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PubMed1998

Bile-esculin test for presumptive identification of enterococci and streptococci: effects of bile concentration, inoculation technique, and incubation time.

The bile-esculin test is used to differentiate enterococci and group D streptococci from non-group D viridans group streptococci. The effects on test performance of the concentration of bile salts, inoculum, and duration of incubation were examined with 110 strains of enterococci, 30 strains of Streptococcus bovis, and 110 strains of non-group D viridans group streptococci. Optimal sensitivity (> 99%) and specificity (97%) of the bile-esculin test can be obtained with a bile concentration of 40%, a standardized inoculum of 10(6) CFU, and incubation for 24 h.

Bile↗
PubMed1998

Effects of different test conditions on MICs of food animal growth-promoting antibacterial agents for enterococci.

The influence of the addition of sheep blood to Mueller-Hinton II agar and the effects of aerobic incubation with or without CO2 and of anaerobic incubation were tested with bacitracin, tylosin, avoparcin, virginiamycin, avilamycin, narasin, and flavomycin on enterococci. The antibacterial activity of bambermycin (Flavomycin) was strongly inhibited by the addition of blood, except with the species Enterococcus faecium, Enterococcus mundtii, Enterococcus hirae, Enterococcus casseliflavus, and Enterococcus gallinarum, which were not susceptible to this antibiotic on blood-free medium. With all other antimicrobials except avoparcin and tylosin, the presence of blood resulted in MIC increases of 1 to 3 log2 differences. Incubation in aerobic or anaerobic atmospheres enriched with CO2 lowered the susceptibility of enterococci to tylosin and increased their susceptibility to avilamycin, narasin, and avoparcin. This effect was most pronounced in tests on blood-free media. Results of susceptibility tests incubated under anaerobiosis and in a CO2-enriched atmosphere did not differ. For all enterococcal species, the preferred conditions for testing the susceptibility are Mueller-Hinton II medium supplemented with blood and incubation in a CO2-enriched atmosphere. However, when only E. faecium and Enterococcus faecalis are being tested, Mueller-Hinton II medium without blood incubated aerobically gives satisfactory results.

Aerobiosis↗
PubMed1998

Detection and differentiation of vanC-1, vanC-2, and vanC-3 glycopeptide resistance genes in enterococci.

The VanC phenotype, as found in Enterococcus gallinarum, E. casseliflavus, and E. flavescens, is characterized by intrinsic low-level resistance to vancomycin. The nucleotide sequences of the vanC-1 gene in E. gallinarum, the vanC-2 gene in E. casseliflavus, and the vanC-3 gene in E. flavescens have been reported, although there is some disagreement as to whether E. flavescens is a legitimate enterococcal species. Previous attempts to differentiate the vanC-2 and vanC-3 genes by PCR analysis have been unsuccessful. The purpose of the present study was to detect and differentiate the three vanC determinants and examine the distribution of these genes in a collection of both typical and atypical enterococci. The 796-bp vanC-1 PCR product was amplified only from E. gallinarum isolates. As expected, due to the extensive homology in the vanC-2 and vanC-3 gene sequences, all of the E. casseliflavus and E. casseliflavus/flavescens isolates produced the 484-bp vanC-2 PCR product, although the E. gallinarum isolates were negative. Only the E. casseliflavus/flavescens isolates produced the 224-bp vanC-3 product. Using the three sets of primers, we were able to detect and distinguish the vanC-1, vanC-2, and vanC-3 genes from both typical and atypical enterococci strains. Antimicrobial susceptibility tests and analysis of genomic DNA by pulsed-field gel electrophoresis were also performed, but the results indicated that they were not able to distinguish among strains possessing the three vanC genotypes.

Anti-Bacterial Agents↗
PubMed2000

Survival of enterococci and staphylococci on hospital fabrics and plastic.

The transfer of gram-positive bacteria, particularly multiresistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), among patients is a growing concern. One critical aspect of bacterial transfer is the ability of the microorganism to survive on various common hospital surfaces. The purpose of this study was to determine the survival of 22 gram-positive bacteria (vancomycin-sensitive and -resistant enterococci and methicillin-sensitive and -resistant staphylococci) on five common hospital materials: smooth 100% cotton (clothing), 100% cotton terry (towels), 60% cotton-40% polyester blend (scrub suits and lab coats), 100% polyester (privacy drapes), and 100% polypropylene plastic (splash aprons). Swatches were inoculated with 10(4) to 10(5) CFU of a microorganism, assayed daily by placing the swatches in nutritive media, and examining for growth after 48 h. All isolates survived for at least 1 day, and some survived for more than 90 days on the various materials. Smaller inocula (10(2)) survived for shorter times but still generally for days. Antibiotic sensitivity had no consistent effect on survival. The long survival of these bacteria, including MRSA and VRE, on commonly used hospital fabrics, such as scrub suits, lab coats, and hospital privacy drapes, underscores the need for meticulous contact control procedures and careful disinfection to limit the spread of these bacteria.

Anti-Bacterial Agents↗
PubMed2000

Evaluation of a new system, VITEK 2, for identification and antimicrobial susceptibility testing of enterococci.

We evaluated the new automated VITEK 2 system (bioMérieux) for the identification and antimicrobial susceptibility testing of enterococci. The results obtained with the VITEK 2 system were compared to those obtained by reference methods: standard identification by the scheme of Facklam and Sahm [R. R. Facklam and D. F. Sahm, p. 308-314, in P. R. Murray et al., ed., Manual of Clinical Microbiology, 6th ed., 1995] and with the API 20 STREP system and, for antimicrobial susceptibility testing, broth microdilution and agar dilution methods by the procedures of the National Committee for Clinical Laboratory Standards. The presence of vanA and vanB genes was determined by PCR. A total of 150 clinical isolates were studied, corresponding to 60 Enterococcus faecalis, 55 Enterococcus faecium, 26 Enterococcus gallinarum, 5 Enterococcus avium, 2 Enterococcus durans, and 2 Enterococcus raffinosus isolates. Among those isolates, 131 (87%) were correctly identified to the species level with the VITEK 2 system. Approximately half of the misidentifications were for E. faecium with low-level resistance to vancomycin, identified as E. gallinarum or E. casseliflavus; however, a motility test solved the discrepancies and increased the agreement to 94%. Among the strains studied, 66% were vancomycin resistant (57 VanA, 16 VanB, and 26 VanC strains), 23% were ampicillin resistant (MICs, >/=16 microgram/ml), 31% were high-level gentamicin resistant, and 45% were high-level streptomycin resistant. Percentages of agreement for susceptibility and resistance to ampicillin, vancomycin, and teicoplanin and for high-level gentamicin resistance and high-level streptomycin resistance were 93, 95, 97, 97, and 96%, respectively. The accuracy of identification and antimicrobial susceptibility testing of enterococci with the VITEK 2 system, together with the significant reduction in handling time, will have a positive impact on the work flow of the clinical microbiology laboratory.

Bacterial Typing Techniques↗
PubMed2000

Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci.

Conditions have been optimized for the use of a multiplex PCR for the detection of vancomycin-resistant enterococci in nosocomial surveillance specimens. Seven primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 Enterococcus faecalis-specific, Enterococcus faecium-specific, and rrs (16S rRNA) were used in one reaction tube. The PCR method developed in the present study is simple and reliable for the rapid characterization of vancomycin-resistant enterococci.

Cross Infection↗
PubMed2001

Accuracy of the VITEK 2 system to detect glycopeptide resistance in enterococci.

We evaluated the accuracy of the VITEK 2 fully automated system to detect and identify glycopeptide-resistant enterococci (GRE) compared to a reference agar dilution method. The sensitivity of vancomycin susceptibility testing with VITEK 2 for the detection of vanA, vanB, and vanC1 strains was 100%. The sensitivity of vancomycin susceptibility testing of vanC2 strains was 77%. The sensitivity of teicoplanin susceptibility testing of vanA strains was 90%. Of 80 vanC enterococci, 78 (98%) were correctly identified by VITEK 2 as Enterococcus gallinarum/Enterococcus casseliflavus. Since the identification and susceptibility data are produced within 3 and 8 h, respectively, VITEK 2 appears a fast and reliable method for detection of GRE in microbiology laboratories.

Anti-Bacterial Agents↗
PubMed2001

Comparison and evaluation of antimicrobial susceptibility testing of enterococci performed in accordance with six national committee standardized disk diffusion procedures.

Studies were conducted to compare and evaluate antimicrobial susceptibility test results for enterococci obtained by six national committee disk diffusion procedures. Variations in the incidence of isolates in resistance categories and errors were associated with the use of ciprofloxacin, gentamicin, nitrofurantoin, rifampin, and teicoplanin in a number of committee procedures. Results indicate that laboratories performing disk diffusion antimicrobial susceptibility testing may have problems correctly identifying resistance in enterococci with agents used to combat infections and that it may be difficult to compare resistance data for surveillance purposes.

Anti-Bacterial Agents↗
PubMed2001

Routine molecular identification of enterococci by gene-specific PCR and 16S ribosomal DNA sequencing.

For 279 clinically isolated specimens identified by commercial kits as enterococci, genotypic identification was performed by two multiplex PCRs, one with ddl(E. faecalis) and ddl(E. faecium) primers and another with vanC-1 and vanC-2/3 primers, and by 16S ribosomal DNA (rDNA) sequencing. For 253 strains, phenotypic and genotypic results were the same. Multiplex PCR allowed for the identification of 13 discordant results. Six strains were not enterococci and were identified by 16S rDNA sequencing. For 5 discordant and 10 concordant enterococcal strains, 16S rDNA sequencing was needed. Because many supplementary tests are frequently necessary for phenotypic identification, the molecular approach is a good alternative.

DNA Primers↗
PubMed2001

Classification and identification of enterococci: a comparative phenotypic, genotypic, and vibrational spectroscopic study.

Rapid and accurate identification of enterococci at the species level is an essential task in clinical microbiology since these organisms have emerged as one of the leading causes of nosocomial infections worldwide. Vibrational spectroscopic techniques (infrared [IR] and Raman) could provide potential alternatives to conventional typing methods, because they are fast, easy to perform, and economical. We present a comparative study using phenotypic, genotypic, and vibrational spectroscopic techniques for typing a collection of 18 Enterococcus strains comprising six different species. Classification of the bacteria by Fourier transform (FT)-IR spectroscopy in combination with hierarchical cluster analysis revealed discrepancies for certain strains when compared with results obtained from automated phenotypic systems, such as API and MicroScan. Further diagnostic evaluation using genotypic methods-i.e., PCR of the species-specific ligase and glycopeptide resistance genes, which is limited to the identification of only four Enterococcus species and 16S RNA sequencing, the "gold standard" for identification of enterococci-confirmed the results obtained by the FT-IR classification. These results were later reproduced by three different laboratories, using confocal Raman microspectroscopy, FT-IR attenuated total reflectance spectroscopy, and FT-IR microspectroscopy, demonstrating the discriminative capacity and the reproducibility of the technique. It is concluded that vibrational spectroscopic techniques have great potential as routine methods in clinical microbiology.

Bacterial Typing Techniques↗
PubMed2003

Diversity of tn1546-like elements in vancomycin-resistant enterococci isolated from humans and poultry in Korea.

To investigate the possible spread of vancomycin resistance among enterococci through horizontal gene transfer between two sources of vancomycin-resistant enterococci (VRE) isolated from farm animals and humans in Korea, molecular characterization of VRE isolated from poultry (20 isolates) and humans (35 isolates) was performed by PCR-restriction fragment length polymorphism typing of the vanA gene cluster (Tn1546). PCR mapping of Tn1546 finally distinguished seven different transposon types (types A to G). Type A was observed only in the poultry isolates, while the other six types were present only in the human isolates. An insertion sequence and deleted sequence were detected in most of the human isolates in the orf2-vanR and vanX-vanY regions in the Tn1546-like element, but not in the poultry isolates. Tn1546-like elements were found in conjugal plasmids of most human VRE, whereas they were detected in the chromosomes of all poultry VRE. Accordingly, no evidence was found of any recent transmission of vancomycin resistance genes between poultry and humans in Korea.

Animals↗
PubMed2004

Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium.

A multiplex PCR for the simultaneous detection of five virulence genes (asa1, gelE, cylA, esp, and hyl) in enterococci was developed. The presence of these genes was investigated in 153 clinical and 118 fecal Enterococcus faecium isolates from inpatients at an increased risk of developing infections (such as patients in intensive care units and hematology wards) from 13 hospitals in eight European countries. Of the 271 E. faecium isolates, 135 were vancomycin resistant E. faecium (VREF) isolates and 136 were vancomycin susceptible E. faecium (VSEF) isolates. Susceptibilities to ampicillin, gentamicin, streptomycin, vancomycin, teicoplanin, ramoplanin, quinupristin-dalfopristin, and linezolid were tested by the microdilution method. Overall, the prevalence of esp was significantly higher (P = 0.03) in clinical VREF isolates (92%) than in fecal VREF isolates (73%). In Italy, the prevalence of esp was significantly higher (P = 0.02) in VREF isolates (91%) than in VSEF isolates (68%), whereas in the United Kingdom, hyl was significantly more prevalent (P = 0.01) in VREF isolates (71%) than in VSEF isolates (29%). No significant differences were found for the other countries. Pulsed-field gel electrophoresis was used to check the clonality among the strains tested and showed the spread of two center-specific (esp-positive) VREF clones in Italy and one center-specific (hyl-positive) clone in the United Kingdom. These clones were resistant to ampicillin, gentamicin, and streptomycin. The multiplex PCR reported in this study is a convenient and rapid method for the simultaneous detection of the virulence genes asa1, gelE, cylA, esp, and hyl in enterococci. Molecular analysis showed the intrahospital spread of esp-positive VREF clones (in Italy) and hyl-positive VREF clones (in the United Kingdom); the role of hyl remains to be elucidated.

Anti-Bacterial Agents↗
PubMed2005

Survivability of vancomycin resistant enterococci and fitness cost of vancomycin resistance acquisition.

AIMS: To investigate the survivability of vancomycin resistant enterococci (VRE) under dry starvation conditions and the fitness cost of vancomycin resistance. METHODS: VRE colonies on cotton swabs were incubated at room temperature in a sterile box and cultured weekly until cultures no longer showed growth. Negative swabs inoculated into brain heart infusion (BHI) broth were subcultured to blood agar after 24, 48, and 72 hours of incubation to resuscitate viable but non-culturable cells. Stability of the vancomycin resistance determinant and of the DNA fingerprint pattern was determined by multiplex polymerase chain reaction (PCR) and repetitive PCR, respectively. Tests for fitness cost were carried out on the same VRE isolates and 28 hospital vancomycin sensitive enterococci (VSE) isolates by incubation and measurement of optical density using a microplate reader and comparing maximum growth rate and lag phase duration between VRE and VSE, using independent samples t tests. RESULTS: Mean maximum time of recovery by primary culture was 8.5 weeks for Enterococcus faecalis VRE and 21.8 weeks for E. faecium VRE. Two of two E. faecalis isolates were resuscitated after 24 hours in BHI broth, and two of five E. faecium isolates after 72 hours. No fitness cost of vancomycin resistance was demonstrated. CONCLUSIONS: VRE can survive for prolonged periods in a dry starvation state, retaining their genetic complement, including vancomycin resistance determinants, and show little or no fitness cost of vancomycin resistance. Thus, the rate of entry required for VRE to become, and remain, endemic in the community is relatively small.

Cross Infection↗
PubMed1977

Aminoglycoside-resistant enterococci.

Thirty-four recent clinical isolates of Streptococcus faecalis were tested for sensitivity to amoxycillin, benzylpenicillin, streptomycin, kanamycin, gentamicin, tobramycin, and amikacin. Amoxycillin was two- to four-fold more active than benzylpenicillin and all strains were inhibited by low concentrations of the penicillins. The aminoglycosides were less active against the enterococci than were the penicillins and a significant number of strains were insensitive or relatively insensitive to one or more of the aminoglycosides. Thus, eight (23%) strains showed a high level of resistance to streptomycin and kanamycin (MIC greater 5000 microng/ml) but were sensitive to gentamicin, tobramycin, and amikacin. In addition, two strains of Strep. faecalis, isolated at different hospitals from patients who had received topical gentamicin therapy, were relatively resistant to gentamicin (MIC250 to 500 microng/ml) and were less sensitive also to the other aminoglycosides. Bactericidal synergy was demonstrated by amoxycillin/aminoglycoside combinations against the enterococci, provided that the test strain of Strep. faecalis was sensitive to the aminoglycoside in the combination. An exception to this was the combination of amoxycillin plus amikacin which was not synergistic against kanamycin-resistant strains of Strep. faecalis although these organisms were sensitive to amikacin in the growth inhibition tests. The gentamicin-resistant strains showed variable responses to amoxycillin/aminoglycoside combinations in tests for bactericidal synergy and were generally less sensitive than typical strains of Strep. faecalis.

Aminoglycosides↗
PubMed1995

Immunoblotting in the diagnosis of culture negative endocarditis caused by streptococci and enterococci.

AIM: To improve the diagnosis of culture negative endocarditis by diagnosing cases due to streptococci and enterococci. METHODS: Serum samples were immunoblotted against extracts of the commonest streptococci and enterococci. They were selected from patients with a cardiac murmur, persistent pyrexia and at least three negative blood cultures. The presence of patterns of endocarditis species specific antigenic bands was measured and correlated with clinical outcome. RESULTS: Negative serology was found in 28 patients where the diagnosis of endocarditis was rejected or, if proved, staphylococcal, yeast, Gram negative, systemic lupus erythematosus, due to Q fever or Chlamydia psittaci or nonbacterial thrombotic. Positive serology was found in 27 of the 34 patients where the response to antibiotics suggested streptococcal or enterococcal infection. In 22 of these there was objective evidence of endocarditis. Positive serology was also found in three of four further patients with vegetations at necropsy. CONCLUSION: The identification of patterns of antibody response on immunoblotting can be used to make a specific diagnosis of streptococcal or enterococcal endocarditis in the absence of positive blood cultures.

Antibodies, Bacterial↗
PubMed1998

Use of yellow-pigmented enterococci as a specific indicator of human and nonhuman sources of faecal pollution.

Antibiotic susceptibility tests and restriction enzyme analysis (REA) of genomic DNA were performed to characterize the relationship between sources of isolates of yellow-pigmented enterococci. Antibiotic susceptibility tests were conducted with 10 therapeutic antibiotics and 54 isolates grouped by source (wild and other) depending on their origin. In three antibiotics, cephalothin, erythromycin, and vancomycin, there was a significant (p < or = 0.05) association between susceptibility and source. Vancomycin resistance was significantly (p < or = 0.001) higher in isolates from wild sources compared with that in isolates from other sources. The REA technique was performed on genomic DNA obtained from 17 Enterococcus mundtii isolates from: human (3), dog (4), horse (4), Canada goose (4), domestic goose (1), and Enterococcus mundtii ATCC 43186. A total of 12 different DNA types (A-L) were identified. Except for type D, 11 DNA types were unique and were distributed among dog (A, B, and C), human (E), horse (F, G, and H), Canada goose (I, J, and K), and domestic goose (L). Results suggested that vancomycin-susceptibility testing of yellow-pigmented enterococci may have potential value in the identification of sources of faecal pollution, especially when combined with traditional quantitative methods.

Animals↗
PubMed2004

Survival of enterococci and Tn916-like conjugative transposons in soil.

The persistence of Enterococcus faecalis, fecal enterococci from swine waste, and Tn916-like elements was determined following inoculation into autoclaved and native soil microcosms. When cells of E. faecalis CG110 (Tn916) were inoculated into native microcosms, enterococcal viability in the soil decreased approximately 5 orders of magnitude (4.8 x 10(5) CFU/g soil to < 10 CFU/g) after 5 weeks. In autoclaved microcosms, the viability of E. faecalis decreased by only 20% in 5 weeks. In contrast, the content of Tn916, based on PCR of DNA extracts from soil microcosms, decreased by about 20% in both native and autoclaved microcosms. Similar results were obtained when the source of fecal enterococci and Tn916-like elements was swine waste. Because the concentration of Tn916-independent E. faecalis DNA (the D-alanine D-alanine ligase gene), based on PCR, decreased to nearly undetectable levels (at least 3 orders of magnitude) after 5 weeks in the native microcosms, the evidence suggests Tn916 stability in the soil results from en masse transfer of the transposon to the normal soil microflora and not survival of E. faecalis DNA in the soil system. Results from denaturing gradient gel electrophoresis suggest that multiple forms of Tn916 occur in swine waste, but only forms most like Tn916 exhibit stability in the soil.

Animals↗
PubMed2004

Vancomycin tolerance in enterococci.

BACKGROUND: Tolerance can be defined as the ability of bacteria to grow in the presence of high concentrations of bactericide antimicrobics, so that the killing action of the drug is avoided but the minimal inhibitory concentration (MIC) remains the same. We investigated vancomycin tolerance in the Enterococcus faecium and Enterococcus faecalis strains isolated from different clinical specimens. METHODS: Vancomycin was obtained from Sigma Chemical Co. We studied 100 enterococci strains. Fifty-six and 44 of Enterococcus strains were idendified as E. feacalis and E. faecium, respectively. To determine MICs and minimal bactericidal concentration (MBC), we inoculated strains from an overnight agar culture to Muller-Hinton broth and incubated them for 4-6 h at 37 degrees C with shaking to obtain a logarithmic phase culture. The inoculum was controlled by performing a colony count for each test. We determined MBC values and MBC/MIC ratios to study tolerance to vancomycin. Vancomycin tolerance was defined as a high MBC value and an MBC/MIC ratio > or =32. RESULTS: Fifty-six and 44 of the Enterococcus strains were identified as E. faecium and E. faecalis, respectively. Thirty-one E. faecium and 48 E. faecalis were found to be susceptible to vancomycin and these susceptible strains were included in this study. The MICs of susceptible strains ranged from < or =1 to 4 mg/l, the MBCs were > or =512 mg/l. Tolerance was detected in all E. faecalis and E. faecium strains. The standard E. faecalis 21913 strain also exhibited tolerance according to the high MBC value and the MBC/MIC ratio. We defined the tolerant strains as having no bactericidal effect and MBC/MIC > or =32. We found that a 100% tolerance was present in susceptible strains. CONCLUSIONS: One of the hypotheses for tolerance is that tolerant cells fail to mobilize or create the autolysins needed for enlargement and division. Our data suggests that tolerance may compromise glycopeptide therapy of serious enterococci infections. To add an aminoglycoside to the glycopeptide therapy unless MBCs are unavailable can be useful in the effective treatment of serious Enterococcus infections.

Agar↗