reveals insertion-deletion events in the ribosomal spacer 1 of enterococci.

The usefulness of 16S-23S (ITS1) and 23S-5S (ITS2) ribosomal spacer nucleotide sequence determination, as a complementary approach to the biochemical tests traditionally used for enterococcal species identification, is shown by its application to the identification of a strain, E27, isolated from a natural bacteria mixture used for cheese production. Using combined approaches we showed, unambiguously, that strain E27 belongs to the Enterococcus faecium species. However, its ITS1 region has an interesting peculiarity. In our previous study of ITS1s from various enterococcal species (NAIMI et al., 1997, Microbiology 143, 823-834), the ITS1s of the two E. faecium strains studied, were found to contain an additional 115-nt long stem-loop structure as compared to the ITS1s of other enterococci, only one out of the 3 ITS1s of E. hirae ATCC 9790, was found to contain a similar 107-nt long stem-loop structure. The ITS1 of strain E27 is 100% identical to that of E. faecium ATCC 19434T, except that the 115-nt additional fragment is absent. This strongly suggests the existence of lateral DNA transfer or DNA recombination events at a hot spot position of the ITS1s from E. faecium and E. hirae. Small and large ITS1 nucleotide sequence determination for strain E27 generalized the notion of two kinds of ITSs in enterococci: one with a tRNA(Ala) gene, one without tRNA gene. To complete strain E27 characterization, its 23S rRNA sequence was established. This is the first complete 23S rRNA nucleotide sequence determined for an enterococcal species.

Base Sequence↗