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PubMed2002

A PCR assay for rapid detection of vancomycin-resistant enterococci.

Since the first report of a vancomycin-resistant enterococcal clinical isolate, these Gram-positive bacteria have emerged as important nosocomial pathogens. Several glycopeptide resistance phenotypes can be distinguished on the basis of the level and inducibility of resistance to vancomycin and teicoplanin. In the present study, we developed a multiplex PCR, which allows the simultaneous identification of enterococci at the genus level and detection of the most frequent glycopeptide resistance genotypes. Five primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 and tuf were used in one reaction tube with bacterial DNA extracted from three to five colonies. This PCR method is suitable for the rapid detection of vancomycin-resistant enterococci.

DNA, Bacterial↗
PubMed1996

Susceptibility of ciprofloxacin-resistant staphylococci and enterococci to trovafloxacin.

The susceptibilities of clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and enterococci obtained over a 13-year period were tested for trovafloxacin, a new fluoroquinolone, and ciprofloxacin. For MRSA, MIC50 values for trovafloxacin increased from 0.03 microgram/ml to 1 microgram/ml from 1984-1985 to 1995-1996, but were lower than those noted for ciprofloxacin, which increased from 0.25 microgram/ml to > 8 micrograms/ml during the same period. Trovafloxacin also showed lower MIC50 values (0.12-0.25 microgram/ml) than ciprofloxacin (MIC50 of 0.5-1 microgram/ml) for E. faecalis isolates from 1985 through 1991, but against E. faecium and E. faecalis isolated in 1995-1996, the MIC50 values for both trovafloxacin and ciprofloxacin were 2 micrograms/ml and the MIC90 values were > or 8 micrograms/ml. Thus, cross-resistance between fluoroquinolones was shown for both enterococci and MRSA. Whether the greater intrinsic activity of trovafloxacin might allow its use in treating MRSA and enterococcal infections remains to be seen.

Anti-Infective Agents↗
PubMed1998

In vitro activity of LY333328 against vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus, and penicillin-resistant Streptococcus pneumoniae.

We report the activity of LY333328 against 35 clinical isolates of vancomycin-resistant enterococci (including organisms carrying the vanA, vanB, vanC-1, and vanC-2/3 genes, as determined by PCR), 33 clinical isolates of methicillin-resistant S. aureus, and 29 clinical isolates of high-level penicillin-resistant S. pneumoniae. All isolates of vancomycin-resistant enterococci were inhibited by 2 micrograms/mL LY333328, and 8 micrograms/mL LY333328 was bactericidal against all isolates tested. All isolates of methicillin-resistant S. aureus were inhibited by 1 microgram/mL LY333328, and 4 micrograms/mL LY333328 was bactericidal against all methicillin-resistant S. aureus isolates tested. All isolates of penicillin-resistant S. pneumoniae were inhibited by < 0.125 microgram/mL LY333328, and 0.25 microgram/mL LY333328 was bactericidal against all S. pneumoniae isolates tested. LY333328 is a promising new glycopeptide antimicrobial agent.

Anti-Bacterial Agents↗
PubMed1999

In vitro activity of linezolid against vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae.

We report the activity of the new oxazolidinone antimicrobial agent linezolid against 37 clinical isolates of vancomycin-resistant enterococci (including organisms carrying the vanA, vanB, vanC-1, and vanC-2/3 genes), 26 clinical isolates of methicillin-resistant S. aureus and 20 clinical isolates of high-level penicillin-resistant S. pneumoniae. All isolates of vancomycin-resistant enterococci were inhibited by < or = 4 ug/ml of linezolid. All isolates of methicillin-resistant S. aureus were inhibited by < or = 8 ug/ml of linezolid. All isolates of penicillin-resistant S. pneumoniae were inhibited by < or = 2 ug/ml of linezolid. Linezolid inhibits strains of multidrug resistant Gram-positive cocci in vitro at concentrations < or = 8 ug/ml.

Acetamides↗
PubMed2000

Comparison of phenotypic methods to identify enterococci intrinsically resistant to vancomycin (VanC VRE).

Clinical laboratories must be able to differentiate between enterococci possessing acquired resistance to vancomycin (vanA and vanB genotypes) from those that are inherently resistant (vanC1 and vanC2/C3 genotypes). We compared several routine phenotypic tests to determine the species identity of clinical isolates of enterococci and a PCR assay for the van ligase genes was used to confirm identification of VanC VRE. The Vitek Gram Positive Identification card identified 53/60 (88%) Enterococcus faecalis and E. faecium isolates and 81/141 (57%) VanC VRE without additional testing. Another 32 of the VanC VRE required additional testing (e.g., motility and pigmentation) for correct identification. However, 7 of these 32 VanC VRE were nonmotile. The rapid ID 32 STREP strips identified 50/60 (83%) E. faecalis and E. faecium isolates and 102/141 (72%) VanC VRE. All E. faecalis and E. faecium isolates were nonmotile and did not acidify 1% methyl-alpha-D-glucopyranoside (MGP). Only 93/115 (81%) E. gallinarum and 21/26 (81%) E. casseliflavus/E. flavescens were motile but all 141 VanC VRE acidified MGP. MGP acidification can accurately differentiate VanC VRE from E. faecalis and E. faecium. Because some VanC VRE isolates are nonmotile, MGP acidification is preferred as a simple and less costly test for identification of these isolates.

Bacterial Proteins↗
PubMed2000

CPT-EIA assays for the detection of vancomycin resistant vanA and vanB genes in enterococci.

Cycling Probe Technology (CPT) was combined with a colorimetric enzyme-immuno assay (EIA) to develop two assays for the detection of vanA and vanB genes in vancomycin resistant enterococci (VRE). The CPT-EIA assay employs a gene-specific fluorescein labeled DNA-RNA-DNA probe that gets cleaved within the probe : target duplex. The cleaved DNA probe fragments dissociate from the target, making it available for further cycling. Following the separation of cleaved probe fragments, anti-fluorescein-horseradish peroxidase antibodies are used for the detection of uncleaved probes. The two CPT-EIA assays were used to screen a collection of 440 clinical isolates (Modrusan et al., 1999). All of the 154 VanA and 131 VanB isolates were correctly identified in the vanA and vanB CPT-EIA, respectively. The VanA and VanB isolates were differentiated from vancomycin sensitive enterococci (VSE) and also from the VanC isolates. In addition, an accurate VRE detection in the CPT-EIA assay was shown with cultures grown on eight different media.

Anti-Bacterial Agents↗
PubMed1995

Species identification and antibiotic susceptibility of enterococci isolated from clinical specimens of hospitalized patients.

Over a 4-month period, a total of 315 enterococci were isolated from various clinical specimens of hospitalized patients. By applying an array of biochemical tests, all strains were accurately identified to the species level, and their susceptibilities to clinically relevant antibiotics were determined by a standardized agar dilution technique. E. faecalis and E. faecium accounted for 87.3% and 9.2% of isolates, respectively. E. avium (1%), E. gallinarum (1%), E. durans (0.6%), E. hirae (0.6%), and E. casseliflavus (0.3%) isolates were also identified. Eleven strains of E. faecium and 1 E. hirae isolate were resistant to ampicillin, but none of the isolates produced beta-lactamase. Twenty-three E. faecium and 3 E. faecalis strains as well as 1 E. hirae isolate revealed imipenem resistance. A total of 25.4% enterococci (60 E. faecalis and 19 E. faecium isolates, 1 E. hirae strain) were erythromycin-resistant. Twelve strains (11 E. faecium and 1 E. avium) exhibited ciprofloxacin resistance. High-level resistance to streptomycin was found in 58 (21.1%) E. faecalis, 9 (31%) E. faecium, and both E. hirae strains, whereas high-level gentamicin resistance (HLGR) was exclusively seen in the species E. faecalis (11.6% of isolates belonging to this species). A simple agar screening test containing 500 micrograms of gentamicin per ml proved to be highly reliable for detection of HLGR. The structural gene coding for HLGR was specifically amplified by the polymerase chain reaction in all isolates showing this resistance trait. Moreover, the gene was specifically detected by a nonradioactively labelled oligonucleotide probe in colony blot hybridization assays, indicating the potential application of these molecular approaches as a diagnostic tool.

Ampicillin↗
PubMed2001

Detection and typing of vancomycin-resistance genes of enterococci from clinical and nosocomial surveillance specimens by multiplex PCR.

Ninety-three clinical isolates of vancomycin-resistant enterococci (VRE) collected from nine hospitals in Taiwan were examined for the presence of vanA, vanB, vanC1, or vanC2/vanC3 genes by a multiplex PCR. Forty-seven of these VRE isolates were vanA positive, 1 contained both vanC1 and vanA, 40 harboured vanB, 2 were vanC1, and 3 were identified to be vanC2/vanC3. Twenty-four vanA isolates were sensitive to teicoplanin and thus did not have a typical VanA phenotype. Five isolates with the VanC phenotype harboured vanB. None of the 40 clinically isolated vancomycin-susceptible E. faecium or E. faecalis and the vancomycin-resistant Leuconostoc and Pediococcus isolates were positive for any of the van genes. While performing nosocomial surveillance, VRE were isolated from 47 of 467 rectal swabs by culture. Compared with the conventional culture method, the sensitivity and specificity of the multiplex PCR for detecting and identifying vancomycin-resistance genes in enterococci directly from culture-positive broth were 97.9% and 100%, respectively. The results suggest that genotypic characterization of vancomycin-resistance is necessary for all clinical VRE isolates and that the multiplex PCR assay can be an alternative method for this purpose.

Bacterial Proteins↗
PubMed2005

High-level aminoglycoside resistant enterococci isolated from swine.

Approximately 42% (187/444) of swine enterococci collected between the years 1999 and 2000 exhibited high-level resistance to gentamicin (MIC > or =500 microg/ml), kanamycin (MIC > or =500 microg/ml), or streptomycin (MIC > or =1000 microg/ml). Eight aminoglycoside resistance genes were detected using PCR, most frequently ant(6)-Ia and aac(6')-Ii from Enterococcus faecium. Twenty-four per cent (45/187) of total high-level aminoglycoside-resistant isolates and 26% (4/15) of isolates resistant to high levels of all three antimicrobials were negative for all genes tested. These data suggest that enterococci isolated from swine contain diverse and possibly unidentified aminoglycoside resistance genes.

Aminoglycosides↗
PubMed2006

Modeling the transport and inactivation of E. coli and enterococci in the near-shore region of Lake Michigan.

To investigate the transport and fate of fecal pollution at Great Lakes beaches and the health risks associated with swimming, the near-shore waters of Lake Michigan and two tributaries discharging into it were examined for bacterial indicators of human fecal pollution. The enterococcus human fecal pollution marker, which targets a putative virulence factor--the enterococcal surface protein (esp) in Enterococcus faecium, was detected in 2/28 samples (7%) in the tributaries draining into Lake Michigan and in 6/30 samples (20%) in Lake Michigan beaches. This was indicative of human fecal pollution being transported in the tributaries and occurrence at Lake Michigan beaches. To understand the relative importance of different processes influencing pollution transport and inactivation, a finite-element model of surf-zone hydrodynamics (coupled with models for temperature, E. coli and enterococci) was used. Enterococci appear to survive longer than E. coli, which was described using an overall first-order inactivation coefficient in the range 0.5-2.0 per day. Our analysis suggests that the majority of fecal indicator bacteria variation can be explained based on loadings from the tributaries. Sunlight is a major contributor to inactivation in the surf-zone and the formulation based on sunlight, temperature and sedimentation is preferred over the first-order inactivation formulation.

Enterococcus↗
PubMed2004

Adhesion properties of enterococci to intestinal mucus of different hosts.

The adhesive capacity of selected enterococci to human, canine and porcine intestinal mucus was investigated in order to select for potential probiotic strains with good adhesive properties for human or animal use. The adhesion to the human intestinal mucus of the tested strains was found to range from log10 3.8 to log10 8.6 cfu per microtitre plate well. The highest adhesion to the human intestinal mucus was found among strains from horse faeces, dog faeces and dog feed. The adhesion to canine mucus was observed to range from log10 3.8 to log10 8.3 cfu/well, with the highest adhesive capacity among strains from dog faeces, horse faeces and dog's feed; on average log10 7.9, 7.3 and 7.0 cfu/well, respectively. Isolates from dogs did not bind at higher levels to canine mucus than to human mucus. A strong correlation was observed for the adhesion to human and canine intestinal mucus (p < 0.0001) and also between porcine and canine or human mucus (p < 0.05 for both). When comparing the adhesion of Enterococcus faecium and E. faecalis, no statistical significant differences were observed for any of the tested mucus types. The tested Enterococcus strains were found to exhibit a strain dependent on in vitro adhesion to human, canine and porcine intestinal mucus and did not exhibit host specificity in their adhesion, though some sources appeared to contain more adhesive strains than others. To our knowledge, this is the first report on the in vivo adhesion to intestinal mucus of a large number of enterococci from different sources.

Animal Feed↗
PubMed2000

Identification and antimicrobial susceptibility of enterococci isolated from the root canal.

Enterococci are occurring in opportunistic infections involving the oral cavity. This study has identified enterococcal species in 29 endodontic infections undergoing treatment with Ca (OH)2 dressings. The in vitro antimicrobial susceptibility of 29 isolated enterococcal strains was determined. Enterococcus faecalis was speciated for 26 isolates and Enterococcus faecium for three isolates. In vitro antimicrobial susceptibility testing revealed enterococcal isolates resistant to benzylpenicillin, ampicillin, clindamycin, metronidazole and tetracycline but sensitive to erythromycin and vancomycin. Due to low sensitivity to antimicrobial agents, enterococci may be selected in root canals undergoing standard endodontic treatment and significantly contribute to endodontic treatment failures.

Ampicillin↗
PubMed2002

The effect of a sewage treatment plant effluent on the faecal coliforms and enterococci populations of the reception river waters.

AIMS: A rural sewage treatment plant and the effect of its effluent on the enterococci and faecal coliforms populations of the receiving river waters was evaluated. METHODS AND RESULTS: The enumeration of bacteria was performed by membrane filtration. Diversity and population similarity were analysed using the PhP-plates system. The treatment plant reduces the number of enterococci and faecal coliforms to values similar to those observed upstream. All water samples showed a high diversity for both bacterial populations. A high similarity in the composition and structure, was detected among all the samples. CONCLUSIONS: The impact of the disposal of treated sewage on the river did not modify the composition of either bacterial populations in the river water. SIGNIFICANCE AND IMPACT OF THE STUDY: The biochemical phenotyping of bacterial populations is a reliable tool for ecological and biodiversity studies. The obtained results provide a better understanding of the sewage treatment process and the impact of the treated sewage effluents in the environment.

Enterobacteriaceae↗
PubMed1997

Susceptibility to vancomycin of enterococci isolated from dairy products.

The antibiotic vancomycin is often used in clinical therapy against multiple antibiotic-resistant bacteria. Twenty strains of enterococci, either Enterococcus faecium or Ent. faecalis, isolated from different cheeses were tested for resistance to vancomycin in liquid medium. Minimum inhibitory concentration (MIC) values ranged from less than 1 to 4 micrograms ml -1. A 399 bp intragenic fragment of the vanA gene was not observed after amplification of total DNA of the strains. It seems, therefore, that resistance to vancomycin, which is a common trait for enterococci of nosocomial origin, is not widespread among dairy isolates.

Anti-Bacterial Agents↗
PubMed1997

Antibacterial activity of enterococci strains against Vibrio cholerae.

Thirty-seven strains of enterococci isolated from milk and milk products from Santa Fe (Argentina) region were tested for antagonistic activity against Vibrio cholerae 01 and non-01. Seven of 17 strains of Enterococcus faecalis, five of 10 strains of Enterococcus faecium and four of 10 strains of Enterococcus durans produced inhibition zones against the indicator species. The activity of the antibacterial compounds was completely destroyed by treatment with trypsin and pronase E in most cases (only the supernatant fluids of a few strains remained weakly active after the treatment), but was resistant to heat treatment at 100 degrees C during 10 and 30 min. When the 10-fold concentrated supernatant fluids were added to a fresh culture of sensitive cells it produced a rapid inactivation. According to these preliminary tests, different strains of enterococci produced compounds with slightly different antivibrio properties, and these compounds were heat-resistant and had a predominantly proteinaceous nature.

Bacteriocins↗
PubMed2003

Anomalies in species identification of enterococci from veterinary sources using a commercial biochemical identification system.

AIMS: A commercial biochemical panel ID kit was used to identify presumptive enterococci isolates of veterinary or agricultural origin obtained during different steps of culture. METHODS AND RESULTS: Fifty isolates identified as enterococci using a genus PCR assay were tested for genus and species identification using the BBL Crystal Identification Gram-Positive ID kit (Becton Dickinson, Sparks, MD, USA). Following sub-culture of the isolates three times, 59% agreement with the original panel ID was obtained. After four and six sub-cultures, percentage agreement increased to 61 and 64%, respectively. Nineteen of the 50 cultures were identified as both Enterococcus faecalis and E. faecium. CONCLUSIONS: As a result of the variability between speciation of isolates following re-culture, additional methods for speciation are warranted. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the identification of the genus and species of non-human enterococcal isolates can vary greatly during successive passages when using this kit.

Animals↗
PubMed1999

Rectal colonization with vancomycin-resistant enterococci among high-risk patients in an Israeli hospital.

The prevalence of rectal carriage of vancomycin-resistant enterococci (VRE) in two high-risk populations--61 patients admitted to ICU and 92 patients on renal dialysis--was studied longitudinally over a period of six months in a 650-bed general hospital. ICU patients were swabbed weekly and dialysis patients monthly. Enterococcal isolates were fully identified using the ATB identification system, and MICs were determined according to the NCCLS recommendations. Enterococci were isolated in 52 (83.6%) ICU patients and 86 (93.4%) dialysis patients. VRE were recovered at least once in 14 (27%) ICU patients and four (4.8%) dialysis patients. All VRE isolates (MIC of vancomycin > or = 256 micrograms/mL) were resistant to teicoplanin (MIC > or = 32 micrograms/mL; vanA phenotype), 87.5% were ampicillin-resistant, and 92% showed high-level resistance to gentamicin; 88% were E. faecium. The main risk factors for acquisition of VRE included duration of hospitalization in the six months preceding entry into the study and during the survey (P = 0.009 and 0.007 respectively, for ICU patients), and duration of antibiotic administration (P = 0.005, for ICU patients). The impact of vancomycin was most prominent (P = 0.005 for receipt and 0.06 for duration of administration, in ICU patients). Six of the 18 VRE carriers developed bacteraemia, six isolates being vancomycin-susceptible and one vancomycin-resistant (one patient had both). In this study, the first in Israel, a low rectal carriage rate occurred in renal dialysis patients and antibiotic use was the most important risk factor for VRE colonization.

Aged↗