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PubMed2004

A rapid antimicrobial susceptibility test based on chemiluminescence assay and its application to screening of genotypes in vancomycin-resistant enterococci.

Minimum inhibitory concentrations (MICs) of vancomycin (VCM) and teicoplanin (TEIC) were measured using a novel susceptibility test based on the chemiluminescence assay (CA) method (Rapid-Lumi Eiken; Eiken Chemicals, Tokyo, Japan) against 33 strains in total: 7, 5, and 10 strains of which are VCM-resistant enterococci (VRE) with vanA, vanB, and vanC genes, respectively, and the other 11 strains are vancomycin-susceptible enterococci (VSE). The results were in good accordance with the values determined by the standard broth dilution method approved by the National Committee for Clinical Laboratory Standards (NCCLS): i.e., 88% (29/33) of consistency for VCM and 97% (32/33) for TEIC, respectively. In addition, genotypes in VRE strains (vanA, vanB, vanC-1, and vanC-2/3 genes) were properly estimated from the results of the CA method and the NCCLS interpretive categories, even though the incubation time was very short (2-4 h). In conclusion, it was found that the new method is reliable and rapid to detect VRE strains in clinical laboratories.

Anti-Bacterial Agents↗
PubMed1993

Emergence of vancomycin-resistant enterococci in New York City.

Enterococci, a common cause of nosocomial infection, are intrinsically resistant to most antimicrobials and readily acquire additional resistance. Vancomycin-resistant enterococci (VRE) have caused clusters of nosocomial infections since 1988. In April, 1991, the New York City Department of Health asked all city laboratories to submit suspected VRE isolates for confirmation. Clinical and epidemiological characteristics of the first 100 patients with VRE were identified, and antimicrobial susceptibility testing, restriction enzyme analysis, and DNA-DNA hybridisation with the vanA gene probe were done. From September, 1989, to October, 1991, 361 patients with VRE were identified at 38 hospitals. The number of hospitals reporting VRE increased from 1 in 1989 to 38 by October, 1991. 98% of 100 VRE infections were nosocomially acquired and 83% patients had received vancomycin and/or a cephalosporin in the 30 days before isolation of VRE. Of 23 isolates from 21 of the first 100 patients, 19 (83%) were resistant to all available antimicrobials. Four vanA probing patterns were noted, and restriction enzyme analysis of the 23 isolates revealed 14 strains. VRE have emerged rapidly in New York City. Molecular analyses suggest that a highly mobile genetic element--eg, a transposon--is responsible for the rapid spread of vancomycin resistance.

Aged↗
PubMed1993

Prevalence in Singapore of enterococci with high-level resistance to aminoglycosides and comparison of three methods for their detection.

The first study of the prevalence in Singapore of enterococci with high-level resistance to aminoglycosides has been made in the National University Hospital. From March to June 1992, 225 isolates were studied by means of three methods: (1) use of brain-heart infusion (BHI) agar incorporating the antibiotic; (2) microdilution of the antibiotic in broth; and (3) use of high content antibiotic discs. The BHI agar was the reference method. Of the total, 49 (22%) isolates were resistant to gentamicin, 86 (38%) to streptomycin, 82 (36%) to kanamycin and 93 (41%) to one or more of the agents. Results of the disc diffusion method correlated completely with the BHI agar method. The broth microdilution method showed a 100, 90 and 96% sensitivity in relation to gentamicin, streptomycin and kanamycin, respectively, and a specificity of 100% for all three aminoglycosides. Statistical significance (P < 0.01 to P < 0.001) was demonstrated in (1) a greater incidence of enterococci with high-level resistance to aminoglycosides among blood isolates; (2) gentamicin resistance predicting kanamycin high-level resistance; and (3) more resistance to gentamicin, streptomycin and kanamycin among Enterococcus faecium isolates compared to those of Enterococcus faecalis. Beta-lactamase activity was not detected.

Aminoglycosides↗
PubMed1990

Histamine formation by enterococci in goat cheese.

Cheeses were made of heat-treated goat milk inoculated with large numbers of a histamine-producing strain of Streptococcus faecium or a non-histamine-producing strain of S. faecalis. Every second week during ripening (91 days) the cheeses were sampled for histamine analyses and bacteriological analyses. The numbers of enterococci remained high throughout the whole period of investigation and the maximum amount of histamine detected was 8.2 micrograms/g in one of the cheeses. The enterococci seem to have no relevance from a histamine intoxication point of view in cheeses of this kind.

Animals↗
PubMed1992

Clinical isolates of enterococci with high-level resistance to currently available aminoglycosides.

Enterococci isolated from different body sites were tested for high-level gentamicin resistance. A total of 139 enterococcal isolates were screened for resistance (minimum inhibitory concentration [MIC] greater than 2000 mg l-1) by a broth-tube method. Twenty-five (18%) were found to exhibit resistance and this was confirmed by agar screening (1000 mg l-1) and agar dilution MIC determinations. The majority of isolates also showed high-level resistance to kanamycin and streptomycin. The remaining isolates showed high-level resistance to gentamicin and kanamycin but not streptomycin. A retrospective clinical review was performed. Most patients had a source of definite or likely infection (78%). Serious infections such as endocarditis or meningitis were not observed during the course of this study. Retrospective clinical data suggest that in cases not involving endocarditis or meningitis, neither infection refractory to therapy nor relapse of infection is a common sequel to infection with gentamicin-resistant enterococci in hospitalized patients.

Anti-Bacterial Agents↗
PubMedDate not supplied

In vitro activity of 43 antimicrobial agents tested against ampicillin-resistant enterococci and gram-positive species resistant to vancomycin.

A total of 57 strains of ampicillin-resistant and -susceptible enterococci representing 10 species and 23 strains of vancomycin-resistant Gram-positive bacteria (Leuconostoc and Pediococcus) were tested to determine their susceptibility to 43 antimicrobial agents by the reference broth microdilution method. The drug MICs for the ampicillin-resistant enterococci were generally similar to those of ampicillin-susceptible strains, that is, highly resistant to cephalosporins, moderately susceptible or resistant to quinolones, and susceptible to "glycopeptides." Some investigational quinolones (PD127391, sparfloxacin, WIN57273), minocycline, and rifampin were highly active. Vancomycin-resistant strains were usually resistant to other "glycopeptides," for which correlation coefficients of MICs ranged from 0.881 to 0.978, except ramoplanin (MICs, 0.008-0.5 micrograms/ml). Most isolates resistant to vancomycin were susceptible to the newer quinolones, penicillins, aminoglycosides, clindamycin, and erythromycin, but highly resistant to cephalosporins. Discrepancies between the MICs and MBCs for glycopeptides were noted (greater than or equal to 8-fold, MBC50/MIC50), but not for ramoplanin. The vancomycin disk test was in 96.1% absolute agreement by identifying resistant strains without contributing false-susceptible, very major error.

4-Quinolones↗
PubMed1993

Susceptibility of beta-lactamase-producing enterococci to piperacillin with tazobactam.

The in vitro activity of piperacillin with and without tazobactam was evaluated against different inocula of 12 clinical isolates of beta-lactamase-producing Enterococcus faecalis obtained from different geographic areas. Minimum inhibitory concentrations (MICs) of piperacillin alone at approximately 10(3) colony-forming units (CFU)/spot ranged from 4 to 8 and from 4 to 8 micrograms/ml with piperacillin plus tazobactam. When approximately 10(7) CFU/spot was used, MICs increased to a range of 128-1024 micrograms/ml piperacillin. This inoculum effect was reversed by the addition of tazobactam to piperacillin at a fixed concentration of 1 microgram/ml or at a ratio of 8 : 1 (piperacillin relative to tazobactam) with an MIC90 of 16/2 micrograms/ml for the combination drug. In time-kill studies, four beta-lactamase-producing (Bla+) isolates were tested and demonstrated a decrease of > or = 2 log10 with 8 or 16 micrograms/ml of piperacillin in combination with 4 micrograms of tazobactam, but not with piperacillin alone. A non-beta-lactamase-producing isolate was equally inhibited by piperacillin alone and piperacillin plus tazobactam. Against a Bla+ isolate, the combination of piperacillin with tazobactam with streptomycin resulted in a synergistic effect relative to that of piperacillin with tazobactam; piperacillin plus streptomycin did not show synergism. Piperacillin in combination with tazobactam is active against enterococci that produce beta-lactamase and, in combination with an appropriate aminoglycoside, could be a viable choice for therapy of enterococci that do not have high-level resistance to all aminoglycosides.

Culture Media↗
PubMed1995

Susceptibility of ciprofloxacin-resistant staphylococci and enterococci to clinafloxacin.

Clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis obtained from the Ann Arbor Veterans Affairs Medical Center within the last decade were tested for susceptibility to ciprofloxacin and clinafloxacin. For MRSA isolates, the minimum inhibitory concentrations (MICs) of ciprofloxacin were several fold higher than those noted with clinafloxacin. Prior to the introduction of the fluoroquinolones (1984-1985), all MRSA isolates were susceptible to ciprofloxacin and clinafloxacin. By 1993, virtually all MRSA isolates were resistant to ciprofloxacin and a 50-fold increase in the MIC50 and MIC90 for clinafloxacin was seen. In 1985-1986, most enterococcal isolates were susceptible to ciprofloxacin and clinafloxacin. By 1993, one-third of all enterococci were resistant to both ciprofloxacin and clinafloxacin. Fluoroquinolone resistance developed more quickly in enterococci that demonstrated high-level gentamicin resistance. Thus, cross-resistance between clinafloxacin and ciprofloxacin was seen; however, the lower MICs of clinafloxacin for MRSA may allow the use of this drug for some MRSA infections.

Anti-Infective Agents↗
PubMed1995

Emerging multiply resistant enterococci among clinical isolates. II. Validation of the etest to recognize glycopeptide-resistant strains.

Accurate, quantitative susceptibility testing of the enterococci for glycopeptide (vancomycin and teicoplanin) activity has become very important to guide chemotherapy as a result of emerging resistance. Reference dilution tests using broth and agar are generally difficult to perform, or in some commercial forms have demonstrated interpretive error. An alternative method, the Etest has promise as a system with qualitative and quantitative accuracy. Nearly 2000 enterococci from 97 laboratories in the United States were tested using broth microdilution, disk diffusion, and Etest methods. The Etest quantitative accuracy (+/- 1 log2 dilution) compared to the broth microdilution minimum inhibitory concentration was 90.1% (teicoplanin) to 94.1% (vancomycin). The qualitative interpretive accuracy of the Etest ranged from 98.7% for vancomycin to 99.9% for teicoplanin (no false-susceptible errors). All three tests were in remarkable agreement, with < or = 0.8% maximal discord by interpretive category. The Etest appears to be an excellent alternative to reference and standardized methods for producing quantitative activity measurements of glycopeptide susceptibility or resistance.

Bacterial Typing Techniques↗
PubMed1994

Detection of vancomycin resistance in enterococci by the Vitek AMS system.

The ability of the Vitek AMS system to detect vancomycin resistance in enterococci was evaluated by comparing the results to an agar dilution screen method. Of 100 strains tested, 43 were resistant and 51 were susceptible by both tests. Two strains were intermediate by Vitek but susceptible by agar screen, one was intermediate by Vitek but resistant by agar screen, and four were susceptible by Vitek but resistant by agar screen. When the Vitek intermediate and resistant results were combined, the false resistant rate was 4.4% and false susceptible rate was 8.5%. In its current format, the Vitek system appears not to have acceptable accuracy for the detection of vancomycin resistance in enterococci.

Colony Count, Microbial↗
PubMed1995

Evaluation of Vitek and API 20S for species identification of enterococci.

The performance of the Vitek System and the API 20S for identifying enterococci to the species level was evaluated in comparison with a conventional reference test scheme. A total of 369 isolates originally derived from a sample from 97 medical centers were tested, and the percentage of correctly identified organisms was (Vitek/API): 97.6%/79.2%, 95.3%/91.2%, and 86.0%/20.7% for Enterococcus faecalis, Enterococcus faecium, and Enterococcus non-faecalis-non-faecium species, respectively. In addition, the predictive values for species identification of the results reported by the systems were 98.1% for Vitek and 85.9% for API. The Vitek System GPI card (software version > or = 8.1) was considered to be very acceptable for enterococci identification, with errors principally among the rare Enterococcus raffinosus isolates.

Bacteriological Techniques↗
PubMed2004

Activity of daptomycin against multi-resistant Gram-positive bacteria including enterococci and Staphylococcus aureus resistant to linezolid.

The activity of daptomycin was assessed against 545 isolates of Gram-positive bacteria including enterococci, coagulase-positive and -negative staphylococci, beta-haemolytic streptococci, viridans streptococci and coryneforms. The panel of isolates was biased towards those with resistance to first-line antimicrobial agents and included linezolid-resistant enterococci and Staphylococcus aureus. MICs of daptomycin were < or =1mg/l for all the staphylococci, streptococci, and coryneforms, except for two isolates of Corynebacterium aquaticum, for which MICs were 8 mg/l. Daptomycin MICs were < or =2 and < or =4 mg/l for Enterococcus faecalis and Enterococcus faecium, respectively.

Acetamides↗
PubMed2006

Antimicrobial resistance and the mechanisms implicated in faecal enterococci from healthy humans, poultry and pets in Portugal.

Antimicrobial resistance and the mechanisms implicated were studied in 440 enterococci (227 Enterococcus faecium, 177 Enterococcus faecalis, 32 Enterococcus hirae and 4 Enterococcus durans) recovered from 220 faecal samples of healthy humans, poultry and pets in Portugal. Higher levels of resistance were detected for ampicillin, tetracycline, erythromycin and chloramphenicol in poultry isolates (10.5%, 97%, 87.5% and 16%, respectively) compared with human isolates (0%, 26%, 31.5% and 5%, respectively); intermediate levels of resistance for these antibiotics were found in pet isolates. Thirty-three per cent of the E. faecium isolates of poultry origin showed quinupristin/dalfopristin resistance. High-level resistance to gentamicin or streptomycin was detected in 1-7% of isolates in our series of enterococci. The aac(6')-aph(2''), aph(3')-IIIa, erm(B) and tet(M) genes were demonstrated in most of the gentamicin-, kanamycin-, erythromycin- and tetracycline-resistant isolates, respectively. The vat(E) gene was found in 39% of the quinupristin/dalfopristin-resistant E. faecium isolates of poultry origin.

Animals↗
PubMed2005

Enterococci from Tolminc cheese: population structure, antibiotic susceptibility and incidence of virulence determinants.

Microbiological analysis of ripened artisanal Tolminc cheese revealed the presence of an enterococcal population in numbers of up to 10(6) per g. All colonies, isolated from the citrate azide tween carbonate (CATC) enterococcal selective medium were Gram positive and coccal-shaped and were analysed with PhenePlate FS system. This system discriminated 10 PhP clusters among the 90 enterococcal isolates. From each cluster the most representative isolate for that particular type was selected for further study. The 10 representative enterococci were catalase negative and grew in the presence of NaCl (2%, 4% and 6.5%) and bile salts (0.06%). Genus specific primers confirmed all 10 enterococcal representatives as Enterococcus members, while species specific primers determined them further as strains of Enterococcus faecalis species. PCR for vanA and vanB genes detection, respectively, amplified no PCR products. The absence of van genes was confirmed with both disc and E-test, as isolates were susceptible to vancomycin according to the National Committee for Clinical Laboratory Standards (NCCLS). The results of disc tests with other antimicrobial agents (ampicillin, vancomycin, kanamycin, penicillin, erythromycin, neomycin, chloramphenicol, clindamycin, rifampin) did not differ much among the tested enterococci: they were all very resistant to clindamycin only. The incidence of enterococcus virulence determinants was as expected: all of the 10 E. faecalis strains tested possessed multiple determinants (between 7 and 11).

Anti-Bacterial Agents↗
PubMed2005

Occurrence of insertion sequences within the genomes and Tn1546-like elements of glycopeptide-resistant enterococci isolated in Brazil, and identification of a novel element, ISEfa5.

Insertion sequences (IS) occur widely within the Tn1546-like elements responsible for VanA glycopeptide resistance in enterococci from several countries. As such insertions can be used as epidemiological markers and for studying horizontal transfer of gene clusters, we investigated the distribution of IS6770, IS1542, IS1216V, IS1476, and IS1251 elements in 26 VanA Enterococcus faecium and 21 VanA Enterococcus faecalis from Brazil. PCR, using genomic DNA as a template, indicated that most of the isolates contained IS6770 (97%), IS1216V (87%) and IS1476 (72%) elements. IS1251 was also detected, but at a higher frequency in E. faecium (80%) than in E. faecalis (14%). None of the isolates harboured IS1542. Only two of 47 isolates had IS elements within their Tn1546-like elements; one possessed IS1251 between vanS and vanH, as reported in the United States; another possessed a novel IS element, designated ISEfa5, located between vanX and van Y. This novel element was found in the genomic DNA of 25 (96%) E. faecium and II (52%) E. faecalis. In stability studies, no IS-mediated changes were detected in the Tn1546-like elements of 25 vancomycin-resistant enterococci (VRE) monitored over 11 months. These results suggest that the occurrence of IS in Brazilian isolates is similar to that reported in American isolates, but that these elements occur rarely within the vanA gene clusters. As patterns of IS carriage did not correlate with the PFGE type of the VRE, the prevalence of IS elements in genomic DNA of VRE is not a useful epidemiological marker. However, the presence of IS-modified Tn1546-like elements, which appear to be rare in Brazil, could be a useful molecular marker in local epidemiological studies to monitor the evolution and horizontal transmission of VanA elements.

Anti-Bacterial Agents↗
PubMed2004

Increasing vancomycin susceptibility in vancomycin resistant enterococci by vanH promoter and ddl transformation.

OBJECTIVES: In vancomycin resistant enterococci (VRE) the vanHAXYZ genes encode a new pathway of enzymes to produce d-alanyl-d-lactate. We investigated the effect of vanH promoter and ddl gene transformation on vancomycin susceptibility in a vanA phenotype of Enterococcus faecalis. METHODS: To construct plasmid pJW1, the vanH promoter was cloned to plasmid pAM401. Plasmid pJW2 was constructed by cloning the ddl gene into pAM401. To construct pJW3, the ddl gene was ligated downstream of the vanH promoter of the plasmid pJW1. The competent VRE was transformed with pJW1, pJW2 and pJW3 using electroporation. The minimal inhibitory concentration (MIC) of vancomycin for VRE and transformed E. faecalis was determined using the broth dilution method. The expression of the vanA and ddl gene of VRE and transformed E. faecalis was evaluated by RT PCR. RESULTS: The transformation of the vanH promoter reduced the vancomycin MIC of VRE. In VRE and transformed E. faecalis, the vanA and ddl genes were expressed. CONCLUSIONS: This study presents a way of altering high-level vancomycin resistance with gene transformation in enterococci. In the future, development of an effective gene delivery system will contribute to the design of new modalities that will help overcome the limitations of antimicrobial therapy.

Bacterial Proteins↗
PubMed2006

Rapid development of secondary sclerosing cholangitis due to vancomycin-resistant enterococci.

Secondary sclerosing cholangitis is a rare complication of acute bacterial cholangitis. The natural course of the disease is a slow progression over years. Here we report about a patient in whom sclerosing cholangitis evolved much faster as a consequence of a persistent biliary infection with vancomycin-resistant enterococci. The infection could be treated successfully with prolonged therapy with linezolid and teicoplanin. This is the first report of sclerosing cholangitis caused by vancomycin-resistant enterococci. It illustrates the rapidity with which this disease can evolve as a fulminant and ultimately fatal complication of acute bacterial cholangitis.

Aged, 80 and over↗
PubMed2005

Use of composite data sets for source-tracking enterococci in the water column and shoreline interstitial waters on Pensacola Beach, Florida.

Sources of Enterococcus faecalis isolates from Pensacola Beach, FL. were identified using a library-based approach by applying the statistical method of average similarity to single and composite data sets generated from separate analyses. Data sets included antibiotic resistance analysis (ARA), rep-fingerprints, and fatty acid methyl ester (FAME) profiles. Use of a composite data set composed of ARA and rep-fingerprints, added to the confidence of the identifications. The addition of FAME data to composite data sets did not add to the confidence of identifications. Source identification was performed to better understand risk associated with higher densities of enterococci found in swash zone interstitial water (SZIW) as compared to adjacent bathing water on Pensacola Beach, FL. The "swash zone" is that area of the beach continually washed over by waves. As the potential sources of enterococci were limited in this environment, only two library units, sea gull and human, were constructed. Identification of the beach isolates using a composite data set indicated a sea gull origin. The clonality of the beach isolates suggested that the beach environment selects certain subspecies of E. faecalis.

Animals↗