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PubMed2001

Quantitative determination of gelatinase activity among enterococci.

Gelatinase, hemolysin and aggregation substance have all been reported to be virulence factors of enterococci. In this study, gelatinase production was investigated in isolates of Enterococcus faecalis (n=93), E. faecium (n=49) and E. avium (n=36) recovered from hospitalized patients. Gelatinase was detected in 45% of E. faecalis isolates, but could not be detected in E. faecium and E. avium. Gelatinase activity was then measured by radial diffusion for the 42 gelatinase-positive E. faecalis isolates. To convert gelatinase activity into proteinase K activity, a standard curve was produced by placing different concentrations of proteinase K into wells in the gelatine plate. Gelatinase activity per E. faecalis colony ranged from 2.6 x 10(-7) to 2.2 x 10(-5) microg/ml, proportionate to the activity of proteinase K. An approximately 84-fold difference in gelatinase concentration was observed between the colony producing the highest amount and that producing the lowest amount. This method may be useful for determining the virulence of given isolates in relation to gelatinase production as it is quick, easy and inexpensive to perform.

Adult↗
PubMed2001

VanA-type vancomycin-resistant enterococci (VRE) remain prevalent in poultry carcasses 3 years after avoparcin was banned.

Avoparcin was used as a growth promoting feed additive in Norwegian broiler and turkey production from 1986 until it was banned in 1995, when an association between vancomycin-resistant enterococci (VRE) and avoparcin use became apparent. A recent study regarding faecal samples documented a continuing high prevalence of VRE among Norwegian poultry 3 years after avoparcin was banned. In the present study, carcasses of broilers and turkeys from farms where avoparcin had previously been in use and carcasses of layer chickens from farms where avoparcin had never been used were examined for the presence of VRE. One carcass from each of 150 different farms was included. By a direct plating method, VRE were isolated from 30 of 100 samples of broilers and turkeys, but not from any samples of layer chickens. When an enrichment step was included, VRE were isolated from a total of 81 of the 100 samples of broilers and turkeys and from nine of the 50 samples of layer chickens. All VRE isolated were highly resistant to vancomycin (MIC > or = 256 microg/ml) and possessed the vanA gene. These results correspond to the prevalence of VRE recently documented in faecal samples from Norwegian poultry. The present study reveals a high prevalence of VRE in broiler and turkey carcasses. Consequently, consumers are exposed to VRE when handling raw poultry meat, although the public health significance of such exposure is unclear.

Animals↗
PubMed2003

Presence of macrolide resistance genes in streptococci and enterococci isolated from pigs and pork carcasses.

Macrolide and lincosamide (ML)-resistant streptococci and enterococci from tonsillar and colon swabs from 33 pigs and 99 pork carcasses swabs from animals originating from different farms in Belgium were isolated, and their ML resistance phenotypes and genotypes were determined by disk diffusion test and PCR assay, amplifying the ermB gene and the mefA gene. From each of the 33 pigs and 88 of the 99 carcasses' swabs, at least one resistant strain was isolated. The predominant phenotype was the constitutively expressed macrolides, lincosamides and streptogramin B (MLS(B)) phenotype. This phenotype was most often encoded by the ermB gene. A minority of the strains showed the M phenotype encoded by the mefA gene in streptococci, or the L or ML phenotype.

Animals↗
PubMed2003

Taxonomy, ecology and antibiotic resistance of enterococci from food and the gastro-intestinal tract.

Apart from genotypic identification methods, there is a need for reliable conventional phenotypic identification schemes for simple and rapid determination of enterococcal species in food or in the gastro-intestinal tract (GIT). Only a limited number of enterococcal species is of importance for the ecology of the GIT or the food microflora, including E. faecalis, E. faecium, E. durans/hirae, E. gallinarum and E. casseliflavus. After genus identification the differentiation within these species can include, e.g. mannitol and arabinose fermentation and growth at 50 degrees C. Widely used commercial identification systems may fail to precisely identify rare species. Ecological aspects should also be taken into account. In the human GIT E. faecium is the most common species whereas in most animal species E. faecalis is at least present in the same amount. Especially in foods of animal origin (cheese, pork meat, beef, poultry meat) also E. faecalis is very frequent. This is of special interest as glycopeptide resistance is most often found in human clinical E. faecium strains as well as in E. faecium from the environment or animal samples and less frequent in E. faecalis strains. EU experts propose as safety criteria for probiotics in feed additives the exclusion of resistances or the lack of transferability. This proposal can also be applied to enterococci in foods. Specific resistances must be excluded, but transferability or acquisition of resistance (e.g. vancomycin) cannot be excluded per se. However, technologically used strains should differ from clinical strains concerning their resistance patterns and transfer rates.

Anti-Bacterial Agents↗
PubMed2003

Species identification and detection of vancomycin resistance genes in enterococci of animal origin by multiplex PCR.

A multiplex PCR assay for the differentiation of species and detection of vancomycin (van) resistance genes in enterococci was established. Three hundred sixty-seven enterococcal strains isolated from food were selected from a sample collection and were examined with regards to the existence of four vancomycin resistance genes (vanA, vanB, vanC1 and vanC2) and to species differentiation (E. faecalis, E. faecium, E. casseliflavus and E. gallinarum) by multiplex PCR. Apart from unambiguous results for the majority of strains, the E. faecium specific primers used here, showed weak points in the specificity and sensitivity, respectively. Despite these rare instances, we succeeded in optimising the multiplex PCR assay by variation of annealing temperature and primer combination. The assay presented here, with its optimised parameters, is therefore suitable for routine use and because of time and material saving, it is an alternative and rapid method in comparison to conventional tests.

Animals↗
PubMedDate not supplied

Antimicrobial resistance from enterococci in a pediatric hospital. Plasmids in Enterococcus faecalis isolates with high-level gentamicin and streptomycin resistance.

BACKGROUND: Enterococcus spp. is an important nosocomial and community-acquired pathogen. Recent studies have documented the increasing importance of this pathogen in children, particularly in the hospital setting. Our objective in this study was to report the frequency of antimicrobial resistance in enterococci and to determine the characteristics of high-level gentamicin resistance (HLGR) plasmids in Enterococcus faecalis clinical isolates. METHODS: Two hundred eighty-nine enterococcal isolates were collected during an 18-month period from a tertiary-care pediatric hospital in Mexico City. Isolates were screened for antibiotic resistance, including HLGR. High-level, gentamicin-resistant E. faecalis strains were selected for pulsed-field electrophoresis (PFGE) typing and plasmid analysis. Transferability of resistance markers was carried out using filter matings. RESULTS: Seventy-six percent of isolates were E. faecalis, 10% were E. avium, 5.2% E. faecium, 5.2% E. raffinossus, 1.38% E. malodoratus, 0.6% E. hirae, and 0.6% E. casseliflavus. Antimicrobial resistance was ampicillin and penicillin 29%, imipenem 17%, and vancomycin 3%, HLGR 5%. The following 15 high-level, gentamicin-resistant isolates were identified: six E. faecalis; four E. avium; three E. faecium, and two E. casseliflavus. Five of the six E. faecalis isolates were different by PFGE and transferred gentamicin and streptomycin resistance on filter membranes. Transfer frequencies ranged from 8.2 x 10(-4) to 6.92 x 10(-5) transconjugants/recipient cell. The plasmid content of donors and transconjugants were homogeneous (one plasmid of 47 kb). CONCLUSIONS: In this pediatric hospital, antimicrobial resistance in Enterococcus spp. is common. Frequency of high-level, gentamicin-resistant strains is low. Mechanism of HLGR appears to be due to a single plasmid dissemination.

Drug Resistance, Microbial↗
PubMed2004

Debate-guidelines for control of glycopeptide-resistant enterococci (GRE) have not yet worked.

Glycopeptide-resistant enterococci (GRE) have become a focus of concern in many countries because options for antimicrobial therapy of GRE infection are limited. Several guidelines for the control and prevention of GRE colonization and infection have been developed for healthcare settings, and occasional journal articles now report "control" (usually relative reduction of incidence or prevalence rate rather than elimination) of GRE infections. Yet, rates of infection and colonization with GREcontinue to climb in many parts of the world, showing that true control has not been achieved. Programmes to control GRE will be effective only when they (1) are less expensive to implement; (2) are shown to be cost-effective despite the fact that they merely reduce prevalence levels rather than eradicating the problem; (3) do not require almost perfect implementation to be effective; (4) are shown to be sustainable; (5) are shown to work in acute-care settings other than selected academic centres; and (6) are shown to work in non-acute care settings. Until then, it is clear that guidelines for control of GRE have not worked. New guidelines that truly control GRE must be developed, and this must be done quickly.

Anti-Bacterial Agents↗
PubMed1997

Gene transfer, gentamicin resistance and enterococci.

Enterococci are versatile pathogens by virtue of their ability to exhibit low-level intrinsic resistance to clinically useful antibiotics and their tolerance to adverse environmental conditions. In the last 20 years these pathogens have become progressively more difficult to treat because of their aptitude for acquiring antibiotic-resistance genes. Of increasing concern is the rapid dissemination of the AAC6'-APH2" bi-functional aminoglycoside modifying enzyme. This enzyme confers high-level resistance to gentamicin and all other related aminoglycosides with the exception of streptomycin. The gene conferring this phenotype has been associated with both narrow and broad host range plasmids, and has recently been found on conjugative transposons. The nature of these conjugative elements raises the possibility of the resistance gene spreading to other pathogenic bacteria.

Anti-Bacterial Agents↗
PubMed1998

Epidemiology and control of vancomycin-resistant enterococci (VRE) in a renal unit.

This study reports an outbreak of infection and colonization caused by vancomycin-resistant enterococci (VRE) in the renal service of a large teaching hospital. The polymerase chain reaction and pulsed-field gel electrophoresis were used to study the epidemiology of 26/34 strains of vancomycin-resistant Enterococcus faecalis and Enterococcus faecium from the outbreak in comparison with five strains from other hospitals in Edinburgh and the Borders, and three from other wards in the Royal Infirmary. The study revealed a heterogeneous population of vancomycin-resistant E. faecalis. Over 60% of E. faecium isolates had matching pulsed-field gel electrophoresis patterns and all of these were of VanA phenotype. These results suggest that clonal spread of VanA phenotype E. faecium within and possibly between hospitals is the major vancomycin-resistant enterococcal problem in Edinburgh. Screening of patients and isolation of colonized and infected patients appear to have been successful in controlling the spread of VRE.

Anti-Bacterial Agents↗
PubMed1998

Selective isolation of vancomycin-resistant enterococci.

Many laboratories are likely to be or to become involved in screening patients for the carriage of vancomycin-resistant enterococci (VRE). A choice has to be made from the numerous formulations described and decisions made on the degree of vancomycin supplementation and the need for an enrichment phase. Few comparative trials and little critical evaluation has been performed to date and there is as yet no clear choice as to the optimal formulation. Laboratories must choose the formulation that most suits their particular requirements and circumstances.

Bacteriological Techniques↗
PubMed1996

Experience with a hospital-wide outbreak of vancomycin-resistant enterococci.

BACKGROUND: Vancomycin-resistant enterococci (VRE) were first detected in our institution in 1991. An outbreak was recognized in late 1992 when there was a sudden rise in the number of patients per month with VRE. Little information exists concerning the natural history of infection with these pathogens, and the effect of antimicrobial therapy is unclear. Recent guidelines emphasize prudent use of vancomycin and prompt institution of barrier precautions to limit the spread of vancomycin resistance. METHODS: Data were obtained by review of microbiologic and clinical records. Patients were categorized according to site of infection, and outcome of therapy was assessed. Hospital antibiotic usage was analyzed to determine any correlation with the outbreak. Infection control measures instituted in 1993 included patient isolation, environmental cleaning, and a reemphasis of barrier precautions. Surveillance cultures were performed to assess the extent of the outbreak in January 1995. RESULTS: VRE were detected in clinical cultures from 159 patients from 1991 through 1994. Mortality rate was 48%, but in most cases death could not be attributed to enterococcal infection. Patients with wound infections healed without specific therapy. Many patients with bacteremia had resolution with ampicillin or without specific therapy. Patients were widely scattered throughout the hospital from the beginning of the outbreak. Hospital usage of cefotaxime correlated with the number of cases. Infection control measures were not successful. Surveillance culture results in January 1995 revealed that 53% of all medical and surgical inpatients had fecal colonization with VRE. Genetic analysis of selected isolates revealed that one strain predominated, but at least seven distinct strains were identified. CONCLUSIONS: Our data suggest that many infections with VRE resolve without specific therapy. The infection control measures we used were ineffective, possibly because of the multiple strains present in our hospital. Isolation of all patients with VRE is impractical when there is widespread fecal carriage.

Adult↗
PubMed1997

Epidemiology and control of vancomycin-resistant enterococci in an adult and children's hospital.

BACKGROUND: The incidence of vancomycin-resistant enterococci (VRE) has reached endemic proportions in many medical centers. To initiate an effective infection control program, an understanding of the epidemiologic attributes of the genus in medical facilities is imperative. METHODS: We studied 138 consecutive cases of VRE from April through December 1995. We created a database to analyze the risk factors for patients in both an adult hospital and a children's hospital and screened all specimens, submitted for routine microbiologic analysis, for VRE. RESULTS: One hundred twenty-three cases (89%) occurred in the adult acute care hospital, and 15 (11%) occurred in the children's hospital. Eighty patients (58%) were colonized with VRE, and 58 (42%) had an infection with VRE. Eighty-three percent of all the cases of VRE were nosocomially acquired. The majority of cases occurred in the medical service. Urine was the most important clinical specimen infected or colonized. Prior use of an antibiotic, other than vancomycin, was the most important risk factor for all nosocomial cases, followed by prior vancomycin use for surgical patients and residence in a unit with other patients infected with VRE for the medical service. Direct admission from another hospital was the most important risk factor for community-acquired cases. Special microbiologic screening of cultures yielded 48% of all VRE identified. Enterococcus faecium was the predominant resistant isolate recovered. CONCLUSIONS: The control of VRE in the hospital setting is difficult for several reasons. Almost half of all patients carrying VRE would not have been identified without special microbiologic screening efforts, as would patients, admitted from the community, who are already colonized with VRE. Controlling antibiotic use both in the hospital and the community is basic for controlling these organisms. Continuous education of all staff about VRE and other nosocomially significant organisms is the key to controlling the spread of these bacteria.

Adolescent↗
PubMed2001

[Risk factors and prevention of the acquisition and transmission of glycopeptide resistant enterococci].

We reviewed the literature concerning the role of antibiotic use as a risk factor for glycopeptide-resistant enterococci (VRE) infection/colonisation, to enable us to develop measures for preventing the acquisition and transmission of VRE. We found that the length of stay, the number of stays in hospital and the transfer of patients between hospitals and units were all risk factors for acquiring VRE infection. However, analysis of group and individual data showed that there was also a clear link between vancomycin and third-generation cephalosporin use and the prevalence of VRE colonisation/infection. Evidence for this link was provided by the consistent association and dose-effect relationship observed, and from the frequently consistent variations observed over time. However, it is difficult to give precise recommendations because very few studies have investigated both intrinsic bacterial factors making specific strains more epidemic and the precise characteristics of the conditions determining antibiotic selection pressure. In the absence of this information, and maintaining the prevention measures against cross-contamination which remain a priority, these results suggest that programs aimed at improving the prescription of antibiotics should be initiated in hospitals.

Anti-Bacterial Agents↗
PubMed2000

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci.

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.

Animals↗
PubMed2004

Analysis of gyrA and parC mutations in enterococci from environmental samples with reduced susceptibility to ciprofloxacin.

The quinolone resistance determining regions of gyrA and parC in four species of enterococci from environmental samples with reduced susceptibility to ciprofloxacin were sequenced. The nucleotide sequence variations of parC could be related to the different enterococcal species. Mutations in Enterococcus faecalis and Enterococcus faecium related to reduced susceptibility were identical to mutations detected in E. faecalis and E. faecium of clinical origin. A minimal inhibitory concentration of 8 microg ml(-1) to ciprofloxacin was not associated with any mutations in the gyrA and parC gene of Enterococcus casseliflavus and Enterococcus gallinarum. These two species may be intrinsically less susceptible to ciprofloxacin.

Amino Acid Sequence↗
PubMed2000

A nested array of rRNA targeted probes for the detection and identification of enterococci by reverse hybridization.

Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.

Base Sequence↗
PubMed2002

Training costs and investment payback of implementing molecular diagnostics for identification of vancomycin resistant enterococci in a clinical microbiology laboratory.

Molecular diagnostics may be a more efficient method to manage resources; but most Microbiology laboratories have not introduced them into routine use due to the specialized training required. Using vancomycin resistant enterococci (VRE) screening during a comparison of a multiplex PCR (MPCR) and conventional biochemical testing (CBT) we studied 3 objectives: 1) to develop a molecular diagnostics in-house training program, 2) to assess the training program outcomes for competency and confidence, and 3) to determine laboratory payback. A training program for 14 technologists using multiple adult learning methods was implemented. Methods to minimize technical errors were introduced and included: use of a calibrated loop to deliver sample; prealiquotting reagents; increasing volume of specimen; addition of gel loading dye directly into reaction tubes; and establishment of an equivocal zone. In our laboratory MPCR costs $7.06 less than CBT, therefore the payback period for training and implementation would be approximately 3 years.

Cost-Benefit Analysis↗
PubMed2002

In vitro susceptibility of vancomycin-resistant enterococci (VRE) to fosfomycin.

We evaluated the in vitro activity of fosfomycin against 75 clinical isolates of vancomycin-resistant enterococci (VRE). Using the NCCLS breakpoint for susceptibility of urinary tract isolates to fosfomycin (MIC > or = 256), 51 out of 52 Enterococcus faecium and all Enterococcus faecalis isolates tested were susceptible or intermediate to fosfomycin.

Anti-Bacterial Agents↗