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PubMed1995

Evaluation of three disk tests for identification of enterococci, leuconostocs, and pediococci.

Simple rapid tests for presumptive identification of catalase-negative non-beta-hemolytic cocci (i.e., enterococci, leuconostocs, and pediococci) have not previously been available. Seven hundred thirty-four strains of aerobic and facultatively anaerobic, catalase-negative, non-beta-hemolytic gram-positive cocci were tested for susceptibility to vancomycin (Vans) by a screening procedure and production of leucine aminopeptidase (LAPase) and pyrrolidonylarylamidase (PYRase) in disk tests. Three unique patterns of activity in response to the three disks (30 micrograms of vancomycin, PYRase, and LAPase) can be used to presumptively identify the vancomycin-resistant (Vanr) enterococci (Vanr and PYRase and LAPase positive), leuconostocs (Vanr and PYRase and LAPase negative), and pediococci (Vanr, PYRase negative, and LAPase positive). The results indicate that, together with Gram stain characteristics and the catalase test, the vancomycin, LAPase, and PYRase disk tests can be used to presumptively identify Vanr strains of enterococci as well as Leuconostoc and Pediococcus strains from human infections.

Aminopeptidases↗
PubMed1996

Evaluation of Alamar colorimetric broth microdilution susceptibility testing method for staphylococci and enterococci.

We compared the results of the Alamar broth microdilution susceptibility testing method with the results of the National Committee for Clinical Laboratory Standards reference broth microdilution method for 119 gram-positive organisms. The strains were tested for their susceptibilities to 20 antimicrobial agents. Only appropriate antimicrobial agents were evaluated for each species of bacteria. Absolute categorical agreement between the reference method and the test method was 91.5% for enterococci, 99.8% for oxacillin-susceptible staphylococci, and 97.4% for oxacillin-resistant staphylococci. Essential agreement (percent complete agreement plus percent minor errors) was > 99% for all organisms tested. The results for enterococci showed no very major errors, one major error with ofloxacin, and numerous minor errors with the quinolones. However, all except one of the minor errors were within +/- 1 log2 dilution of the reference result. For staphylococci, only 2 very major errors (one each with chloramphenicol and oxacillin), 1 major error (chloramphenicol), and 15 minor errors (multiple drugs) were observed. The Alamar colorimetric system was easy to use and the results were easy to read. It appears to be an acceptable method for antimicrobial susceptibility testing of staphylococci and enterococci.

Colorimetry↗
PubMed1996

Selective isolation of vancomycin-resistant enterococci.

Broth formulations of two media selective for enterococci, Enterococcel, M-Enterococcosel broths were supplemented with 6 micrograms of vancomycin per ml and evaluated for isolation of vancomycin-resistant enterococci (VRE). Each broth was challenged with various concentrations of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and vancomycin-susceptible and vancomycin-resistant enterococci and with 193 perianal specimens obtained from patients at risk in our institution for VRE colonization. Both the Enterococcosel and M-Enterococcus broths with vancomycin detected as few as 1 to 9 CFU of VRE while inhibiting growth of the other organisms tested. Enterococcus faecium organisms (MIC, > 256 micrograms/ml) were recovered from 66 perianal swab cultures in the enterococcosel-vancomycin broth, and VRE were recovered from 62 perianal swab cultures in the M-Enterococcus-vancomycin broth. Enterococcosel-vancomycin broth detected VRE in perianal specimens 48 h earlier than did M-Enterococcus-vancomycin broth. Enterococcosel broth with 6 micrograms of vancomycin per ml can be used for the rapid and selective isolation of VRE from surveillance specimens.

Anti-Bacterial Agents↗
PubMed1997

Detection of vancomycin-resistant enterococci in fecal samples by PCR.

Surveillance cultures for vancomycin-resistant enterococci (VRE) are time-consuming and expensive for the laboratory to perform. Therefore, we investigated the use of PCR as an alternative method of detecting and identifying VRE directly in fecal samples. PCR primers directed to vanA, vanB, vanC1, vanC2, and enterococcal ligase genes were used to detect and identify VRE in fecal material obtained by rectal or perirectal swabbing. Although PCR-inhibitory substances were present in DNA prepared directly from the swabs, the inhibitory substances could be reduced by processing the nucleic acid with two commercially available DNA preparation columns. Fecal material from 333 swabs was cultured on several selective agar media before and after broth enrichment. DNA was extracted from the fecal material and was analyzed by PCR. By using all four primer sets, only 59 (67.8%) of the samples were positive for vanA. However, after retesting the negative samples with only the vanA primer set, 77 (88.5%) of 87 specimens that were culture positive for Enterococcus faecium containing vanA were positive by PCR. One specimen was PCR positive for the vanA gene but culture negative for enterococci. The specificity of the vanA assay was 99.6%. PCR analysis of enrichment broth samples with all four primers sets after 15 to 18 h of incubation detected 74 (85.1%) of the 87 culture-positive specimens. The specificity of the vanA assay after the enrichment step was 100%. No vanB-containing enterococci were recovered by culture. Since 16 samples can be tested by PCR in 4 h (including electrophoresis), identification of VRE is possible within 8 h of specimen submission at a cost of approximately $10.12/assay. Thus, PCR may be a cost-effective alternative to culture for surveillance of VRE in some hospitals.

Anti-Bacterial Agents↗
PubMed1997

Molecular epidemiology and antibiotic susceptibility of enterococci in Cincinnati, Ohio: a prospective citywide survey.

To determine patterns of antimicrobial susceptibility among enterococci and to assess molecular characteristics of vancomycin-resistant enterococci, 157 clinical blood isolates of enterococci from 10 hospitals in Cincinnati, Ohio, were prospectively collected during a 6-month period from February to July 1995. The isolates included 108 (69%) E. faecalis isolates, 46 (29%) E. faecium isolates, and 1 isolate each of E. avium, E. durans, and E. gallinarum. The E. faecalis and E. faecium isolates differed in their susceptibilities to ampicillin (100 versus 20%), ampicillin-sulbactam (100 versus 13%), vancomycin (100 versus 57%), imipenem (94 versus 2%), and high levels of gentamicin (59 versus 83%). Supplemental susceptibility testing of the 21 vancomycin-resistant isolates showed that 21 (100%) were susceptible to chloramphenicol and that only 7 (33%) were susceptible to doxycycline. Nineteen (90%) of the vancomycin-resistant E. faecium isolates were of the VanB phenotype, with vanB resistance genes detected by PCR and hybridization with gene-specific probes; and the E. gallinarum isolates demonstrated the VanC phenotype with the vanC1 gene. One vancomycin-resistant E. faecium isolate was highly resistant to both teicoplanin and vancomycin, corresponding to the VanA phenotype; however, it was found to have the vanB gene. Pulsed-field gel electrophoresis (PFGE) revealed that all of the 19 E. faecium isolates with the VanB phenotype had identical to closely related banding patterns. Hybridization of restriction enzyme-digested DNA separated by PFGE with a vanB gene probe demonstrated differences in the locations of vanB genes that corresponded closely to the PFGE banding patterns. Our study has documented that the emerging vancomycin resistance in our city was mainly due to the clonal dissemination of a single strain of E. faecium VanB.

Anti-Bacterial Agents↗
PubMed1998

Use of molecular and reference susceptibility testing methods in a multicenter evaluation of MicroScan dried overnight gram-positive MIC panels for detection of vancomycin and high-level aminoglycoside resistances in enterococci.

Modified MicroScan gram-positive MIC no. 8 panels (PM-8) were analyzed for their improved ability to detect vancomycin resistance (VR) and high-level aminoglycoside resistance (HLAR) in enterococci. A validation study design that utilized selected challenge strains, recent clinical isolates, and reproducibility experiments in a multicenter format was selected. Three independent medical centers compared the commercial panels to reference broth microdilution panels (RBM) and Synergy Quad Agar (QA). Resistance was verified by demonstration of VR and HLAR genes by PCR tests. The study was conducted in three phases. (i) In the challenge phase (CP), two well-characterized sets of enterococci were obtained from the Centers for Disease Control and Prevention; one set contained 50 isolates for VR testing and one contained 48 isolates for HLAR testing. In addition, a set of 47 well-characterized isolates representing diverse geographic areas, obtained from earlier national surveillance studies, was tested at the University of Iowa College of Medicine (UICM). (ii) In the efficacy phase (EP), each laboratory tested 50 recent, unique clinical isolates by all methods. (iii) In the reproducibility Phase (RP), each laboratory tested the same 10 strains by all methods in triplicate on three separate days. All isolates from the EP were sent to the UICM for molecular characterization of vanA, -B, -C1, -C2-3, and HLAR genes. In the CP, the ranking of test methods by error rates (in parentheses; very major and major errors combined, versus PCR results) were as follows: for high-level streptomycin resistance (HLSR), QA (12.0%) > PM-8 (5.2%) > RBM (1.6%); for high-level gentamicin resistance (HLGR), RBM (3.7%) > PM-8 (3.1%) > QA (2.6%); and for VR, RBM = QA (3.0%) > PM-8 (1.2%). In the EP, agreement between all methods and the reference PCR result was 98.0% for HLSR, 99.3% for HLGR, and 98. 6% for VR. In the RP, the percentages of results +/- 1 log2 dilution of the all-participant mode were as follows: for VR, 100% (PM-8), 98.9% (QA), and 90.0% (RBM); for HLSR, 99.6% (RBM), 98.5% (PM-8), and 82.2% (QA); and for HLGR, 99.6% (RBM), 99.3% (PM-8), and 98.1% (QA). The ability of the PM-8 to detect VR and HLAR in enterococci was comparable to those for reference susceptibility and molecular PCR methods and was considered acceptable for routine clinical laboratory use.

Anti-Bacterial Agents↗
PubMed1998

Epidemiologic evaluation of antimicrobial resistance in community-acquired enterococci.

Fecal samples from 200 consecutive patients admitted to a community hospital yielded 107 enterococci. High-level gentamicin resistance occurred in 10 (14%) of the Enterococcus faecalis isolates. Ampicillin resistance occurred in two (3%) of the E. faecalis isolates and six (23%) of the Enterococcus faecium isolates. There were no vancomycin-resistant enterococci. Risk factors for enterococci with high-level aminoglycoside (gentamicin) or ampicillin resistance included prior hospitalization and previous antibiotic use.

Adult↗
PubMed1998

Emergence of vancomycin-resistant enterococci in Australia: phenotypic and genotypic characteristics of isolates.

Enterococci with resistance to glycopeptides have recently emerged in Australia. We developed multiplex PCR assays for vanA, vanB, vanC1, and vanC2 or vanC3 in order to examine the genetic basis for vancomycin resistance in Australian isolates of vancomycin-resistant Enterococcus faecium and E. faecalis (VRE). The predominant genotype from human clinical E. faecium isolates was vanB. The PCR van genotype was consistent with the resistance phenotype in all but six cases. One vanA E. faecalis isolate had a VanB phenotype, one vanB E. faecium isolate had a VanA phenotype, and four E. faecalis isolates were consistently negative for vanA, vanB, vanC1, and vanC2 or vanC3, even though they exhibited a VanB phenotype. These four isolates were subsequently examined for the presence of vanD by published methods and were found to be negative. No vancomycin-susceptible strains produced a PCR product. On the basis of our findings the epidemiology of VRE in Australia appears to be different from that in either the United States or Europe. Our multiplex PCR assays gave a rapid and accurate method for determining the genotype and confirming the identification of glycopeptide-resistant enterococci. Rapid and accurate methods are essential, because laboratory-based surveillance is critical in programs for the detection, control, and prevention of the transmission of glycopeptide-resistant enterococci.

Anti-Bacterial Agents↗
PubMed2004

Emergence of vanA Genotype Vancomycin-Resistant Enterococci with Low or Moderate Levels of Teicoplanin Resistance in Korea.

In Korea, vancomycin-resistant enterococci have become important nosocomial pathogens since the late 1990s, and most vancomycin-resistant enterococcal isolates have been VanA phenotype-vanA genotype strains. In 2001, we experienced an outbreak of VanB phenotype-vanA genotype vancomycin-resistant enterococci at a university hospital. This is the first report of VanB-vanA vancomycin-resistant enterococci from humans in Korea.

Adolescent↗
PubMed2005

In vitro activity of daptomycin versus linezolid and vancomycin against gram-positive uropathogens and ampicillin against enterococci, causing complicated urinary tract infections.

OBJECTIVES: The existing therapeutic options for complicated urinary tract infections (UTI) caused by gram-positive uropathogens are not always optimal. Therefore, newer antimicrobials have to be assessed. METHODS: The antimicrobial activity of daptomycin was tested versus linezolid, vancomycin, and ampicillin (enterococci on ly), against pathogens from three different collections: (1) Uropathogens from hospitalized urological patients with complicated and/or hospital-acquired UTIs of the Urologic Clinic, Hospital St. Elisabeth, Straubing. (2) Uropathogens from a multicenter study comprising 37 urological centers throughout Germany. (3) Methicillin-resistant Staphylococcus aureus (MRSA) isolates of patients and staff within the Hospital St. Elisabeth, Straubing. Genotyping of the latter isolates was performed by pulsed-field gel electrophoresis. The minimal inhibitory concentrations (MIC) of daptomycin, linezolid, vancomycin, and ampicillin (only tested against enterococci) were determined by an agar dilution method using a multipointer with an inoculum of 10(4) CFU per point. RESULTS: For all methicillin-susceptible Staphylococcus aureus (n = 25), MRSA (n = 49), methicillin-susceptible coagulase-negative staphylococci (n = 129), methicillin-resistant coagulase-negative staphylococci (n = 33), for Enterococcus faecalis (n = 289), and for Enterococcus faecium (n = 4) the MICs ranged up to 2 mg/l (daptomycin, linezolid), up to 4 mg/l (vancomycin), and up to 8 mg/l (ampicillin, enterococci only) indicating that all strains were susceptible to the antibiotics tested. CONCLUSIONS: According to the in vitro activity daptomycin may be considered a promising antibacterial agent for the treatment of complicated UTI caused by gram-positive uropathogens. Thus, daptomycin should be evaluated in a clinical study.

Acetamides↗
PubMed2005

The resazurin microplate method for rapid detection of vancomycin resistance in enterococci.

Vancomycin-resistant enterococci (VRE) are a serious challenge for physicians because of the limited treatment options for infections caused by this organism. Prevention of VRE transmission in hospitals requires early detection of infected or colonized patients. Therefore rapid and correct detection of vancomycin resistance is essential. In this study, we use the resazurin microplate method (RMM), which is a modification of the NCCLS and BSAC broth microdilution methods to rapidly determine the susceptibilities of clinical enterococci isolates to vancomycin. The alteration in the RMM was relevant to the final bacterial count. In this method, inoculum that was 10-fold higher than standard methods was used. A total of 80 enterococci, including 11 VRE isolates and 6 vancomycin intermediate isolates, were screened with this modified colorimetric broth microdilution method. After 4 h of incubation 30 microl of 0.01% resazurin solution were added to each well and the plates were reincubated for color change for 5-10 min. The MICs were obtained at the 4th h. The results were in exact agreement with the NCCLS and the BSAC microdilution methods. Absolute and essential agreements were 100% and there were no minor, major or very major errors. In conclusion, this modified colorimetric broth microdilution method can be used as a reliable, easy, cheap and rapid method for early detection of VRE. Moreover, this method has the potential of being used to test the susceptibilities of different bacteria to other antibiotics.

Colony Count, Microbial↗
PubMed2001

Vancomycin resistance reversal in enterococci by flavonoids.

The development of clinical vancomycin-resistant strains of enterococci (VRE) is a major cause for concern. Here we show that a combination of galangin or 3,7-dihydroxyflavone with vancomycin may be used to sensitize resistant strains of Enterococcus faecalis and Enterococcus faecium to the level of vancomycin-sensitive strains. Minimum inhibitory concentrations (MICs) and viable counts were determined in Iso-sensitest broth using a microtitre method. MICs of vancomycin against 67% of resistant clinical isolates and a type strain of enterococci were lowered from > 250 microg mL(-1) to < 4 microg mL(-1) in the presence of galangin (12.5 microg mL(-1)) or 3,7-dihydroxyflavone (6.25 microg mL(-1)). Viable counts for type culture E. faecalis ATCC 51299 showed the flavonoids alone significantly lowered numbers of colony forming units (CFUs). CFUs were maintained at low levels (< 10(3) CFU mL(-1)) for 24 h by vancomycin/flavone combinations. This combinational action in reversing vancomycin resistance of enterococci highlights novel drug targets and has importance in the design of new therapeutic regimes against resistant pathogens.

Anti-Bacterial Agents↗
PubMed2000

Control of glycopeptide-resistant enterococci in an oncology unit.

Enterococci are responsible for 10-12% of nosocomial infections. Since 1987 the incidence of glycopeptide-resistant enterococci (GRE; termed vancomycin-resistant enterococci in the United States) has increased dramatically. The mechanism of GRE is well understood and involves mutation of a single terminal amino acid in a peptidoglycan precursor leading to reduced vancomycin affinity. Studies implicated antibiotic selection pressure as a major risk factor for GRE infection and colonization. In the hematology unit of a London hospital, GRE emerged in December 1993, with 38% of patients positive in a point prevalence study. Between December 1993 and June 1995, 13 patients acquired GRE bacteremia, five of whom died. Between 1995 and 1996 a prospective sequential study was undertaken to determine the effect of changing antibiotic treatment of febrile neutropenia (FN). All patients admitted to the hematology unit were enrolled in the study. In phase 1, treatment continued with ceftazidime monotherapy. In phase 2, piperacillin-tazobactam replaced ceftazidime, and enhanced infection control measures were encouraged. In phase 3, therapy returned to ceftazidime. In phase 1, 57% of patients became colonized or infected with GRE. Phase 2, which was divided into two 4-month cohorts, showed a significant reduction in GRE acquisition, falling to 8% colonization and no clinical infections in the second 4 months. In phase 3, despite heightened infection control procedures, the acquisition rate rose to 36%.

Anti-Bacterial Agents↗
PubMed2006

Detection of genes encoding virulence factors and bacteriocins in fecal enterococci of poultry in Portugal.

Seventy-six Enterococcus isolates (43 E. faecalis, 30 E. faecium, two E. durans, and one E. hirae) recovered from fecal samples of poultry in a slaughterhouse (one isolate per fecal sample and one fecal sample per lot of animals) were studied for bacteriocin production and for the presence of genes encoding bacteriocins and virulence factors. The presence of genes encoding virulence factors (cpd, geE, fsr, ace, agg, and esp) and bacteriocins (entA, entB, entP, entQ, entAS-48, entL50A/B, cyl, and bac31) were studied by polymerase chain reaction in all enterococci. At least two virulence genes were detected in all 43 E. faecalis isolates, cpd and gelE being the most frequently detected genes (97.7%) followed by ace (62.8%), agg (39.5%), fsr (27.9%), and esp (2.3%). No virulence genes were detected in the other enterococcal species with the exception of one E. faecium and one E. durans isolates that harbored the gelE gene. Antimicrobial activity against eight indicator bacteria (including Listeria monocytogenes) was assayed in the enterococci, and 23 (30.3%) showed inhibitory activity against L. monocytogenes, the other 22 enterococci showing activity against indicator bacteria other than L. monocytogenes. Only the entA, entB, and cyl genes were detected in our study (entA + entB in nine E. faecium isolates and the cyl gene in seven E. faecalis isolates). A wide variety of virulence genes have been detected in fecal E. faecalis isolates from poultry, but not in the other enterococcal species. However, the presence of known bacteriocin structural genes is associated more with the E. faecium species.

Animals↗
PubMed2006

Analysis of enterococci using portable testing equipment for developing countries--variance of Azide NutriDisk medium under variable time and temperature.

This report compares the enterococci count on samples obtained with Azide NutriDisk (AND) (sterile, dehydrated culture medium) and Slanetz and Bartley (SB) medium when exposed to a variable in incubation time and temperature. Three experiments were performed to examine the recovery of enterococci on AND and SB media using membrane filtration with respect to: (a) incubation time; (b) incubation temperature; and (c) a combination of the two. Presumptive counts were observed at 37, 41, 46 and 47 degrees C and at 20, 24, 28 and 48 h. These were compared to AWWA standard method 9230 C (44 degrees C, 44 h). Samples were confirmed using Kanamycin Aesculin Azide (KAA) agar. Friedman's ANOVA and Students t-test analysis indicated higher enumeration of enterococci when grown on AND (p = 0.45) than SB (p = < 0.001) at all temperatures with a survival threshold at 47 degrees C. Significant results for AND medium were noted at 20 h (p = 0.021), 24 h (p = 0.278) and 28 h (p = 0.543). The study concluded that the accuracy of the AND medium at a greater time and temperature range provided flexibility in incubator technology making it an appropriate alternative to SB medium for monitoring drinking water using field testing kits in developing countries.

Colony Count, Microbial↗
PubMedDate not supplied

Comparison of E. coli, enterococci, and fecal coliform as indicators for brackish water quality assessment.

Escherichia coli (E. coli), enterococci, and fecal coliform data were collected and compared as potential indicators for swimmablility assessment of a brackish waterbody (Lake Pontchartrain, Louisiana). These indicators were measured during lake background conditions, in stormwater runoff (before dilution with lake water), and in the outfall plume within the lake following storm events. Microbial indicator titers associated with suspended particles and lake-bottom sediments were also investigated. Overall reduction rate constants for fecal coliform, E. coli, and enterococci in lake water and sediment were measured and reported. Attachment of microbial indicators to suspended matter and subsequent sedimentation appeared to be a significant fate mechanism. A slower reduction rate of indicator organisms in sediment further suggested that bottom sediment may act as a reservoir for prolonging indicator organism survival and added concern of recontamination of overlaying waters due to potential solids resuspension. Results indicated that enterococci might be a more stable indicator than E. coli and fecal coliform and, consequently, a more conservative indicator under brackish water conditions.

Enterobacteriaceae↗
PubMedDate not supplied

Diversity among multidrug-resistant enterococci.

Enterococci are associated with both community- and hospital-acquired infections. Even though they do not cause severe systemic inflammatory responses, such as septic shock, enterococci present a therapeutic challenge because of their resistance to a vast array of antimicrobial drugs, including cell-wall active agents, all commercially available aminoglycosides, penicillin and ampicillin, and vancomycin. The combination of the latter two occurs disproportionately in strains resistant to many other antimicrobial drugs. The propensity of enterococci to acquire resistance may relate to their ability to participate in various forms of conjugation, which can result in the spread of genes as part of conjugative transposons, pheromone-responsive plasmids, or broad host-range plasmids. Enterococcal hardiness likely adds to resistance by facilitating survival in the environment (and thus enhancing potential spread from person to person) of a multidrug-resistant clone. The combination of these attributes within the genus Enterococcus suggests that these bacteria and their resistance to antimicrobial drugs will continue to pose a challenge.

Animals↗
PubMedDate not supplied

Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future.

Enterococci, leading causes of nosocomial bacteremia, surgical wound infection, and urinary tract infection, are becoming resistant to many and sometimes all standard therapies. New rapid surveillance methods are highlighting the importance of examining enterococcal isolates at the species level. Most enterococcal infections are caused by Enterococcus faecalis, which are more likely to express traits related to overt virulence but--for the moment--also more likely to retain sensitivity to at least one effective antibiotic. The remaining infections are mostly caused by E. faecium, a species virtually devoid of known overt pathogenic traits but more likely to be resistant to even antibiotics of last resort. Effective control of multiple-drug resistant enterococci will require 1) better understanding of the interaction between enterococci, the hospital environment, and humans, 2) prudent antibiotic use, 3) better contact isolation in hospitals and other patient care environments, and 4) improved surveillance. Equally important is renewed vigor in the search for additional drugs, accompanied by the evolution of new therapeutic paradigms less vulnerable to the cycle of drug introduction and drug resistance.

Anti-Bacterial Agents↗