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PubMedDate not supplied

Penicillin resistance and aminoglycoside-penicillin synergy in enterococci.

Susceptibility to penicillin, vancomycin, imipenem, streptomycin, kanamycin and gentamicin was tested in 130 clinical isolates of Enterococcus spp. by an agar dilution method. Penicillin resistance (MIC > 8 mg/l) was only observed among strains of Enterococcus faecium and Enterococcus raffinosus. Thirty-nine percent of the penicillin-resistant enterococci showed low-level resistance to at least one of the three aminoglycosides tested (gentamicin, kanamycin and streptomycin). Six Enterococcus strains (5 E. faecium and 1 E. raffinosus) with low-level resistance to gentamicin and different MICs for penicillin were tested for antibiotic synergy using time-killing curves. When penicillin concentrations equal to or higher than the MICs were used, synergism was established, even when highly penicillin-resistant strains (MIC > 200 mg/l) were tested. No synergy was observed when penicillin concentrations were below the MICs.

Aminoglycosides↗

PubMed1999

Biochemical and genetic evidence of enterocin P production by two Enterococcus faecium-like strains isolated from fermented sausages.

Two bacteriocin-producing Enterococcus faecium-like strains were independently isolated from fermented sausages. Bacteriocins were purified to homogeneity by ammonium sulfate precipitation, gel filtration, cationic exchange, hydrophobic interaction, and reverse-phase liquid chromatography. Two peptide inhibitory fractions were purified from each strain, denominated A and B for E. faecium AA13, and C and D for E. faecium G16. Fraction B was blocked for amino acid sequencing by Edman degradation, while the amino acid sequences obtained from peptides A, C, and D contained the YGNGV consensus motif in positions 5 to 9, and the ATRS sequence in positions 1 to 4. By use of PCR techniques and nucleotide sequencing, the structural gene of enterocin P was found both in E. faecium AA13 and E. faecium G16. Metabolic and genetic features of the two strains suggest that they are slightly different, they may produce more than one bacteriocin, and both produce enterocin P.

Amino Acid Sequence↗

PubMed2006

Inhibitory effects of Enterococcus strains obtained from a probiotic product on in vitro growth of Salmonella enterica serovar enteritidis strain IFO3313.

Enterococcus faecium and Enterococcus gallinarum strains were isolated from a commercial probiotic product and the effects of these strains on the growth of Salmonella enterica serovar Enteritidis strain IFO3313 were investigated. Viable cell counts of Salmonella Enteritidis in mixed cultures with the probiotic product isolate of E. faecium were significantly (P < 0.05) lower than those in pure cultures after 6, 8, and 24 h when the cultures were incubated in heart infusion broth at 37 and 41 degrees C. Significant differences in viable cell counts of Salmonella Enteritidis in mixed cultures with the probiotic product isolate of E. gallinarum and those in pure cultures were also observed after 8 and 24 h at 37 and 41degrees C. Similar observations were shown in mixed cultures of Salmonella Enteritidis with the reference strains of E. faecium GIFU8355 and E. gallinarum ATCC 49573. Significant differences in viable cell counts of these enterococcal strains were not shown among pure and mixed cultures with Salmonella Enteritidis. The pH values in pure and mixed cultures were 7.0 or 7.5 throughout the experiments. E. faecium strains were found to harbor the genes encoding enterocins A and B and showed inhibitory zones with a diameter of 4 to 6 mm against growth of Salmonella Enteritidis in the enterocin production assays. However, the E. gallinarum strains possessed neither of the enterocin genes tested and exhibited no inhibition zone in the enterocin production assays. These results indicated that enterococcal strains exhibit inhibitory effects on the growth of Salmonella Enteritidis and these effects were due to both enterocin and nonenterocin factors.

Antibiosis↗

PubMed2006

Identification of Tn5397-like and Tn916-like transposons and diversity of the tetracycline resistance gene tet(M) in enterococci from humans, pigs and poultry.

OBJECTIVES: To analyse the sequence diversity of the tetracycline resistance gene tet(M) and its location on mobile elements in Enterococcus faecium and Enterococcus faecalis from humans, pigs and poultry in Denmark. METHODS: A total of 76 isolates were screened for Tn916/Tn1545-like and Tn5397-like transposons using PCR. tet(M) was sequenced in 15 of the isolates and compared with tet(M) sequences submitted to GenBank (phylogenetic analysis and signs of recombination). Plasmids were extracted, filter-mating experiments were performed and Tn5397-like transposons were further characterized in selected isolates. RESULTS: In 8 of 13 isolates of E. faecium from broilers, tet(M) was present on Tn5397-like transposons, whereas tet(M) was predominantly associated with Tn916/Tn1545-like transposons in E. faecium from pigs and humans, as well as in E. faecalis from humans, pigs and broilers (50 of 63 isolates). The tet(M) genes were divided into three major subgroups according to the phylogenetic analysis. Subgroup I consisted of tet(M) from Clostridium difficile and E. faecium associated with Tn5397-like elements, subgroup II consisted of tet(M) located on Tn916/Tn1545 family transposons and subgroup III consisted of tet(M) associated with composite elements containing several resistance genes. We found evidence of recombination both within and between these groups. Moreover, we identified an E. faecium isolate with both Tn916/Tn1545-like and Tn5397-like elements. CONCLUSIONS: This study showed that enterococci contain diverse tet(M) genes present on different mobile elements, which may suggest that enterococci play an important role in the evolution and horizontal spread of mobile elements carrying tet(M). This is the first report of Tn5397-like elements in enterococci.

Animals↗

PubMed2006

In vitro development of resistance to DX-619 and other quinolones in enterococci.

OBJECTIVES: To investigate the molecular events involved in the development of quinolone resistance in enterococci. METHODS: Clinical isolates of Enterococcus faecium and Enterococcus faecalis were exposed to inhibitory and subinhibitory concentrations of DX-619, ciprofloxacin, levofloxacin, gatifloxacin and moxifloxacin. Mutational frequencies were calculated and susceptibility changes were determined. The quinolone resistance determining regions (QRDRs) of gyrA and parC in less-susceptible mutants were amplified by PCR and sequenced. RESULTS: Single-step mutants of E. faecalis and E. faecium were selected with all drugs. There were no differences in the frequencies of mutant selection among drugs, with frequencies ranging from 10(-5) to 10(-8). All single-step mutants were inhibited by 0.03-1 mg/L DX-619, 0.25-8 mg/L moxifloxacin, 0.5-8 mg/L gatifloxacin, 1-16 mg/L levofloxacin and 1-32 mg/L ciprofloxacin. No QRDR changes were observed in single-step mutants. Less-susceptible mutants selected after five passages on agar containing subinhibitory quinolone concentrations were inhibited by 0.12-8 mg/L DX-619, 1-64 mg/L moxifloxacin, 2-64 mg/L gatifloxacin and 2-128 mg/L levofloxacin and ciprofloxacin. QRDR changes were detected in only 9 of the 20 fifth-passage mutants, suggesting that mutations outside the purported QRDRs and/or other resistance mechanisms were also involved. CONCLUSION: The relatively high frequencies at which single-step mutants were selected with all drugs indicate that caution is necessary if quinolones are to be considered for monotherapy of serious enterococcal infections. DX-619, the most potent quinolone, may have potential as an anti-enterococcal agent if sufficient concentrations can be safely attained in vivo.

Anti-Bacterial Agents↗

PubMedDate not supplied

In vitro activity of LY333328, a new glycopeptide, against extracellular and intracellular vancomycin-resistant enterococci.

The objectives of the study were to observe the activity of LY333328, a new semisynthetic glycopeptide, compared to that of vancomycin against six strains of Enterococcus faecium and Enterococcus faecalis, including four vancomycin-resistant strains. Bacteria ingested by polymorphonuclear leukocytes (PMN) as well as extracellular bacteria were studied using a colony count method. The activity against intracellular bacteria was tested with the drugs present in the extracellular medium, as well as after preincubating the PMN and removal of the drugs. LY333328 is active against the tested enterococci, regardless of their susceptibility to vancomycin, with MICs of 1-2 mg/l. It is bacteriostatic against extracellular enterococci at concentrations of 2 microg/ml and above regardless of their resistance to vancomycin. After 4 h incubation at 10 MIC, vancomycin-resistant strains of E. faecium and E. faecalis located intracellularly were reduced by 55% and 90%, respectively. Even after preincubation and removal of the drug, LY333328 had an effect at 10 MIC with a 20-30% reduction in the inoculum. The results suggest that in contrast to vancomycin, LY333328 is active against intracellular vancomycin-resistant enterococci, particularly E. faecalis, even after removal of the extracellular drug.

Anti-Bacterial Agents↗

PubMed1995

Comparison of pigment production and motility tests with PCR for reliable identification of intrinsically vancomycin-resistant enterococci.

Forty-eight clinical isolates identified as either Enterococcus faecium or Enterococcus faecalis with the MicroScan system (Dade International, MicroScan Inc., West Sacramento, Calif.) were further characterized by two supplementary biochemical tests (pigment production and motility). Twenty isolates (42%), all initially identified as E. faecium, were motile. Of these 20, 8 isolates (17%) produced yellow pigment and were identified as Enterococcus casseliflavus and the remaining 12 (25%) were nonpigmented and were identified as Enterococcus gallinarum. Identical identification results were obtained when PCR amplification of regions of the vanC gene was used as a technique for differentiating these organisms. The results of this study indicate that motility and pigment production tests together with commercial test systems are sufficient for reliable identifications of E. faecium, E. casseliflavus, and E. gallinarum.

Bacteriological Techniques↗

PubMed2006

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food.

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.

Drug Resistance, Bacterial↗

PubMed1995

Evaluation of Vitek and API 20S for species identification of enterococci.

The performance of the Vitek System and the API 20S for identifying enterococci to the species level was evaluated in comparison with a conventional reference test scheme. A total of 369 isolates originally derived from a sample from 97 medical centers were tested, and the percentage of correctly identified organisms was (Vitek/API): 97.6%/79.2%, 95.3%/91.2%, and 86.0%/20.7% for Enterococcus faecalis, Enterococcus faecium, and Enterococcus non-faecalis-non-faecium species, respectively. In addition, the predictive values for species identification of the results reported by the systems were 98.1% for Vitek and 85.9% for API. The Vitek System GPI card (software version > or = 8.1) was considered to be very acceptable for enterococci identification, with errors principally among the rare Enterococcus raffinosus isolates.

Bacteriological Techniques↗

PubMed2006

Sources and pathways of spread of vancomycin-resistant enterococci in hemato-oncological patients.

The presented study aims at analyzing an increasing prevalence of vancomycin-resistant enterococci (VRE) isolated from various kinds of clinical material obtained from patients in the Department of Hemato-oncology (DHO), University Hospital in Olomouc, Czech Republic. Between January 1 and March 31, 2005, enterococci were isolated by standard microbiological procedures using both clinical material obtained from hospitalized patients and samples from the department environment. Resistance to vancomycin and teicoplanin was determined by a standardized microdilution method. Phenotype determination of resistance to vancomycin was verified by PCR detection of vanA and vanB genes. In VanA Enterococcus faecium, macrorestriction analysis was performed by pulsed-field gel electrophoresis. During the monitored period, a total of 128 Enterococcus sp. strains were isolated, of which 38 (30 %) isolates from 22 different patients were determined as VRE. Dominating were Enterococcus faecium VanA (63 %) and Enterococcus casseliflavus VanC (16 %) strains. At the same time, one Enterococcus faecium VanA strain was acquired from a bed-side table used by a patient in whom a similar strain had been isolated repeatedly from various clinical materials including a rectal swab taken in 2004. Based on the macrorestriction analysis of genome DNA in 24 vancomycin-resistant Enterococcus faecium VanA strains isolated from the patients' clinical material, one strain from the bed-side table surface and one strain isolated from stools in 2004, 8 unique restriction profiles with similarity ranging from 90 % to 100 % were identified, which could be classified into 3 clonal types. Thus, we can assume not only the endogenous origin of the VRE in hemato-oncological patients and their potential selection caused by therapy with broad-spectrum antibiotics but also the ability of the strains to survive in a hospital setting and, subsequently, to be spread clonally by various vectors.

Cross Infection↗

PubMed2004

[Complex therapy of chronic hepatic encephalopathy supplemented with probiotic: comparison of two studies].

BACKGROUND: Several reports about usefulness of probiotics, including Enterococcus faecium in the treatment of chronic hepatic encephalopathy in patients with liver cirrhosis have been recently published. The results obtained by the administration of Enterococcus faecium M-74 + selenium will be evaluated and compared with those published by Loguercio et al. for Enterococcus faecium SF 68. METHODS AND RESULTS: Fifteen patients with liver cirrhosis, portal hypertension and chronic hepatic encephalopathy were treated, beside the standard therapy, also with one capsule of probiotic "Enterococcus faecium M-74 + Selenium" per day in three four-week periods, separated by two fortnight pauses. During the treatment, every patient was examined 9-times. The severity of chronic hepatic encephalopathy was evaluated with the aid of the portal systemic encephalopathy index, calculated from 5 parameters (mental stage, asterixis, number connecting test, blood ammonia, EEG). The indexes of initial and final values were compared. The time course of blood ammonia levels and that of results from number connecting test in either study were mutually compared on the basis of interval estimates of quadratic regression function. Significant improvement of the portal systemic encephalopathy index after the treatment was found: it decreased in average by 70% (55 to 85%, interval 95% confidence interval). The mental stage improved and the asterixis disappeared. The blood ammonia levels as well as the results from the number-connecting test after 8-9 weeks significantly approached the normal pattern. EEG findings improved and they were often normalised. Our starting blood ammonia levels (increased by 31% above norm) and results from the connecting test (increased by 60%) were significantly lower accordingly the Loguercio et al. (increased by 243 and 238%, respectively). During the treatment, the values in our study decreased by 25% and 30%, in the compared study by 50% and 70%. CONCLUSIONS: Our results proved the hypothetical favourable effect of probioticum Enterococcus faecium M-74 + Selenium on chronic hepatic encephalopathy. Interestingly, a markedly higher relative therapeutic effect has been achieved in more serious disorder in comparison with the less severe disorders in the present study.

Adult↗

PubMed2006

Occurrence and preliminary study of antimicrobial resistance of enterococci isolated from dairy goats in Spain.

The present study performed in Spain was designed to investigate the occurrence and antimicrobial resistance of enterococci in faecal and bulk tank milk samples from 222 healthy Murciano-Granadina dairy goats reared on 12 farms. Enterococci were isolated in 54.5% and 63.6% of the faecal and bulk tank milk samples, respectively. Enterococci were detected more frequently from goat kids (70%) than from replacement animals (44.4%) and adults (56.4%). Seven species were found in the faecal samples but the most common species detected were Enterococcus faecium (32.3%), Enterococcus faecalis (27.6%) and Enterococcus hiriae (22%). In contrast, only E. faecium and E. faecalis were found in the bulk tank milk samples. According to NCCLS (2002), of the 134 strains studied, 17 (12.7%) were resistant to at least 7 antimicrobials and 14 of these strains were resistant to vancomycin.

Animals↗

PubMed1996

Intrinsic penicillin resistance in enterococci.

Penicillin resistance development in enterococci has been associated with overproduction of a low-affinity penicillin-binding protein (PBP) that is a normal component of the PBP pattern of these bacteria and is apparently able to substitute the functions of the other PBPs. In resistant mutants of Enterococcus hirae ATCC 9790 the low-affinity PBP (PBP5) overproduction was associated with a deletion in a genetic element, located 1 kb upstream of the pbp5 gene, which negatively controlled PBP5 synthesis. Hypersusceptibility to penicillin was associated with a point mutation in the pbp5 gene, which causes premature termination of translation. Structural homologies between low-affinity PBPs of the different enterococcal species have been suggested by cross-reactivity of antibodies raised against E. hirae PBP5 with PBP5 of Enterococcus faecium and Enterococcus faecalis. Acquisition of a high-level ampicillin resistance in E. faecium was associated with overproduction of PBP5, which, compared with PBP5 of moderately resistant strains, appeared to be modified in its penicillin-binding capability. The modified phenotype of PBP5 was found to be associated to some amino acid substitutions in the region between the SDN and KTG motifs. In particular, the substitution converting a polar residue (T) in a nonpolar one (A or I) could play an important role in remodeling the penicillin-binding domain and determining the decrease in penicillin affinity.

Amino Acid Sequence↗

PubMed2004

Evaluation of the prevalence and risk factors for colonization by vancomycin-resistant Enterococcus among patients on dialysis.

BACKGROUND: Vancomycin-resistant Enterococcus (VRE) has been reported among long-term dialysis patients, although risk factors for VRE colonization are not well defined. This study aims to appraise the prevalence and risk factors for VRE colonization among patients on long-term dialysis therapy, as well as the mechanisms for dissemination of vancomycin resistance. METHODS: This is a cross-sectional survey of 320 patients on long-term dialysis therapy at 2 hospitals of the Federal University of São Paulo from June 2001 to March 2003. Fecal samples were collected from each patient once a week for 1 month. Samples with positive test results for VRE were submitted to molecular typing through automated ribotyping. RESULTS: VRE prevalence was 14.4%. There were significant associations between VRE and dialysis type (hemodialysis, P = 0.04), number of hospitalizations (P = 0.03), low hemoglobin level (P = 0.03), and leukocytosis (P = 0.05). Among samples with VRE (n = 56), 25% were Enterococcus faecium; 10.7%, Enterococcus casseliflavus; 57.1%, Enterococcus gallinarum; and 3.6%, Enterococcus faecalis. All samples isolated were sensitive to teicoplanin, except for E faecium samples, which were strongly resistant, although 9 of 14 patients with this isolate presented the same ribogroup (111-S-4). Typing of 6 samples from 8 dialysis patients with E gallinarum was performed, showing a predominant ribogroup (112-S-4). CONCLUSION: Hospital environment, hemodialysis, anemia, and leukocytosis appear to be associated with VRE colonization. These results suggest that dissemination of these bacteria among patients on long-term dialysis therapy may be taking place.

Aged↗

PubMed2001

Antimicrobial resistance of Enterococci in Lebanon.

There is little information on the types of Enterococcus spp and their antibiotic resistance patterns in Lebanon. One hundred and fifty-three consecutive clinical enterococcal isolates collected between 1998 and 1999 were tested against 11 antimicrobial agents using disk diffusion and the Etest. The isolates were identified by conventional methods and API-Strep and were found to consist of Enterococcus faecalis (72.5%), Enterococcus faecium (22.9%), Enterococcus avium (3.2%) and Enterococcus gallinarum (1.3%). The percent of resistant strains of E. faecalis and E. faecium respectively were, ampicillin 0.9 and 14%, erythromycin 59% and 40%, tetracycline 72% and 34%, chloramphenicol 32 and 11%, rifampin 36% and 57%, ciprofloxacin, 23% and 34%, norfloxacin 22 and 8%. High level aminoglycoside (HLA) resistance was found in 19% E. faecalis and 9% E. faecium for gentamicin and 36% and 26% for streptomycin. Excellent correlation was observed between the high level disk tests and the Etest in the detection of HLA resistance but not with the regular disks. None of the isolates showed resistance to vancomycin or teicoplanin except for one E. gallinarum isolate which showed intermediate resistance (MIC 16 mg/l) to vancomycin. These variable antimicrobial rates of resistance suggest a surveillance programme for antimicrobial resistance in this country would be helpful to help control infection, guide empirical antibiotic therapy and implement a policy of antibiotic usage.

Aminoglycosides↗

PubMed2003

Antibiotic resistance in animals.

There is currently no systematic surveillance or monitoring of antibiotic resistance in Australian animals. Registration of antibiotics for use in animals is tightly controlled and has been very conservative. Fluoroquinolones have not been registered for use in food producing animals and other products have been removed from the market because of human health concerns. In the late 1970s, the Animal Health Committee coordinated a survey of resistance in Salmonella and Escherichia coli isolates from cattle, pigs and poultry and in bovine Staphylococcus aureus. Some additional information is available from published case reports. In samples collected prior to the withdrawal of avoparcin from the market, no vancomycin resistant Enterococcus faecium or Enterococcus faecalis were detected in samples collected from pigs, whereas some vanA enterococci, including E. faecium and E. faecalis, were found in chickens. No vanB enterococci were detected in either species. Virginiamycin resistance was common in both pig and poultry isolates. Multiple resistance was common in E. coli and salmonellae isolates. No fluoroquinolone resistance was found in salmonellae, E. coli or Campylobacter. Beta-lactamase production is common in isolates from bovine mastitis, but no methicillin resistance has been detected. However, methicillin resistance has been reported in canine isolates of Staphylococcus intermedius and extended spectrum beta-lactamase producing E. coli has been found in dogs.

Animal Diseases↗

PubMed2004

Lack of correlation between phenotypic techniques and PCR-based genotypic methods for identification of Enterococcus spp.

A total of 123 genetically-unrelated strains of Enterococcus spp. strains (51 Enterococcus faecalis, 57 Enterococcus faecium, 10 Enterococcus gallinarum, and 5 Enterococcus casseliflavus) were phenotypically identified by biochemical profiles and by using an automated method. The strains were also analyzed by a PCR assay to assess the accuracy of the phenotypically-based methods for identification of Enterococcus spp. With this aim, a PCR assay using different cell targets, which allows simultaneous detection of glycopeptide-resistant genotypes as well as identification to the species level by means of different gene targets, was used as the gold standard method. All 51 strains of E. faecalis were correctly identified, whereas 48 of 57 strains (84.2%) of E. faecium, were correctly identified. All of the strains of E. gallinarum and 3 out of 5 strains of E. casseliflavus were also correctly identified. The overall results showed that it is possible to identify Enterococcus spp. at the molecular level in less than 30 hours, compared with the 48-96 hours required for the phenotypically-based methods. The excellent accuracy of the PCR assay in identifying these species, particularly E. faecium, must also be emphasized. These findings may have implications for the routine clinical identification of enterococci species.

Bacterial Typing Techniques↗

PubMed2000

Presence of variations in ribosomal protein L16 corresponding to susceptibility of enterococci to oligosaccharides (Avilamycin and evernimicin).

Fragments (414 bp) of the gene-encoding ribosomal protein L16 from Enterococcus faecium and Enterococcus faecalis that were resistant and susceptible to the oligosaccharide antibiotics avilamycin and evernimicin (SCH 27899) were sequenced and compared. The susceptible E. faecalis and E. faecium isolates had sequences that were similar to those of the type strains. All resistant E. faecalis isolates contained the same base pair variation [CGT (Arg-56) --> CAT (His-56)]. The same variation and two additional variations [ATC (Ile-52) --> ACC (Thr-52) and ATC (Ile-52) --> AGC (Ser-52)] were found in the resistant E. faecium isolates. This study indicated that resistance to the oligosaccharides in enterococci is associated with variations in the ribosomal protein L16.

Aminoglycosides↗