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Linezolid-resistant Enterococcus faecium isolated from a patient without prior exposure to an oxazolidinone: report from the SENTRY Antimicrobial Surveillance Program.

A patient case report describes an Enterococcus faecium strain isolated from a blood culture that was resistant to linezolid (MIC, 8 microg/mL; G2576U mutation of 23S rRNA). Co-resistances were identified for vancomycin, ampicillin, macrolides, fluoroquinolones, chloramphenicol, rifampin, gentamicin (high-level), nitrofurantoin and trimethoprim/sulfamethoxazole. Etest (AB BIODISK, Solna, Sweden) and disk diffusion results also detected the oxazolidinone resistance pattern. Laboratories should be aware of the rare possibility of these strains occurring during linezolid therapy or spontaneously (this case) in contemporary practice, and have in vitro susceptibility methods available capable of detecting oxazolidinone resistance.

Acetamides↗

The bactericidal activity of magainins against Pseudomonas aeruginosa and Enterococcus faecium.

The antimicrobial activities of two synthetic magainins, MSI 94 and MSI 93, were investigated in vitro against 20 clinical isolates each of Pseudomonas aeruginosa and Enterococcus faecium. The ranges of the MICs of both agents were 6.25-50 mg/L for P. aeruginosa and 3.13-12.5 mg/L for E. faecium. In time-kill studies, the magainins demonstrated rapid, concentration-dependent bactericidal activity against selected isolates of both species. This bactericidal effect was inoculum-dependent for P. aeruginosa in both the logarithmic and stationary phases of growth. Bacterial regrowth was regularly observed after 24 h of incubation in the time-kill studies; this was due to loss of antimicrobial activity during overnight incubation rather than to the development of resistance. More rapid and sustained bactericidal activity was noted when the magainins were combined with either silver nitrate (AgNO3) or gentamicin against P. aeruginosa and with gentamicin against E. faecium. Neither agent produced a post-antibiotic effect on P. aeruginosa.

Animals↗

Prolonged colonization with vancomycin-resistant Enterococcus faecium in long-term care patients and the significance of "clearance".

Little is known about the persistence of colonization with vancomycin-resistant Enterococcus faecium (VRE) in the nononcologic, non-intensive care unit patient. We studied all patients who had VRE isolated on > or =2 occasions of > 1 year apart (Study A) and those who had been "cleared" of VRE colonization after 3 negative stool cultures (Study B). Twelve patients had stored VRE isolates recovered > 1 year apart (Study A), and 58% of paired isolates were genotypically related according to pulsed field gel electrophoresis patterns. In Study B, stool samples were obtained weekly from 21 "cleared" patients for 5 weeks, which revealed that 24% were VRE positive. For these culture-positive patients, 72% of the cultures failed to detect VRE. Recent antibiotic use was significantly more common in the culture-positive patients, as compared with culture-negative patients (P=.003). Colonization with VRE may persist for years, even if the results of intercurrent surveillance stool and index site cultures are negative. Cultures for detection of VRE in stool samples obtained from patients declared "cleared" are insensitive.

Culture Media↗

Linkage of determinants for streptogramin A, macrolide-lincosamide-streptogramin B, and chloramphenicol resistance on a conjugative plasmid in Enterococcus faecium and dissemination of this cluster among streptogramin-resistant enterococci.

A new streptogramin A resistance gene, satG (= vatE), has been recently identified in Enterococcus faecium UW1965 (Werner and Witte 1999. Antimicrob. Agents Chemother. 43: 1813-1814). Further sequence analysis of this plasmid revealed that vatE is in a cluster together with other resistance genes. The identified ORFs were nearly identical with the already known genes ermB and cat. The ermB fragment exhibited more than 99% identity with a resistance region from the streptococcal plasmid pIP501, whereas the cat fragment also contained a truncated rep gene homologue with more than 99% identity to sequences in small staphylococcal plasmids. The cat-rep and the ermB-vatE segments were linked by an IS1216V insertion sequence widely distributed among enterococci. PCR analysis of additional 76 streptogramin-resistant isolates possessing vatE and ermB revealed a linkage of both genes in 45 isolates (59%); 15 of them with a gene arrangement, cat-repU-IS1216V-ermB-vatE, identical to the reference strain UW1965. An identical linkage of IS1216V-ermB-vatE was found among isolates from poultry manure, poultry meat, stool samples of humans, and hospital patients indicating a possible spread of the resistance gene cluster via the food chain to humans.

Anti-Bacterial Agents↗

Drug resistance of Enterococcus faecium clinical isolates and the conjugative transfer of gentamicin and erythromycin resistance traits.

Drug resistance and the transferability of resistance were examined in 218 Enterococcus faecium clinical isolates obtained from in-patients of a Japanese university hospital between 1990 and 1999. One hundred and sixty one isolates (73.9%) were drug-resistant and 127 (58.2%) isolates were resistant to two or more drugs. Vancomycin resistant E. faecium (VRE) was not isolated. The transferability of drug-resistance to an E. faecium strain was examined by broth or filter mating. Six (12.5%) of the 48 gentamicin resistance traits, and fifty (50%) of the 101 erythromycin resistance traits were transferred by filter mating. The gentamicin resistance traits of five isolates and the erythromycin resistance traits of four isolates were transferred to the recipient strains by both broth mating and filter mating at a frequency of about 10(-6) and 10(-5) per donor cell, respectively. The five gentamicin resistant strains were shown to harbor pMG1-like plasmids on the basis of their Southern hybridization with pMG1 (65.1 kbp, Gm(r)), which transfers efficiently between enterococci by broth mating. Each of the four erythromycin resistant transconjugants obtained by broth mating harbored a large conjugative plasmid (more than 100 kbp). The plasmids showed no homology with well-characterized enterococcal conjugative plasmids such as pAD1, pPD1, pAM(beta)1, pIP501 and pMG1 by Southern hybridization. Of the erythromycin resistance traits that transferred only by filter mating, it was found that the erythromycin resistance trait was conferred by a 47-kbp transposable element that transferred from the chromosome of the donor strain to different sites within the pheromone responsive plasmid pAD1 (60 kbp) of the recipient strain, suggesting that the erythromycin resistance trait was encoded on a conjugative transposon, which was named Tn950.

Anti-Bacterial Agents↗

Bactericidal and inhibitory activity of quinupristin/dalfopristin against vancomycin- and gentamicin-resistant Enterococcus faecium.

There is a need for new agents, or combinations of agents, for the treatment of infections caused by vancomycin- and gentamicin-resistant Enterococcus faecium (VGREF) that may be resistant to all available antimicrobial agents. The early in-vitro activity of quinupristin/dalfopristin (30:70)--an injectable streptogramin--encouraged us to test this agent against VGREF. By broth dilution, the MICs of quinupristin/dalfopristin against 38 isolates of VGREF ranged from 0.06 mg/L to 2.0 mg/L (mode 0.12 mg/L). The addition of 0.5 mg/L of ciprofloxacin significantly reduced the modal MIC of quinupristin/dalfopristin to 0.015 mg/L (P = 5.75 x 10(-8)). Although the addition of 8.0 mg/L of teicoplanin or 4 mg/L of tetracycline did not significantly reduce the modal MIC, the lowest concentration of the MIC range was reduced from 0.06 to 0.015 mg/L. In broth, quinupristin/dalfopristin had slow bactericidal activity against the four strains tested over 48 h, with a 1-2 log10 cfu/mL reduction after 24 h in > 1 mg/L of quinupristin/dalfopristin for two strains and > 8 mg/L for the two other strains. A mixture of quinupristin/dalfopristin in a 70:30 ratio was more bactericidal: against one of the four strains 4-32 mg/L of the combination produced a further 0.5-1.0 log10 reduction in cfu/mL after 24 h and there was a reduction of 6.0 log10 cfu/mL after 48 h for another. By ultracentrifugation, the binding of 32 mg/L quinupristin/dalfopristin to human plasma protein was 90%, and in plasma broth, 32 mg/L of quinupristin/dalfopristin maintained bacteriostatic but not bactericidal activity. There is some useful synergy with ciprofloxacin and tetracycline, and the activity of quinupristin/dalfopristin may be enhanced against some strains by reversing the concentrations of its two components, quinupristin and dalfopristin, as that may occur in vivo.

Anti-Bacterial Agents↗

[Lactic acid production and urease activity in strains of Enterococcus faecium found in the rumen and their genetic stability].

Lactic acid production, urease activity and genetic stability were investigated in five selected rumen strains of Enterococcus faecium. The average value of urease activity in the tested strains was 16.5 +/- 0.953 nkat per ml, two strains were urease-negative. The values of E. faecium strains produced lactic acid ranged from 1.087 +/- 0.134 to 1.787 +/- 0.213 mmol per 1 l. Cultivation of the strains in ethidium bromide (EB) eliminated urease activity of these strains already in the first subculture, but the elimination effects of sodium dodecyl sulphate (SDS) and acridine orange (AO) were manifested later on (1-8 subcultures). Lactic acid production was eliminated in all strains from 1st to 8th subculture after cultivation in SDS, EB and also AO.

Animals↗

Detection of oxazolidinone-resistant Enterococcus faecalis and Enterococcus faecium strains by real-time PCR and PCR-restriction fragment length polymorphism analysis.

A real-time PCR assay identified linezolid-resistant Enterococcus faecalis and Enterococcus faecium isolates with a G2576U rRNA mutation. PCR-restriction fragment length polymorphism analysis of ribosomal DNA amplicons with NheI also detected this mutation. Both assays detected isolates heterozygous at this position. Recognition of isolates with what is presently the most frequent oxazolidinone resistance mutation may aid surveillance and individual case management.

Anti-Bacterial Agents↗

Evaluation of a vanA-specific PCR assay for detection of vancomycin-resistant Enterococcus faecium during a hospital outbreak.

We investigated the use of PCR as an alternative to culture of fecal samples for detection of vanA-containing Enterococcus faecium during a recent hospital outbreak. Rectal swabs collected consecutively from 223 patients were analyzed by culture with and without enrichment broth and by vanA-specific PCR of enrichment broth samples. Fifty-five specimens were positive for vanA-containing E. faecium by at least one method. The sensitivities of the vanA-specific PCR assay and agar culture with and without enrichment broth were 94.5, 98, and 89%, respectively. All three methods were 100% specific. Final results were obtained much more rapidly by PCR (within 24 to 30 h of specimen submission) than by the culture methods (4 to 5 days). Thus, PCR is an accurate and rapid alternative to culture for detection of vancomycin-resistant enterococci during hospital outbreaks.

Anti-Bacterial Agents↗

Conjugal transfer of aminoglycoside and macrolide resistance between Enterococcus faecium isolates in the intestine of streptomycin-treated mice.

The purpose was to study conjugal transfer of resistance genes between a multi-resistant Enterococcus faecium isolate and a sensitive E. faecium isolate. Co-transfer of erm(B)-Tn5405-like element and aac(6')-Ie-aph(2'')-Ia was obtained in both in vivo and in vitro. Plasmid profiles and Southern blots showed that both the erm(B)-Tn5405-like element and aac(6')-Ie-aph(2'')-Ia were placed on the same large plasmid (>147 kb). These data show to our knowledge the first co-transfer of the erm(B)-Tn5405-like element and aac(6')-Ie-aph(2'')-Ia. The in vivo study also indicates that transfer of resistance genes between enterococci might occur under natural conditions in the gut of animals.

Aminoglycosides↗

Detection of clonally related vanB2-containing Enterococcus faecium strains in two Spanish hospitals.

The aim of this study was to characterize the resistance mechanism in four clinical and five intestinal vancomycin-resistant Enterococcus faecium strains with VanB phenotype recovered from unrelated patients confined in two Spanish hospitals and to determine their clonal relationships. MIC values for vancomycin and teicoplanin were 16-32 and 0.5 microg ml-1, respectively. The mechanism of vancomycin resistance, as well as the genetic environment of the implicated gene, was analysed by PCR and sequencing. The vanB2 gene was detected in all nine E. faecium strains and the intergenic vanSB-YB region showed the characteristic mutations of the vanB2 subtype. Two possibly related PFGE patterns, A (seven strains) and B (two strains), were distinguished among these enterococci. The vanXB-ORFC intergenic region was amplified in the nine strains and two amino acid changes were detected in the protein encoded by the vanXB gene in strains of pattern A with respect to those of pattern B. The vanB2 gene cluster was integrated into Tn5382 in all nine strains, being pbp5 gene-linked to this transposon. The ant(6')-Ia, aph(3')-IIIa and erm(B) genes were also detected in all of the strains. Both isolates with PFGE pattern B contained the esp gene. In summary, vanB2-containing E. faecium strains with indistinguishable PFGE patterns were recovered from seven patients from two Spanish hospitals.

Anti-Bacterial Agents↗

Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.

Sequence determination of the flanking regions of the vancomycin resistance van gene cluster carried by pIP816 in Enterococcus faecium BM4147 revealed similarity to transposons of the Tn3 family. Imperfect inverted repeats (36 of 38 bp) delineated a 10,851-bp element designated Tn1546. The 4-kb region located upstream from the vanR gene contained two open reading frames (ORF) transcribed in opposite directions. The deduced amino acid sequence of ORF1 (988 residues) displayed, respectively, 56 and 42% identity to those of the transposases of Tn4430 from Bacillus thuringiensis and of Tn917 from Enterococcus faecalis. The product of ORF2 (191 residues) was related to the resolvase of Tn917 (33% amino acid identity) and to the Res protein (48%) of plasmid pIP404 from Clostridium perfringens. Tn1546 transposed consecutively in Escherichia coli from plasmid pUC18 into pOX38 and from pOX38 into various sites of pBR329. Transposition was replicative, led to the formation of cointegrates, and produced a 5-bp duplication at the target site. Southern hybridization and DNA amplification revealed the presence of Tn1546-related elements in enterococci highly resistant to glycopeptides. Analysis of sequences surrounding these elements indicated that transposition plays a role in dissemination of the van gene cluster among replicons of human clinical isolates of E. faecium.

Alanine↗

Screening and electronic labelling of ward contacts of vancomycin-resistant Enterococcus faecium vanB carriers during a single-strain hospital outbreak and after discharge from hospital.

A large single-strain outbreak of vancomycin-resistant Enterococcus faecium (VREF) vanB occurred in Royal Perth Hospital from July to December 2001. When a VREF-carrying patient was discovered on a ward, all patients on the ward were screened with rectal swabs. A total of 172 patients were colonised, four with infections, but no deaths were attributable to VREF. The number of rectal swabs required to detect each carrier was recorded. On average four rectal swabs, each collected on separate days, were needed to detect more than 90 per cent of the 172 VREF carriers who were epidemiologically linked to the Royal Perth Hospital outbreak. An electronic alert system (Micro-Alert) was used to identify ward contacts of VREF carriers and enabled those who had not been screened before discharge to be followed-up and screened. Ninety-six contacts were actively followed-up in October 2001 and 32 (33.3%) were found to be VREF carriers. From 28 September 2001 to 30 April 2002, a total of 1,977 ward contacts were screened after discharge from hospital and 54 (2.73%) were found to be carrying VREF. We conclude that during single-strain outbreaks of vancomycin-resistant enterococci in hospitals, patient contacts need to be screened on more than three occasions in order to detect most of the carriers and control the outbreak. Secondly, electronic labelling and active follow-up of ward contacts of VREF carriers resulted in a significant number of carriers being detected who otherwise posed a risk of initiating further outbreaks in hospitals if they were readmitted.

Carrier State↗

Performance, diarrhea incidence, and occurrence of Escherichia coli virulence genes during long-term administration of a probiotic Enterococcus faecium strain to sows and piglets.

As part of an interdisciplinary research project, the performance response of sows and their litters to the probiotic strain Enterococcus faecium NCIMB 10415, as well as some health characteristics of the piglets, were studied. Gestating sows (n = 26) were randomly allotted into 2 groups. The probiotic was administered by dietary supplementation to 1 group of sows and their respective litters (probiotic group), whereas the second group (control group) received no probiotic supplementation. The duration of the treatment was nearly 17 wk for sows (d 90 ante partum until d 28 postpartum) and 6 wk for piglets (d 15 to 56). Body weight and feed consumption were recorded weekly. The frequency of 4 toxin and 5 adhesion genes of putative pathogenic Escherichia coli was monitored weekly (d 7 to 35) by multiplex PCR assays, and fecal consistency of weaned piglets was studied daily. Probiotic treatment of lactating sows led to an overall pre-weaning mortality of 16.2% compared with 22.3% in the control group (P = 0.44). Animal losses during the first 3 d of the suckling period were decreased in the probiotic group (P = 0.09). For piglets (n = 153), which were weaned at 28 d, there were no overall treatment differences in BW gain, feed intake, or feed efficiency. Probiotic supplementation, however, led to nearly a 40% reduction (P = 0.012). The actual percentage of piglets with postweaning diarrhea in the probiotic group was 21% compared with 38% in the control group (P = 0.05). The study on virulence factors of dominant fecal E. coli isolates revealed a high diversity with varying frequency and distribution of each single pathogenicity gene. The 440 isolates carried 29 different pathogenicity gene combinations as well as each of the 9 pathogenicity genes alone. Altogether, isolates with more than 2 pathogenicity genes were quite rare (< or = 10%), and up until d 28 isolates without any pathogenicity gene occurred most frequently. Depending on the time of sampling, one-third or more of all isolates contained est2 or est1b as single gene or in combination with other pathogenicity genes.

Animal Feed↗

Copper resistance in Enterococcus faecium, mediated by the tcrB gene, is selected by supplementation of pig feed with copper sulfate.

The tcr gene cluster mediates in vitro copper resistance in Enterococcus faecium. Here we describe the selection of tcr-mediated copper resistance in E. faecium in an animal feeding experiment with young pigs fed 175 mg copper/kg feed (ppm), which is the concentration commonly used for piglets in European pig production. tcr-mediated copper resistance was not selected for in a control group fed low levels of copper (6 ppm). We also show coselection of macrolide- and glycopeptide-resistant E. faecium in the animal group fed the high level of copper. Finally, we identify the tcr genes in the enterococcal species E. mundtii, E. casseliflavus, and E. gallinarum for the first time.

Animal Feed↗

Enterococcus faecium F58, a bacteriocinogenic strain naturally occurring in Jben, a soft, farmhouse goat's cheese made in Morocco.

AIMS: Characterization of Ent F-58 produced by Enterococcus faecium strain F58 isolated from Jben, a soft, farmhouse goat's cheese manufactured without starter cultures. METHODS AND RESULTS: E. faecium strain F58 was isolated because of its broad inhibitory spectrum, including activity against food-borne pathogenic and spoilage bacteria. The antimicrobial substance was produced during the growth phase, with maximum production after 16-20 h of incubation at 30 degrees C, and was stable over a wide pH range (4-8) and at high temperatures (5 min at 100 degrees C). The enterocin was purified to homogeneity using cation exchange and hydrophobic interaction on C-18 and reverse-phase high-performance liquid chromatography. The activity was eluted as two individual active fractions (F-58A and F-58B) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed masses of 5210.5 and 5234.3 Da respectively. Both peptides were partially sequenced by Edman degradation, and amino-acid sequencing revealed high similarity with enterocin L50 (I). PCR-amplified fragments containing the structural genes for F-58 A and B were located in a 22-kb plasmid harboured by this strain. We verified that it also holds the structural gene for P-like enterocin. CONCLUSION: E. faecium strain F58 from Jben cheese, a producer of enterocin L50, exerts an inhibitory effect against strains of genera such as Listeria, Staphylococcus, Clostridium, Brochothrix and Bacillus. Enterocin was characterized according to its functional and biological properties, purification to homogeneity and an analysis of its amino acid and genetic sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium strain F58 is a newly discovered producer of enterocin L50, the biotechnological characteristics of which indicate its potential for application as a protective agent against pathogens and spoilage bacteria in foods.

Amino Acid Sequence↗

Diversity of structures carrying the aac(6')-aph(2") gene in clinical Enterococcus faecalis and Enterococcus faecium strains isolated in Tunisia.

The diversity of structures carrying the aac(6')-aph(2") gene was studied in 46 high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium clinical strains recovered in a Tunisian hospital during the period 2000-2003. The inclusion of the aac(6')-aph(2") gene within the Tn4001 composite element or in its truncated forms (lacking the IS256 at the right, the left or at both sides of the aac(6')-aph(2") gene) was investigated by PCR and sequencing. The aac(6')-aph(2") gene was included in the composite Tn4001 element in 19 of 34 high-level gentamicin-resistant E. faecalis strains (56%) and in 1 of 12 E. faecium strains (12%). A truncated form of Tn4001 lacking IS256 at the left-hand (in 10 E. faecalis and 8 E. faecium), at the right-hand (3 E. faecalis and 2 E. faecium) or at both sides of the aac(6')-aph(2") gene (in 2 E. faecalis and 1 E. faecium) was also detected in 26 of our enterococci. The transference by conjugation of the aac(6')-aph(2") gene, associated with other resistance genes, was demonstrated in seven of the high-level gentamicin-resistant E. faecalis strains.

Acetyltransferases↗

Cloning and heterospecific expression of the resistance determinant vanA encoding high-level resistance to glycopeptides in Enterococcus faecium BM4147.

Fragments of plasmid pIP816, which confers high-level glycopeptide resistance in Enterococcus faecium BM4147, were cloned into a conjugative gram-negative-gram-positive shuttle vector. The resulting hybrids were transferred by conjugation from Escherichia coli to Enterococcus faecalis and Bacillus thuringiensis. A 4-kilobase EcoRI fragment from pIP816 was found to confer vancomycin resistance in these hosts but not in E. coli or Bacillus subtilis.

Cloning, Molecular↗