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PubMed2000

Characterization of transposon Tn1549, conferring VanB-type resistance in Enterococcus spp.

Transfer of VanB-type resistance to glycopeptides among enterococci has been reported to be associated with the movement of large chromosomal genetic elements or of plasmids. The authors report the characterization of the 34 kb transposon Tn1549 borne by a plasmid related to pAD1 and conferring vancomycin resistance in clinical isolates of Enterococcus spp. Tn1549 contained 30 ORFs and appeared to be organized like the Tn916 family of conjugative transposons into three functional regions: (i) the right end, implicated in the excision-integration process; (ii) the central part, in which the vanB2 operon replaces the tet(M) gene; and (iii) the left extremity, in which eight of the 18 ORFs could be implicated in the conjugative transfer.

Bacterial Proteins↗
PubMed2003

Application of the fluorogenic probe technique (TaqMan PCR) to the detection of Enterococcus spp. and Escherichia coli in water samples.

A recent PCR detection technique (TaqMan) based on the 5'-3'-exonuclease activity of the Taq DNA polymerase was applied to the detection of indicator organisms in water samples. In this technique, an increasing fluorescence signal is measured online which enables direct assessment of results after PCR without additional detection steps. The test is completed within about 5 h. Two sets of primers and probes were designed and tested: a genus-specific assay for the detection of Enterococcus spp. based on 23S rRNA sequence and an Escherichia coli-specific assay based on the uidA gene sequence. Specificity of the assays was confirmed by testing strains of target bacteria and potential interfering microorganisms. Application of the tests to 55 natural water samples showed the need of an overnight enrichment step to achieve compliance with detection limits of existing regulations. Compared with a parallel microbiological examination of the samples, agreement was 96% with the Enterococcus assay and 98% with the E. coli assay. The rapidity and feasibility of the method point to benefits in drinking water analysis, particularly in emergency situations and, thus, to improved public health management.

Enterococcus↗
PubMed2005

Virulence, phenotype and genotype characteristics of endodontic Enterococcus spp.

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Phenotypic and genotypic evidence of potential virulence factors were identified in endodontic Enterococcus spp., specifically production of gelatinase and response to pheromones.

Adult↗
PubMed1999

Recovery of Escherichia coli Biotype I and Enterococcus spp. during refrigerated storage of beef carcasses inoculated with a fecal slurry.

Three beef front quarters/carcasses were inoculated with a slurry of cattle manure. During storage at 4 degrees C, two sponge samples from each of three sites (i.e., 100 cm2 from each of two fat surfaces and 100 cm2 from a lean surface) were taken from each of the three carcasses on days 0, 1, 3, 7, and 10 after inoculation. The initial numbers of Escherichia coli averaged 2.0 log10 CFU/cm2 (1.21 to 2.47 log10 CFU/cm2) using the Petrifilm method and 2.09 log10 most probable number (MPN)/cm2 (0.88 to 2.96 log10 MPN/cm2) using the MPN method. The initial numbers of enterococci averaged 3.34 log10 CFU/cm2 (3.07 to 3.79 log10 CFU/cm2) using kanamycin esculin azide agar. In general, an appreciable reduction in the numbers of E. coli occurred during the first 24 h of storage; for the Petrifilm method an average reduction of 1.37 log10 CFU/cm2 (0.69 to 1.71 log10 CFU/cm2) was observed, and for the MPN method an average reduction of 1.52 log10 MPN/cm2 (0.47 to 2.08 log10 MPN/cm2) was observed. E. coli were not detected (<-0.12 log10 CFU/cm2) using Petrifilm on day 7 of the storage period on two (initial counts of 1.21 and 2.29 log10 CFU/cm2) of the three carcasses. However, viable E. coli cells were recovered from these two carcasses after a 24-h enrichment at 37 degrees C in EC broth. Viable E. coli cells were detected at levels of -0.10 log10 CFU/cm2 on the third carcass (initial count of 2.47 log10 CFU/cm2) after 7 days at 4 degrees C. No significant difference in recovery of viable cells was observed between the MPN and Petrifilm methods on days 0, 1, and 3 (P > 0.05). However, viable E. coli cells were recovered from all three carcasses by the MPN method on day 7 at an average of -0.29 log10 MPN/ cm2 (-0.6 to -0.1 log10 MPN/cm2). On day 10, viable cells were recovered by the MPN method from two of the three carcasses at -0.63 and -0.48 log10 MPN/cm2 but were not recovered from the remaining carcass (<-0.8 log10 MPN/cm2). Similar to E. coli, the greatest reduction (average of 1.26 log10 CFU/cm2, range = 1.06 to 1.45 log10 CFU/cm2) in the numbers of enterococci occurred during the first 24 h of storage. Because of higher initial numbers and a slightly slower rate of decrease, the numbers of Enterococcus spp. were significantly higher (P < 0.017) than the numbers of E. coli Biotype I after 3, 7, and 10 days of storage. These results suggest that enterococci may be useful as an indicator of fecal contamination of beef carcasses.

Abattoirs↗
PubMed2000

Macrolide resistance genes in Enterococcus spp.

Seventy-eight isolates of different Enterococcus species (E. faecalis, n = 27; E. faecium, n = 23; E. durans, n = 8; E. avium, n = 6; E. hirae, n = 9; E. gallinarum, n = 3; and E. casseliflavus, n = 2) with a variety of erythromycin resistance phenotypes were examined for the presence of macrolide resistance genes (ermA, ermB, ermC, ermTR, mefA/E, and msrA). Positive PCR amplifications of ermB were obtained for 39 of 40 highly erythromycin-resistant Enterococcus isolates (MICs, >128 microg/ml) of different species; the remaining highly resistant E. faecium isolate was positive for PCR amplification of ermA but was negative for PCR amplification of the ermB and ermC genes. For all enterococcal strains for which erythromycin MICs were < or =32 microg/ml PCRs were negative for erm methylase genes. For all E. faecium isolates PCR amplified products of the expected size of 400 bp were obtained when msrA primers were used, with the results being independent of the erythromycin resistance phenotype. All the other enterococcal species gave negative results by msrA PCRs. Sequencing of the msrA PCR products from either erythromycin-susceptible, low-level-resistant, or highly resistant E. faecium strains showed that the amplicons did not correspond to the msrA gene described for Staphylococcus epidermidis but corresponded to a new putative efflux determinant, which showed 62% identity with the msrA gene at the DNA level and 72% similarity at the amino acid level. This new gene was named msrC.

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

Flow cytometric determination of Enterococcus spp. susceptibility to four antimicrobial agents.

Two Enterococcus strains (E. faecalis and E. faecium) isolated from 2 patients in an intensive care unit (blood and drain, respectively) were analyzed for susceptibility to 4 antibiotics (penicillin, vancomycin, gentamicin, streptomycin) by agar dilution standard method (MICs), time-kill and flow cytometry. We compared the data from classical methods of antibiotic susceptibility detection, that are compulsory 24 hrs long and flow cytometry results at 5 and 24 hrs cultivation. The results from both classical and flow cytometric analyses were highly cogent and revealed the fact that flow cytometry is very useful in early diagnosis of bacterial resistance to antibiotics.

Drug Resistance, Microbial↗
PubMed2000

Aminoglycoside-modifying enzymes in high-level streptomycin and gentamicin resistant Enterococcus spp. in Spain.

Aminoglycoside resistance was evaluated in 690 enterococcus strains isolated from different clinical sources originating from patients at the University Clinic Hospital of Zaragoza (Spain). The enterococci obtained from clinically significant samples (blood, urine, or exudates) showed more high-level resistance to gentamicin and streptomycin (65 and 42%, respectively) than those isolated from faecal samples (49 and 23%, respectively). Aminoglycoside-modifying enzymes (AME) from 119 of these high-level gentamicin and streptomycin resistant enterococcus strains were studied. The most frequent AMEs found were APH(3') and AAC(6')-APH(2"). More than one enzyme was detected in 71% of the strains (four different enzymes in 5% of the strains). Three Enterococcus faecalis strains had ANT(4')(4") enzymatic activity. Different enzymatic expressions of the bifunctional enzyme AAC(6')-APH(2") were demonstrated in strains in which the complete aac(6')-aph(2") gene was detected by PCR and hybridization: (i) AAC(6') + APH(2") activity; (ii) AAC(6') only; (iii) APH(2") only; and (iv) no activity of AAC(6') or APH(2").

Anti-Bacterial Agents↗
PubMed1994

vanA-mediated vancomycin-resistant Enterococcus spp. in sewage.

Three high-level vancomycin-resistant Enterococcus strains (two Enterococcus faecium and one Enterococcus durans) were recovered from three of eight sewage samples taken from the general sewage collector at Logroño (Northern Spain). The strains were present in the sewage samples at estimated concentrations of ten resistant bacteria/mL, corresponding to about 0.4% of the enterococcal population. The VanA protein was detected in each strain by immunoblotting of membrane extracts of the vancomycin-induced cells, and the vanA gene was demonstrated in the wild strains and their transconjugants by DNA-DNA hybridization. This is the first, confirmed report of vanA mediated vancomycin resistance in E. durans.

Bacterial Proteins↗
PubMed1995

Resistance to aminoglycosides and other antibiotics among clinical isolates of Enterococcus spp.

We conducted a study on 81 clinical isolates of enterococci (65 Enterococcus faecalis and 16 Enterococcus faecium) collected consecutively over a one year period at our University Hospital. Of them, 10 (15%) Enterococcus faecalis and 6 (38%) Enterococcus faecium showed high level resistance to aminoglycosides, were all acquired in surgical units and in five cases they were responsible for invasive infections. Ampicillin and imipenem were active only against Enterococcus faecalis. Vancomycin and teicoplanin were bacteriostatic; however, teicoplanin MICs were lower and in 45% of cases its MBC values were within clinically achievable serum concentrations. Ciprofloxacin and cotrimoxazole were active in vitro.

Aminoglycosides↗
PubMed2003

Heat resistance in liquids of Enterococcus spp., Listeria spp., Escherichia coli, Yersinia enterocolitica, Salmonella spp. and Campylobacter spp.

The aim of the work was to collect, evaluate, summarize and compare heat resistance data reported for Campylobacter, Enterococcus, Escherichia, Listeria, Salmonella and Yersinia spp. The work was limited to resistance in liquids with pH values 6-8. Results obtained under similar experimental conditions were sought. Thermal destruction lines for the various bacterial groups studied were constructed using log10 D values and treatment temperatures. There was a good linear relationship between log10 D and temperature with Escherichia coli, listerias and salmonellas. For campylobacters, enterococci and yersinias the relationships were weaker but, nevertheless, present. Using the slopes of the lines and their 95% confidence limits, z values and their 95% confidence limits were calculated. z values were compared with z values obtained from reports. The equations for the lines were also used for calculation of predicted means of D values at various treatment temperatures. 95% confidence limits on predicted means of D values and on predicted individual D values were also calculated. Lines and values are shown in figures and tables. Differences in heat resistance noted between and within the bacterial groups studied are discussed.

Bacteria↗
PubMed1993

Detection of ampicillin resistance in Enterococcus spp. by disk diffusion and two commercial automated systems.

A total of 154 Enterococcus faecium and 48 Enterococcus faecalis isolates, 55% of which were ampicillin-resistant, were used to evaluate two commonly used automated systems, Vitek's GPS-TA card (bioMérieux, USA) and MicroScan's POS MIC Type 6 panel (Baxter Health-care, USA), and the NCCLS disk diffusion method for the detection of ampicillin resistance. MicroScan panels were read by the Walk/Away system and by visual inspection. The results were as follows: disk diffusion, 100% sensitive and specific; Vitek, 100% sensitive and 95% specific; and MicroScan, when read by the Walk/Away, 85% sensitive and 98% specific. When MicroScan was interpreted by visual inspection, however, sensitivity increased to 96%.

Ampicillin Resistance↗
PubMed1988

Laboratory detection of high-level aminoglycoside-aminocyclitol resistance in Enterococcus spp.

Methods for detection of high-level resistance to aminoglycoside-aminocyclitol antibiotics were evaluated using 104 blood isolates of enterococci (97 Enterococcus faecalis and 7 Enterococcus faecium). Kanamycin was used to predict resistance to amikacin. Discrepancies between methods were resolved by time-kill studies. Four methods (MicroScan, macrotube, microtiter, and disk diffusion) for detecting resistance to gentamicin and streptomycin were compared, using 51 consecutive strains. There were 13 gentamicin-resistant strains, all of which were detected by macrotube, microtiter, and disk diffusion. MicroScan detected 2 (15%) of the 13. Of the 18 streptomycin-resistant strains, 17 (93%) were detected by disk diffusion, 16 (89%) by microtiter, 9 (50%) by macrotube, and 6 (33%) by MicroScan. An additional 53 consecutive strains were examined only by disk diffusion and microtiter for resistance to gentamicin, streptomycin, and kanamycin. The entire population of 104 strains contained 35 gentamicin-, 22 streptomycin-, and 54 kanamycin-resistant enterococcal isolates. All 35 gentamicin-resistant strains were detected by both methods. Of the 22 streptomycin-resistant strains, 1 was detected only by microtiter, 2 only by disk diffusion, and 19 by both methods. Of the 54 kanamycin-resistant strains, 1 was detected only by microtiter, 2 only by disk diffusion, and 51 by both methods. One additional strain which was resistant only by disk diffusion was susceptible to amikacin plus penicillin by time-kill studies. Disk diffusion is a suitable method for detection of high-level aminoglycoside-aminocyclitol resistance in E. faecalis and is well suited for sporadic testing. Additional data are necessary to determine the suitability of these tests for E. faecium.

Anti-Bacterial Agents↗
PubMed2000

Isolation and characterization of Carnobacterium, Lactococcus, and Enterococcus spp. from cooked, modified atmosphere packaged, refrigerated, poultry meat.

The microbiota of commercially produced, cooked and modified atmosphere packaged poultry meat was followed during storage at 3.5 degrees C for up to 7 weeks. The dominant microbiota consisted of Lactococcus raffinolactis (117 isolates), Carnobacterium divergens (61 isolates), Carnobacterium piscicola (11 isolates), Lactococcus garvieae (four isolates), Lactococcus lactis (one isolate) and Enterococcus faecalis (three isolates). All isolates were screened for production of bacteriocins. Only C. piscicola isolates produced an inhibitory substance active against other lactic acid bacteria and against several Listeria spp. Species-specific polymerase chain reaction (PCR) primers were used for the differentiation of Carnobacterium, L. raffinolactis, L. lactis, and L. garvieae strains associated with the modified atmosphere packaged poultry products. No false PCR products were observed with other closely related bacterial species.

Animals↗