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Yield of vancomycin-resistant enterococci and multidrug-resistant Enterobacteriaceae from stools submitted for Clostridium difficile testing compared to results from a focused surveillance program.

It has been suggested that a method of performing surveillance for vancomycin-resistant enterococci (VRE) is to screen specimens submitted for Clostridium difficile testing. We compared this approach to our focused surveillance program of high-risk units during October 1997 to compare the yield of VRE and multidrug-resistant Enterobacteriaceae (MDRE) with both methods. Of the stools submitted for C. difficile testing, 14% were positive for VRE or MDRE, whereas rectal swabs from routine surveillance yielded 11% VRE- or MDRE-positive results. Although stools submitted for C. difficile testing resulted in a higher percentage of positive cultures, 14 VRE- and 2 MDRE-positive patients from our high-risk population were missed because many patients had no stool submitted for C. difficile testing. Therefore, while screening stools submitted for C. difficile testing cannot replace our focused surveillance program, it appears advantageous to assess these stools at various intervals to detect new patient reservoirs of drug-resistant organisms that may benefit from routine surveillance cultures.

Clostridioides difficile↗

Molecular characterization of vancomycin-resistant Enterococci repopulating the gastrointestinal tract following treatment with a novel glycolipodepsipeptide, ramoplanin.

We characterized baseline and repopulating stool isolates recovered during a phase II trial of ramoplanin for the treatment of patients with stool carriage of vancomycin-resistant enterococci (VRE). Repopulation with a strain with a related genotype was found in 74, 60, and 53% of individuals in groups treated with placebo, 100 mg of ramoplanin, and 400 mg of ramoplanin, respectively. All ramoplanin-treated patients with a culture positive for VRE at day 7 had a relapse caused by a genotypically related isolate. In ramoplanin-treated patients, antibiotics with activities against anaerobic organisms were associated with positive cultures on day 7 (relative risk [RR] = 8.8; P = 0.004), and the avoidance of such antibiotics was significantly associated with culture negativity through day 21 (RR = 0.16; P = 0.02).

Anti-Bacterial Agents↗

Laboratory contamination of specimens with quality control strains of vancomycin-resistant enterococci in Ontario.

Cross-contamination with laboratory control strains of vancomycin-resistant Enterococcus faecalis was documented in 15 clinical specimens from nine clinical microbiology laboratories in Ontario, Canada. Laboratories should be alert to the possibility of contamination of specimens with vancomycin-resistant enterococci from the laboratory environment. Molecular typing of strains may assist in elucidating the source of such contamination.

DNA, Bacterial↗

Comparative study using type strains and clinical and food isolates to examine hemolytic activity and occurrence of the cyl operon in enterococci.

The hemolytic ability, the presence of cyl genes, and the diagnostic accuracy of cytolysin molecular detection were investigated in the genus Enterococcus by using 164 strains from 20 different species (26 reference strains, 42 clinical isolates from human and veterinary origin, and 96 isolates from ewe cheese and milk). Hemolysis was assayed with sheep and horse erythrocytes and under aerobic or anaerobic conditions. Screening of cytolysin genes (cylL(L), cylL(S), cylM, cylB, and cylA) was performed with new specific primers and the anaerobic assay of beta-hemolysis was used as the "gold standard" for the evaluation of cyl gene-based PCRs. Since beta-hemolysis and cyl genes were found in 10 and 14 species, respectively, the hemolytic ability seems to be spread throughout the genus ENTEROCOCCUS: Beta-hemolysis was observed in 6 of 26 (23%) reference strains, 14 of 42 (33%) clinical isolates, and 6 of 96 (6%) food isolates. The presence of cyl genes was detected in 15 of 26 (58%) reference strains, 37 of 42 (88%) clinical isolates, and 67 of 96 (70%) food isolates. These data indicate a virulence potential in food isolates, reinforcing the need of their safety assessment. Analysis of phenotypic-genotypic congruence suggests a divergent sequence evolution of cyl genes and the effect of environmental factors in the regulation of cytolysin expression. Evaluation of the diagnostic accuracy of cytolysin molecular detection points to cylL(L)-based PCR and cylL(L)L(S)MBA-based PCR as the most reliable approaches. Nevertheless, the low sensitivity (46%) and gene variability indicated by our study strongly recommend the phenotypic assay for the assessment of hemolytic ability in enterococci, followed by the molecular screening of cyl genes in nonhemolytic strains to evaluate their virulence potential.

Animals↗

Detection of the van alphabet and identification of enterococci and staphylococci at the species level by multiplex PCR.

A multiplex PCR assay was developed for detection of the six types of glycopeptide resistance characterized in enterococci and for identification of Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis at the species level. Primers targeting the genes vanA, vanB, vanC, vanD, vanE, vanG, and ddl of E. faecium and E. faecalis and nuc of S. aureus and a chromosomal portion specific to S. epidermidis were designed to allow amplification of fragments with various sizes. This specific and sensitive technique allows detection of glycopeptide-resistant strains, in particular methicillin-resistant S. aureus, that may escape phenotype-based automated rapid methods.

Anti-Bacterial Agents↗

Comparison of the Roche LightCycler vanA/vanB detection assay and culture for detection of vancomycin-resistant enterococci from perianal swabs.

We compared the performance characteristics of a real-time PCR method, the LightCycler vanA/vanB detection assay (Roche Diagnostics Corporation, Indianapolis, Ind.) to that of Enterococcosel agar (BBL, Sparks, Md.) for direct detection of vancomycin-resistant enterococci (VRE) from 894 perianal stool swabs. For 421 of 894 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing vancomycin at 6 microg/ml; for the remaining 473 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing 8 microg/ml vancomycin. The LightCycler method produced considerably more positive results than either the Enterococcosel plate containing vancomycin at 6 microg/ml (n = 25 versus n = 11; sensitivity, 100%; specificity, 97%; positive predictive value [PPV], 42%; negative predictive value [NPV], 100%) or the Enterococcosel plate containing vancomycin at 8 microg/ml (n = 31 versus n = 10; sensitivity, 100%; specificity, 95%; PPV, 32%; NPV, 100%). When possible, additional testing, including culture, LightCycler PCR, and/or a conventional PCR method (PCR-restriction fragment length polymorphism assay), were performed on either the original specimens or original cultures or subsequent specimens for cases in which the original specimen was positive by LightCycler PCR but the Enterococcosel plate was negative. This additional testing demonstrated positive results for 7 of 14 (50%) evaluable discordant specimens which initially tested as LightCycler PCR positive but culture negative using the Enterococcosel plate containing vancomycin at 6 microg/ml and 12 of 17 (71%) evaluable discordant specimens which initially tested as LightCycler positive but culture negative using the Enterococcosel plate containing vancomycin at (8 microg/ml). These results demonstrate that the LightCycler VRE detection assay is considerably more sensitive than the standard culture method for detecting VRE directly from perianal swab specimens. The LightCycler assay also provides results much faster than culture (approximately 3.5 versus > or =72 h). The use of this test could have important implications for the effective control and prevention of nosocomial outbreaks of VRE.

Anal Canal↗

Epidemiology and molecular analysis of intestinal colonization by vancomycin-resistant enterococci in greek hospitals.

From 1,246 specimens collected from 13 Greek hospitals, 266 vancomycin-resistant enterococci strains were isolated from 255 patients (20.5%). The VanA phenotype was present in 82 (30.8%) strains, the VanB phenotype in 17 (6.4%) strains, the VanC1 phenotype in 152 (57.1%) strains, and the VanC2/C3 phenotypes in 15 (5.6%) strains. When only VanA and VanB phenotypes were considered, the overall prevalence was 7.5%. Eighty-six isolates exhibiting the VanA or VanB phenotype were analyzed by pulsed-field gel electrophoresis (PFGE), and 46 PFGE groups were found.

Anti-Bacterial Agents↗

Phylogeny and identification of Enterococci by atpA gene sequence analysis.

The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed > 99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci.

Animals↗

Action and interaction of penicillin and gentamicin on enterococci.

The action and interaction of benzylpenicillin and gentamicin on Streptococcus faecalis was studied using mainly turbidimetric methods. The minimum antibacterial concentration (MAC) of each antibiotic lay considerably below the conventionally determined minimum inhibitory concentration, and levels of the two agents exceeding the MAC were necessary in order to obtain a synergic interaction. Evidence was obtained that gentamicin interfered with bacterial lysis induced by penicillin, and this suggests that the aminoglycoside is responsible for the bactericidal activity of the combination, the role of the penicillin being solely to facilitate access of the aminoglycoside to its target site. Our findings do not, however, fully support the generally held view that the increased permeability of enterococci to aminoglycosides is due to penicillin-induced cell wall damage. 'Persisters'--cells surviving prolonged exposure to the optimum lethal concentration of penicillin--were not killed by subsequent exposure to gentamicin if the penicillin was removed but were killed if the penicillin remained present.

Dose-Response Relationship, Drug↗

Gentamicin-resistant enterococci and endocarditis.

We report a case of endocarditis associated with a highly gentamicin-resistant enterococcus that failed to respond to conventional antibiotic combination therapy. Clinical resolution was achieved following cardiac surgery and antimicrobial treatment dictated by in vitro testing of the patient's organism. Laboratories should test blood culture isolates of enterococci for high level gentamicin resistance. Management of such cases requires individual assessment and extensive laboratory investigation.

Aged↗

Evidence of thermonuclease production by Bacillus spp. and enterococci in naturally contaminated cheese.

Of 105 thermonuclease-positive (TNase-positive) cheese samples comprising 13 types, 92 (87.6%) contained coagulase-positive staphylococci, whereas 9 (8.6%) contained microorganisms other than staphylococci as the major contaminants. Of the latter group, six samples contained Bacillus spp. comprising three species (B. cereus, B. licheniformis, and B. subtilis), and three contained mainly enterococci (Streptococcus faecalis), which were proven to be TNase producers. The organisms responsible for TNase production in the other four samples (3.8%) are not known, because isolates from these samples failed to produce the enzyme. Unlike staphylococcal TNase, a greater part of nonstaphylococcal TNase remains in the cheese homogenate after extraction of the enzyme at pH 3.8 instead of pH 4.5.

Bacillus↗

Increasing prevalence of high-level gentamicin resistance among Enterococci isolated in Greece.

High-level gentamicin-resistant enterococci (HLGRE) (MIC >512 microg/ml) were frequently recovered in the AHEPA University Hospital, Thessaloniki, Greece, during a 15-month period (67/158, 42.4%). PCR testing showed that high-level gentamicin resistance was attributable to the presence of the aac(6')-aph(2") gene, while the recently described genes aph (2")-Ic and aph(2")-Id were not detected. High-level gentamicin resistance could be transferred with varying frequency from 41 of 57 Enterococcus faecalis and 5 of 10 Enterococcus faecium isolates. All transconjugants acquired high-level resistance to streptomycin. HLGRE cause therapeutic problems in our region, since none of the clinically available aminoglycosides can be used in severe infections.

Anti-Bacterial Agents↗

Inhibitory and bactericidal effects of telithromycin (HMR 3647, RU 56647) and five comparative antibiotics, used singly and in combination, against vancomycin-resistant and vancomycin-susceptible enterococci.

The inhibitory and bactericidal effects of telithromycin (HMR 3647, RU 66647) were compared with those of gentamicin, ampicillin, erythromycin, azithromycin and vancomycin against 74 strains of enterococci (34 Enterococcus faecalis and 40 Enterococcus faecium) by agar dilution, broth dilution, time kill assays and postantibiotic effect (PAE). The telithromycin MIC(90) for vancomycin-sensitive (VSE) E. faecalis strains tested using the agar dilution method was 8 microg/ml. For a different group of VSE E. faecalis strains tested using the broth dilution method it was 0.06 microg/ml The telithromycin MIC(90)s for vancomycin-resistant (VRE) and VSE E. faecium strains, determined using the agar dilution method, were 4 and 8 microg/ml, respectively, while for a different set of VRE and VSE E. faecium strains tested using the broth macrodilution method, they were 32 and 16 microg/ml, respectively. Telithromycin MBC(90)s for E. faecalis were 4-6 tubes higher and for E. faecium 3-5 tubes higher, respectively, than the MIC(90)s. In time kill assays, telithromycin had bactericidal activity against only 1 of 7 E. faecium strains; for all other E. faecium and E. faecalis strains, only inhibitory activity was demonstrated. Neither synergy nor drug interference was observed when telithromycin was used in combination with ampicillin, vancomycin or gentamicin. At 10 times the MIC, the PAE of telithromycin against E. faecalis was 2.8 h, while for E. faecium it was 1.6 h. Telithromycin should be evaluated for therapy of enterococcal infections, including those caused by VRE organisms. However, because of the strain-to-strain variability in susceptibility to telithromycin, MIC determinations are important, especially for erythromycin-resistant strains.

Anti-Bacterial Agents↗

Outbreak of vancomycin-resistant enterococci (VRE) in a hematological oncology ward and hygienic preventive measures. A long-term study.

BACKGROUND: We report on two endemic outbreaks of VRE (vancomycin-resistant enterococci, vanA type Enterococcus faecium) on a hematological oncology ward in a university hospital and a new strategic concept to fight against spread of VRE. PATIENTS AND METHODS: During management of the outbreak, a total of 1,124 patients and 1,700 specimens were investigated from June 1999 to December 2001. Protective measures were instituted. Intensive prophylactic infection control was practiced until December 2003. RESULTS: Of the 1,124 patients, 44 were VRE-positive. From 1,700 environment specimens, 110 VRE isolates were obtained from the inanimate environment, 5 from the living environment. Molecular biological typing identified 5 different clones which had spread on the ward in different locations. CONCLUSION: The study shows the need for prophylactic microbiological screening investigations in the risk group of hematological oncology patients and consistent implementation of protective hygiene measures to prevent the spread of multiresistant infectious pathogens on risk wards. It is also shown that intervention can to control such an endemic outbreak.

Anti-Bacterial Agents↗

Comparison of in vitro inhibitory and bactericidal activities of daptomycin (LY 146032) and four reference antibiotics, singly and in combination, against gentamicin-susceptible and high-level-gentamicin-resistant enterococci.

The in vitro inhibitory and bactericidal activities of daptomycin and reference antibiotics were determined by serial macrobroth dilution for 23 gentamicin-susceptible (MIC: < 2,000 mg/l) and 21 high-level-gentamicin-resistant (HLGR) Enterococcus faecalis, Enterococcus faecium, and Enterococcus avium isolates. The activities of daptomycin, vancomycin, and teicoplanin were independent of the gentamicin susceptibility profile and species tested. For all the isolates, the inhibitory activity of daptomycin (MIC90: 2 mg/l) was comparable to vancomycin (MIC90: 2 mg/l), but less than that of teicoplanin (MIC90: 0.5 mg/l). Daptomycin demonstrated excellent bactericidal activity against all enterococci tested (MBC90: 8 mg/l). In contrast, all microorganisms were tolerant to vancomycin and teicoplanin. Ampicillin and ciprofloxacin MICs and MBCs were dependent on enterococcal gentamicin resistance profile and species, with MICs and MBCs that ranged between 1 and > 64 mg/l. By time-kill curves, the combination of daptomycin plus ampicillin demonstrated synergistic bactericidal activity against gentamicin-susceptible and HLGR E. faecalis. Daptomycin, singly and in combination, may be useful in treating enterococcal infections, including those caused by HLGR microorganisms.

Ampicillin↗