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PubMed1997

Potential for gene transfer among enterococci from a single patient and the possibility of confounding typing results.

The results of typing methods used in a study of the epidemiology of enterococci in burn patients showed inconsistencies. The possibility that these inconsistencies were the result of gene acquisition or loss was investigated by using 12 isolates of Enterococcus faecalis from a single patient. In vivo and in vitro exchange and loss of genes were observed. The study showed that the typing results for isolates from this patient can be modified by known and demonstrated genetic elements; as a result, the isolates could be divided into between three and seven strains. In the present study, the SmaI digestion patterns gave the most consistent results, correctly identifying the transconjugants as indistinguishable from recipient strain 196R.

Bacterial Typing Techniques↗
PubMed1997

Rapid characterization schemes for surveillance isolates of vancomycin-resistant enterococci.

Surveillance cultures for vancomycin-resistant enterococci (VRE) and subsequent characterization of the isolates can be extremely burdensome and difficult. Therefore, efficient and reliable schemes for the characterization of surveillance isolates are needed. In this study, a commercial agar (bile esculin azide agar with 6 microg of vancomycin per ml [BEAA]; Remel, Lenexa, Kans.) was used in the initial screening step to establish relatively rapid (i.e., in < or = 24 h from the time of isolation) phenotype-based and PCR-based schemes for the detection and characterization of VRE. The phenotype-based scheme included Gram staining of growth on BEAA and subculture of cocci on sheep blood agar plates for vancomycin disk diffusion and pyrazinamidase (PYR) testing. For the PCR scheme, inocula for van gene detection were taken directly from the BEAA plates. The phenotypic approach was applied to 378 surveillance cultures that yielded growth on BEAA. Gram staining quickly eliminated gram-positive bacilli from further testing, and a negative PYR test classified 25 additional isolates as probable pediococci. A positive PYR test reliably identified 121 single-patient VRE isolates that included 83 Enterococcus faecium, 33 E. gallinarum, and 5 E. casseliflavus strains. The vancomycin inhibition zone size clearly distinguished VanA and VanB strains from VanC strains within 24 h of BEAA isolation. All VanA and VanB strains failed to produce zones of >6 mm, while only one VanC strain produced a zone of < 15 mm. Challenging this phenotypic scheme with 47 stock VRE isolates produced similar findings. In direct PCR analyses, false-negative vanA and vanB results occurred with 12% of the specimens. Many of the false-negative reactions also failed to produce an internal control product, which underscores the need for including control primers when a PCR scheme is used. In summary, the phenotype- and the PCR-based schemes provide efficient methods for characterizing VRE within 24 h of isolation.

Anti-Bacterial Agents↗
PubMed1998

Comparison of eight methods to detect vancomycin resistance in enterococci.

A collection of genetically unrelated vancomycin-resistant enterococci (VRE) including 50 vanA, 15 vanB, 50 vanC1, and 30 vanC2 VRE were used to evaluate the accuracy of eight currently available susceptibility test methods (agar dilution, disk diffusion, E-test, agar screen plate, Vitek GPS-TA and GPS-101, and MicroScan overnight and rapid panels). vanA VRE were detected by all methods. vanB VRE were often not detected by Vitek GPS-TA and MicroScan rapid (sensitivities, 47 and 53%, respectively), though the new Vitek GPS-101 was found to be a significant improvement. E-test and the agar screen were the only two methods detecting all VRE, including the vanC1/C2 VRE.

Anti-Bacterial Agents↗
PubMed1998

Characterization of glycopeptide-resistant enterococci from a Swiss hospital.

Between August 1994 and September 1996, 28 glycopeptide-resistant enterococci (GRE) were isolated from 8 infected patients and 11 intestinal carriers hospitalized at the University Hospital of Geneva. Identification to the species was made by both phenotypic (API 20 STREP and Rapid ID 32 STREP systems, and Vitek Gram Positive Identification Card) and genotypic methods using a multiplex PCR assay developed also for the determination of the genotype of glycopeptide resistance (vanA, vanB, vanC1, and vanC2-C3 genes). Fifteen isolates were identified as Enterococcus faecium, 8 as E. gallinarum, 4 as E. faecalis, and 1 as E. hirae. All of the phenotypic identification methods failed to differentiate some isolates of E. gallinarum from E. faecium, or vice versa. Both vanA (n = 18) and vanB (n = 4) glycopeptide resistance genotypes were found. For the first time, the vanB determinant was found in two isolates of E. gallinarum. Two patients were colonized by two different species containing the vanA gene and one by two different species containing the vanB gene. All vanA isolates were highly resistant to both vancomycin and teicoplanin except for three isolates which were susceptible to teicoplanin. Molecular typing by pulsed-field gel electrophoresis showed identical or similar patterns among E. faecium isolates with the vanA gene in five patients for whom the epidemiological link could not be always elucidated. This study emphasizes the necessity of utilizing both phenotypic and genotypic methods to characterize GRE.

Anti-Bacterial Agents↗
PubMed1998

Molecular characterization of vancomycin-resistant enterococci from hospitalized patients and poultry products in The Netherlands.

Vancomycin-resistant enterococci (VRE) pose an emerging health risk, but little is known about the precise epidemiology of the genes coding for vancomycin resistance. To determine whether the bacterial flora of consumer poultry serves as a gene reservoir, the level of contamination of poultry products with VRE was determined. VRE were genotyped by pulsed-field gel electrophoresis (PFGE), and transposon structure mapping was done by PCR. The vanX-vanY intergenic regions of several strains were further analyzed by sequencing. A total of 242 of 305 (79%) poultry products were found to be contaminated with VRE. Of these VRE, 142 (59%) were high-level-vancomycin-resistant Enterococcus faecium strains (VREF). PFGE revealed extensive VREF heterogeneity. Two genotypes were found nationwide on multiple occasions: type A (22 of 142 VREF [15%]) and type B (14 of 142 VREF [10%]). No PFGE-deduced genetic overlap was found when VREF from humans were compared with VREF from poultry. Two vanA transposon types were identified among poultry strains. In 59 of 142 (42%) of the poultry VREF, the size of the intergenic region between vanX and vanY was approximately 1,300 bp. This transposon type was not found in human VREF. In contrast, all human strains and 83 of 142 (58%) of the poultry VREF contained an intergenic region 543 bp in size. Sequencing of this 543-bp intergenic vanX-vanY region demonstrated full sequence conservation. Though preliminary, these data suggest that dissemination of the resistance genes carried on transposable elements may be of greater importance than clonal dissemination of resistant strains. This observation is important for developing strategies to control the spread of glycopeptide resistance.

Animals↗
PubMed1999

Comparison of simple and rapid methods for identifying enterococci intrinsically resistant to vancomycin.

Three different methodologies, reduction of litmus milk (LM) and acidification of arabinose (ARA), acidification of methyl-alpha-D-glucopyranoside (MGP), and rapid motility (RM), for differentiating isolates of Enterococcus casseliflavus and Enterococcus gallinarum (intrinsically vancomycin-resistant enterococci [IVRE]) from Enterococcus faecalis and Enterococcus faecium were evaluated. All 33 isolates of E. faecalis tested reduced LM within 4 h and were negative in all other tests, while the 53 isolates of E. faecium were ARA positive only. In contrast, 45 of 46 (98%) IVRE isolates examined (26 E. casseliflavus and 20 E. gallinarum isolates) acidified MGP, 41 of 46 (89%) were LM and ARA positive, and 45 of 46 (98%) were RM positive. Acidification of MGP was therefore the single most useful test for differentiating IVRE from vancomycin-resistant E. faecium and E. faecalis; however, a combination of LM-ARA and RM testing enabled the correct designation of organisms without the need for overnight incubation.

Anti-Bacterial Agents↗
PubMed1999

Variation in structure and location of VanA glycopeptide resistance elements among enterococci from a single patient.

Forty-six VanA glycopeptide-resistant enterococci (GRE) from a single patient were investigated for variation in structure and location of VanA resistance elements. Together with identification to species level and pulsed-field gel electrophoresis, these data divided the GRE into 10 groups and subgroups. Combining data in this manner appears helpful when investigating the epidemiology of GRE.

Bacterial Proteins↗
PubMed1999

Comparison of direct plating and broth enrichment culture for the detection of intestinal colonization by glycopeptide-resistant enterococci among hospitalized patients.

The results of prevalence studies on glycopeptide-resistant enterococci (GRE) in the intestine may be influenced by the detection methods applied. In most studies different media, different concentrations of antibiotics, and different methods are used, and these differences result in differences in recovery rates. In this cross-sectional study on the carrier state of GRE among patients at the University Hospital Antwerp, Antwerp, Belgium, performed on 21 May 1996, direct plating and broth enrichment were compared by using the same media. Stool samples (n = 213) or rectal swabs (n = 122) were plated directly on Enterococcosel agar (bioMérieux) and after enrichment in Enterococcosel broth. The prevalence of GRE was 12.8%. Direct plating recovered 53.4% of the GRE isolates, and broth enrichment recovered an additional 46.5% of them; in the latter test the isolates were thus present at less than 10(3) CFU per g of feces. The prevalence of GRE among dialysis patients was higher than among the other patients, but the difference was not significant (P = 0.06), possibly as a result of the small numbers of dialysis patients examined. The GRE species isolated included 19 E. gallinarum (44.2%), 13 E. faecium (30.2%), 6 E. faecalis (13.9%), and 5 E. casseliflavus (11.6%) isolates. All E. faecalis and E. faecium strains isolated carried the vanA gene, and E. gallinarum and E. casseliflavus carried the vanC1 and vanC2 gene, respectively. The majority of isolates were polyclonal. Our data indicate that the rate of detection of GRE from both stool samples and rectal swabs is significantly increased with enrichment cultures.

Anti-Bacterial Agents↗
PubMed1999

Survey of antibiotic resistance among enterococci in North Rhine-Westphalia, Germany.

A surveillance study on antibiotic resistance of enterococcal isolates (n = 730) was carried out in North Rhine-Westphalia, Germany, in 1997. Resistance rates to ampicillin (7.4%), high-level gentamicin (15.0%), high-level streptomycin (27.9%), ciprofloxacin (37.9%), vancomycin (1.5%), and teicoplanin (1.5%) were determined. All vancomycin-resistant enterococci (VRE) carried the vanA gene. SmaI and ApaI macrorestriction patterns indicated an intra- and interhospital spread of VRE.

Bacterial Proteins↗
PubMed1999

Detection of clinically relevant genotypes of vancomycin-resistant enterococci in nosocomial surveillance specimens by PCR.

This study evaluated a PCR method for the rapid detection of clinically significant genotypes of vancomycin-resistant enterococci (VRE) in nosocomial surveillance specimens. Detection of the vanA and vanB genes by multiplex PCR using 657 specimens that showed presumptive growth of VRE on bile esculin azide agar containing 6 mg of vancomycin/liter was compared to the conventional method. The diagnostic values for the PCR compared to the phenotypic method were as follows: 99.8% specificity, 95.4% sensitivity, 98.8% positive predictive value, and 99.3% negative predictive value. The average cost per test for PCR is $8.26, compared to $9.45 for the phenotypic method. The average turnaround time for detecting a VRE is 48 h for PCR, compared to 96 h for the conventional method.

Cross Infection↗
PubMed2000

Comparison of the Rodac imprint method to selective enrichment broth for recovery of vancomycin-resistant enterococci and drug-resistant Enterobacteriaceae from environmental surfaces.

We compared the Rodac imprint technique to selective enrichment broth for detecting vancomycin-resistant enterococci (VRE) and multidrug-resistant Enterobacteriaceae (MDRE) on surfaces. Rodac plates contained tryptic soy agar with 5% sheep blood, vancomycin (6 microg/ml), ceftazidime (2 microg/ml), amphotericin B (2 microg/ml), and clindamycin (1 microg/ml). Two types of broth were used: brain heart infusion (BHI) and BHI plus vancomycin (6 microg/ml) and ceftazidime (2 microg/ml) (BHIVC). Of the 46 surfaces cultured for VRE, 12 (26%) were positive. Of the 12 VRE-positive surfaces, 11 (92%) grew from Rodac, 8 (67%) grew from BHIVC, and 7 (58%) grew from BHI. A larger study is needed for MDRE, as only 4 of 43 surfaces were MDRE positive. The Rodac imprint technique successfully recovered VRE from environmental surfaces.

Culture Media↗
PubMed2000

Prevalence of vancomycin-resistant enterococci in fecal samples from hospitalized patients and nonhospitalized controls in a cattle-rearing area of France.

Vancomycin-resistant enterococci (VRE) have emerged as nosocomial pathogens over the last decade, but little is known about their epidemiology. We report on the prevalence of VRE fecal colonization on the basis of a prospective study among patients hospitalized in a hematology intensive care unit and among nonhospitalized subjects living in the local community. A total of 243 rectal swabs from hematology patients and 169 stool samples from the control group were inoculated onto bile-esculin agar plates with and without 6 mg of vancomycin per liter and into an enrichment bile-esculin broth supplemented with 4 mg of vancomycin per liter. A total of 37% of the hospitalized patients and 11.8% of the subjects from the community were found to be VRE carriers. A total of 65 VRE strains were isolated: 12 (18.5%) E. faecium, 46 (70.7%) E. gallinarum, and 7 (10.8%) E. casseliflavus strains. No E. faecalis strains were detected. All the E. faecium strains were of the vanA genotype. Molecular typing by pulsed-field gel electrophoresis revealed a different pattern for each vanA VRE strain that originated from an individual subject. To our knowledge, this is the first study to be carried out in a cattle-rearing region of France. It reports a higher VRE prevalence than that reported in previous European or U.S. studies. A partial explanation is the use of an enrichment broth step which enabled detection of strains which would otherwise have been missed, but the fact that subjects and patients were recruited from a predominantly agricultural area where vancomycin-related antibiotics have recently been used in animal husbandry could also contribute to the high levels of VRE in patients and subjects alike.

Adolescent↗
PubMed2000

Epidemiology of glycopeptide-resistant enterococci colonizing high-risk patients in hospitals in Johannesburg, Republic of South Africa.

Recent cases of infections caused by glycopeptide-resistant enterococci (GRE) have highlighted the emergence of these organisms in the Republic of South Africa. During May 1998 we conducted a prevalence study in four hospitals in Johannesburg and obtained 184 rectal swabs from patients identified as being at high risk for GRE colonization. Twenty enterococcal isolates showing various glycopeptide resistance genotypes were recovered: 3 Enterococcus faecium vanA isolates, 10 E. faecium vanB isolates, 6 E. gallinarum vanC1 isolates, and 1 E. avium vanA isolate. Macrorestriction analysis was used to demonstrate the clonal spread of GRE strains within hospitals. Evidence also demonstrated the likely persistence of the original E. faecium vanA isolate associated with the first confirmed death contributed to by GRE infection in South Africa in March 1997.

Adolescent↗
PubMed2000

Isolation and characterization of glycopeptide-resistant enterococci from hospitalized patients over a 30-month period.

In February 1996, a Hospital Infection Control Practices Advisory Committee-style screening program was commenced to isolate and subsequently characterize glycopeptide-resistant enterococci (GRE) from patients at a hospital trust in Glasgow, Scotland. Over the next 30 months, GRE were isolated from 154 patients. GRE were isolated from patients in traditionally high-risk areas such as the renal unit and intensive care unit and also in areas considered to be lower risk, including medical wards and associated long-stay geriatric hospitals. The majority (90%) of isolates were Enterococcus faecium vanB. The remaining isolates consisted of seven E. faecalis (vanA), three E. gallinarum (vanC), and a further six E. faecium (five vanA, one both vanA and vanB) isolates. Analysis of SmaI-digested DNA by pulsed-field gel electrophoresis revealed that 34 of 40 (85%) VanB E. faecium isolates were identical or closely related, while 11 of 13 (85%) VanA GRE were distinct. High-level aminoglycoside resistance was seen in less than 8% of isolates. VanB E. faecium isolates were almost uniformly resistant to ampicillin and tetracycline. In this study, GRE have been isolated over a prolonged period from a broad range of patients. Glycopeptide resistance within the study hospital trust appeared to be mainly due to the clonal dissemination of a single strain of E. faecium VanB.

Aminoglycosides↗
PubMed2000

Limitations of vitek GPS-418 cards in exact detection of vancomycin-resistant enterococci with the vanB genotype.

The susceptibilities of 20 strains of vancomycin-resistant enterococci (VRE) with the vanB genotype obtained by using Vitek GPS-418 cards were compared with those obtained by the broth dilution method of the National Committee for Clinical Laboratory Standards (NCCLS) (approved standard M7-A4) and with those obtained by the agar screen method using bile esculin azide agar containing 6 microgram of vancomycin per ml. Although both the broth dilution and agar screen methods disclosed no discordance, Vitek GPS-418 cards yielded a very major error compared with the results obtained by the reference broth dilution method of the NCCLS. Vitek GPS-418 cards were therefore found to have considerable room for improvement for the accurate detection of vanB VRE strains.

Bacterial Proteins↗
PubMed2001

Contamination of the clinical microbiology laboratory with vancomycin-resistant enterococci and multidrug- resistant Enterobacteriaceae: implications for hospital and laboratory workers.

We surveyed environmental surfaces in our clinical microbiology laboratory to determine the prevalence of vancomycin-resistant enterococci (VRE) and multidrug-resistant Enterobacteriaceae (MDRE) during a routine working day. From a total of 193 surfaces, VRE were present on 20 (10%) and MDRE were present on 4 (2%) of the surfaces tested. In a subsequent survey after routine cleaning, all of the 24 prior positive surfaces were found to be negative. Thus, those in the laboratory should recognize that many surfaces may be contaminated by resistant organisms during routine processing of patient specimens.

Colony Count, Microbial↗
PubMed2001

Selection of a teicoplanin-resistant Enterococcus faecium mutant during an outbreak caused by vancomycin-resistant enterococci with the vanB phenotype.

Vancomycin-resistant enterococci (VRE) have recently become an increasing problem in hospitals in Poland, being responsible for a growing number of nosocomial outbreaks. In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. It was caused by clonal dissemination of a single strain of vancomycin-resistant Enterococcus faecalis (VRES) and horizontal transmission of vancomycin resistance genes among several vancomycin-resistant Enterococcus faecium (VREM) strains. Two similar restriction fragment length polymorphism types of the vanB gene cluster characterized VRES and VREM isolates, and they both contained the same vanB2 variant of the vanB gene. Two vancomycin-susceptible E. faecium (VSEM) isolates, recovered from the same wards during the outbreak, proved to be related to certain VREM isolates and could represent endemic strains that had acquired vancomycin resistance. One VSEM and four VREM isolates, all identified in the same patient, belonged to a single clone, although they revealed remarkable diversity in terms of susceptibility, PFGE patterns, plasmid content, and number of vanB gene cluster copies. Most probably they reflected the dynamic evolution of an E. faecium strain in the course of infection of a single patient. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its vanB gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the vanB gene cluster, including the regulatory genes vanR(B) and -S(B), and the vancomycin-inducible promoter P(YB). Expression of the resistance genes vanH(B), -B, and -X(B) was constitutive in the mutant, and this phenomenon was responsible for its unusual phenotype.

Anti-Bacterial Agents↗