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PubMed2000

Characterization of vancomycin-resistant and vancomycin-susceptible Enterococcus faecium isolates from humans, chickens and pigs by RiboPrinting and pulsed-field gel electrophoresis.

Forty-eight vancomycin-resistant and 35 vancomycin-sensitive Danish Enterococcus faecium isolates obtained from pigs, chickens and humans, as well as the human vanA reference isolate BM4147, were characterized by EcoRI RiboPrinting and SmaI pulsed-field gel electrophoresis. RiboPrinting of the 84 isolates yielded 40 types whereas PFGE-typing yielded 57 types discriminated by differences in more than three bands. By molecular typing, both clonal spread of E. faecium as well as horizontal transmission of Tn1546 between animals and humans was supported. Furthermore, it was found that the population of E. faecium spreads freely between the animal and human reservoir.

Animals↗

PubMed2001

Vancomycin-resistant Enterococcus faecium strain carrying the vanB2 gene variant in a Polish hospital.

About 2.5 years after the first isolation of the VanA phenotype of vancomycin-resistant Enterococcus faecium (VREM) in Poland, the first VREM strains with the VanB phenotype have emerged independently in two different Warsaw hospitals. In one of these the VREM strain was selected during the long-term antimicrobial treatment of a patient with a wide variety of infection risk factors who died after 3 months of hospitalization. The strain was found to contain the transferable vanB2 gene cluster variant of the polymorphic type that was identified earlier in vancomycin-resistant enterococci from several different countries. In the course of infection the strain underwent genetic diversification due to DNA recombination.

Acute Disease↗

PubMed1995

Successful treatment of persistent bacteremia due to vancomycin-resistant, ampicillin-resistant Enterococcus faecium.

Emergence of vancomycin-resistant enterococci has become an increasing problem in many medical centers. We report a liver transplant recipient with vancomycin-resistant Enterococcus faecium bacteremia who was successfully treated using very high dose continuous infusion ampicillin/sulbactam, plus gentamicin after he remained bacteremic on high dose ampicillin and gentamicin. At our institution, 83% of E. faecium isolates from 1994 were inhibited by ampicillin/sulbactam compared to 66% for ampicillin at an MIC < or = 64 micrograms/ml. None of these strains produced beta-lactamase, suggesting sulbactam may have an unexplained beneficial effect against some enterococci. Although an MIC of < or = 8 micrograms/ml is required for ampicillin to be considered active against enterococci, much higher levels of ampicillin or ampicillin/sulbactam are safely achievable. The response of our patient and the reported in vivo data have implications for future treatment of this pathogen, and may necessitate a reevaluation of susceptibility interpretation guidelines by clinical laboratories, and therapeutic drug dosing by clinicians.

Aged↗

PubMed1999

Influence of a probiotic adjunct culture of Enterococcus faecium on the quality of cheddar cheese.

Cheddar cheese has previously been shown to be an effective vehicle for delivery of viable cells of a probiotic Enterococcus faecium strain to the gastrointestinal tract. The particular strain, E. faecium PR88, has proven efficacy in the treatment of irritable bowel syndrome, and in this study it was evaluated for suitability as a starter adjunct for Cheddar cheese manufacture. When added to cheesemilk at an inoculum of 2 x 10(7) cfu/mL, the enterococcal adjunct maintained viability in Cheddar cheese at levels of up to 3 x 10(8) cfu/g during 9 months of ripening. Increased proteolysis and higher levels of some odor-active volatile compounds were observed in Cheddar cheeses containing the PR88 adjunct compared with the control throughout the ripening period. In addition, the enterococcal adjunct strain did not affect cheese composition. Although sensory evaluation showed no significant difference in flavor/aroma and body/texture scores between control and experimental cheeses, repeated comments by the commercial grader consistently described the cheeses containing PR88 as 'more advanced than the control' and as having 'better flavor'. These findings indicate that the presence of the PR88 adjunct strain in Cheddar cheese at levels of >/=10(8) cfu/g may positively influence Cheddar flavor.

Cheese↗

PubMed2005

Relationship between copper, glycopeptide, and macrolide resistance among Enterococcus faecium strains isolated from pigs in Denmark between 1997 and 2003.

A significant relationship between copper resistance (tcrB), glycopeptide resistance (Tn1546), and macrolide resistance [erm(B)] in Enterococcus faecium isolated from pigs was found. The tcrB gene was located closely upstream of the Tn1546 element. However, the continued use of copper sulfate has not been able to maintain high levels of macrolide and glycopeptide resistance.

Animals↗

PubMed2005

Characterization of vancomycin-resistant Enterococcus faecium isolates from broiler poultry and pig farms in England and Wales.

This study aimed to investigate the occurrence and molecular epidemiology of vancomycin-resistant Enterococcus faecium (VREF) isolates on poultry and pig farms in England and Wales. A total of 217 VREF isolates were obtained from fresh feces and environmental swabs collected from conventional and organic farms. A predominant pulsed-field gel electrophoresis (PFGE) profile was found for each VREF-positive farm, together with less frequent types. All isolates presented the vanA genotype and were esp negative. Seventy-six percent of the VREF isolates were additionally resistant to nine or more antimicrobials, presenting a diverse range of resistance phenotypes. The multiresistance traits did not appear to be specific to individual farms or sample types (i.e., environmental or fecal), nor did they correlate with any specific PFGE type. Ninety-three percent of the isolates were resistant to penicillin, 89% were resistant to tetracycline, 87.5% were resistant to erythromycin, and 50% were resistant to quinupristin-dalfospristin (Synercid). The lack of clonality among these populations may suggest the horizontal transfer of resistance genes and/or a dynamic replacement of clonal lines rather than persistence.

Animal Husbandry↗

PubMed2005

Vancomycin-resistant Enterococcus faecium: catheter colonization, esp gene, and decreased susceptibility to antibiotics in biofilm.

To evaluate the molecular characteristics and antibiotic susceptibility in biofilm of vancomycin-resistant Enterococcus faecium (VREF) organisms that had caused catheter-related VREF bacteremia (VREF-CRB), we compared 22 isolates causing bacteremia obtained from patients with VREF-CRB with 30 isolates from control patients with gastrointestinal colonization by VREF. Using pulsed-field gel electrophoresis, we identified 17 unique strains among the 22 VREF-CRB isolates and 23 strains among the gastrointestinal isolates. The esp gene was detected in 53% (9 of 17) of the VREF-CRB and 61% (14 of 23) of the control strains (P = 0.6). VREF-CRB produced heavier biofilm colonization of silicone disks than did control organisms (P < 0.001). Daptomycin, minocycline, and quinupristin-dalfopristin were each independently more active than linezolid in reducing biofilm colonization by VREF-CRB (P < 0.01), with daptomycin being the most active, followed by minocycline. In conclusion, the esp gene in VREF is not associated with heavy biofilm colonization or catheter-related bacteremia. In biofilm, daptomycin and minocycline were the most active antibiotics against VREF, and linezolid was the least active.

Anti-Bacterial Agents↗

PubMed2001

Role of penicillin-binding protein 5 in expression of ampicillin resistance and peptidoglycan structure in Enterococcus faecium.

The contribution of penicillin-binding protein 5 (PBP 5) to intrinsic and acquired beta-lactam resistance was investigated by constructing isogenic strains of Enterococcus faecium producing different PBP 5. The pbp5 genes from three E. faecium clinical isolates (BM4107, D344, and H80721) were cloned into the shuttle vector pAT392 and introduced into E. faecium D344S, a spontaneous derivative of E. faecium D344 highly susceptible to ampicillin due to deletion of pbp5 (MIC, 0.03 microg/ml). Immunodetection of PBP5 indicated that cloning of the pbp5 genes into pAT392 resulted in moderate overproduction of PBP 5 in comparison to wild-type strains. This difference may be attributed to a difference in gene copy number. Expression of the pbp5 genes from BM4107 (MIC, 2 microg/ml), D344 (MIC, 24 microg/ml), and H80721 (MIC, 512 microg/ml) in D344S conferred relatively low levels of resistance to ampicillin (MICs, 6, 12, and 20 microg/ml, respectively). A methionine-to-alanine substitution was introduced at position 485 of the BM4107 PBP 5 by site-directed mutagenesis. In contrast to previous hypotheses based on comparison of nonisogenic strains, this substitution resulted in only a 2.5-fold increase in the ampicillin MIC. The reversed-phase high-performance liquid chromatography muropeptide profiles of D344 and D344S were similar, indicating that deletion of pbp5 was not associated with a detectable defect in cell wall synthesis. These results indicate that pbp5 is a nonessential gene responsible for intrinsic resistance to moderate levels of ampicillin and by itself cannot confer high-level resistance.

Ampicillin Resistance↗

PubMed2000

In-vitro synergistic activity of the combination of ampicillin and arbekacin against vancomycin-and high-level gentamicin-resistant Enterococcus faecium with the aph(2")-Id gene.

The combination of ampicillin plus arbekacin produced synergistic killing against 8 of 13 vancomycin-, ampicillin-, gentamicin-, and streptomycin-resistant Enterococcus faecium isolates that possess the high-level gentamicin resistance gene, aph(2")-Id. This combination may prove useful in treating infections caused by multiresistant enterococci.

Aminoglycosides↗

PubMed2006

Quinupristin-dalfopristin resistance in Enterococcus faecium isolates from humans, farm animals, and grocery store meat in the United States.

Three hundred sixty-one quinupristin-dalfopristin (Q-D)-resistant Enterococcus faecium (QDREF) isolates were isolated from humans, turkeys, chickens, swine, dairy and beef cattle from farms, chicken carcasses, and ground pork from grocery stores in the United States from 1995 to 2003. These isolates were evaluated by pulsed-field gel electrophoresis (PFGE) to determine possible commonality between QDREF isolates from human and animal sources. PCR was performed to detect the streptogramin resistance genes vatD, vatE, and vgbA and the macrolide resistance gene ermB to determine the genetic mechanism of resistance in these isolates. QDREF from humans did not have PFGE patterns similar to those from animal sources. vatE was found in 35%, 26%, and 2% of QDREF isolates from turkeys, chickens, and humans, respectively, and was not found in QDREF isolates from other sources. ermB was commonly found in QDREF isolates from all sources. Known streptogramin resistance genes were absent in the majority of isolates, suggesting the presence of other, as-yet-undetermined, mechanisms of Q-D resistance.

Animals↗

PubMed1996

Treatment of experimental endocarditis due to multidrug-resistant Enterococcus faecium with clinafloxacin and penicillin.

Clinafloxacin, a new quinolone antibiotic with enhanced activity against Gram-positive bacteria, has demonstrated in-vitro activity against multidrug-resistant Enterococcus faecium, particularly when combined with penicillin. Rabbits with experimental endocarditis due to a multidrug-resistant strain of E. faecium were treated with clinafloxacin and/or penicillin. After three days of therapy, significant reduction of bacterial concentrations were found in vegetations, kidneys, and spleens of animals treated with clinafloxacin. The combination of clinafloxacin and penicillin was significantly better in reducing vegetation bacterial concentrations compared to the other groups (-4.4 log10 cfu/g compared with control). Serum levels of clinafloxacin consistently exceeded the MIC of the strain, and clinafloxacin-resistant isolates could not be detected. Clinafloxacin demonstrated promising activity in vivo against multidrug-resistant E. faecium, and further studies are warranted.

Animals↗

PubMed1999

Genomic relationships between Enterococcus faecium strains from different sources and with different antibiotic resistance profiles evaluated by restriction endonuclease analysis of total chromosomal DNA using EcoRI and PvuII.

Forty-seven Enterococcus faecium strains from different sources were evaluated by restriction endonuclease analysis (REA) of total chromosomal DNA. Strains from chicken, pork, and humans were clearly divided into separate clusters, whereas strains from different countries, strains with different antibiotic resistance profiles, or clinical and healthy-subject strains were not.

Animals↗

PubMed2000

Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q.

Enterococcus faecium L50 grown at 16 to 32 degrees C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47 degrees C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Hâvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63:4321-4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47 degrees C and maximally detected at 47 and 37 to 47 degrees C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25 degrees C and are not detected at 37 degrees C or above.

Amino Acid Sequence↗

PubMed1996

Nosocomial infections with vancomycin-resistant Enterococcus faecium in liver transplant recipients: risk factors for acquisition and mortality.

The risk factors for acquisition of and mortality due to nosocomial infection with vancomycin-resistant Enterococcus faecium (VREF) in orthotopic liver transplant (OLT) recipients were studied at a tertiary care hospital; 32 VREF-infected OLT patients (cases) were compared with 33 randomly selected OLT recipients (controls). More antibiotics were administered preoperatively to cases (mean, 4 antibiotics per patient for 474 antibiotic-days) than to controls (mean, 1.8 antibiotics per patient for 131 antibiotic-days). Cases were more likely than controls to have received vancomycin therapy preoperatively and to have been hospitalized in the intensive care unit (ICU) preoperatively. Logistic regression revealed that the risk factors for acquisition of VREF infection were surgical reexploration and a prolonged stay in the surgical ICU postoperatively. In the cases, the risk factors for mortality were admission to the ICU preoperatively and hemodialysis. The mortality rate associated with polymicrobial bloodstream infections was 100% despite appropriate therapy. Sixteen and 18 cases received parenteral chloramphenicol and doxycycline, respectively, for treatment of VREF infection. There were no hematologic adverse effects attributed to chloramphenicol treatment. DNA analysis of selected E. faecium isolates suggested that infections were due to multiple clones. In summary, the source of VREF infection in OLT patients is the gastrointestinal tract. Antibiotic selective pressure may contribute to colonization. Infection with VREF is a predictor of morbidity and mortality in OLT patients.

Anti-Bacterial Agents↗

PubMed1995

Vancomycin-dependent Enterococcus faecium isolated from stool following oral vancomycin therapy.

The isolation of clinical strains of enterococci requiring vancomycin for growth has only recently been reported. We describe the isolation of Enterococcus faecium requiring vancomycin for growth from the stool of a patient who had completed oral vancomycin therapy. Growth of the vancomycin-dependent E. faecium was supported by ristocetin and D-alanyl-D-alanine but not by daptomycin, teicoplanin, or D,L-alanine. Spontaneous revertants not requiring vancomycin occurred at a rate of 1 in 10(6). Both the vancomycin-dependent E. faecium and the revertant hybridized with a vanB gene probe and had identical contour-clamped homogeneous electrophoresis patterns. The majority of revertant colonies were resistant to teicoplanin, suggesting constitutive production of the vanB ligase. We believe the vancomycin-dependent E. faecium evolved from a vancomycin-resistant, vancomycin-independent E. faecium in the presence of high concentrations of vancomycin in the intestine.

Aged↗

PubMed2006

A multifactorial design for studying factors influencing growth and tyramine production of the lactic acid bacteria Lactobacillus brevis CECT 4669 and Enterococcus faecium BIFI-58.

A central composite face design was used to study growth and tyramine production of two strains of lactic acid bacteria, Lactobacillus brevis CECT 4669 and Enterococcus faecium BIFI-58. The effects of five physicochemical factors (incubation temperature and time, environmental pH, added tyrosine concentration, and pyridoxal-5-phosphate (PLP) supplementation) on cell growth and tyramine production were analyzed under aerobic and anaerobic conditions. The parameters of the quadratic model for each response variable were estimated by multiple linear regression (MLR), and statistical analysis of the results led to the elucidation of mathematical models capable of predicting the behavior of the responses as a function of the main variables involved in the process. Incubation time was found to be the most important variable influencing growth in L. brevis, while pH showed the highest contribution in E. faecium. The production of tyramine was dependent on the added tyrosine concentration and incubation time. The proposed MLR model predicted the optimum conditions that gave maximum responses for L. brevis and E. faecium growth and tyramine production. In both strains, this model predicted that the anaerobic condition at acidic pH (4.4) in the presence of a high tyrosine concentration favors tyramine production.

Culture Media↗

PubMed2005

Population structure of Enterococcus faecium causing bacteremia in a Spanish university hospital: setting the scene for a future increase in vancomycin resistance?

Over an 8-year period (1995 to 2002), 86 Enterococcus faecium blood isolates from 84 patients, of which 54 were ampicillin resistant (AREF) and 32 were ampicillin susceptible (ASEF), were studied in a university hospital (1,200 beds; serving a population of 600,000) in Spain, a country characterized by a near-absence of resistance to vancomycin and very high rates of ampicillin resistance among enterococci. Clonal relatedness by pulsed-field gel electrophoresis (PFGE), antibiotic susceptibility, presence of the virulence/epidemicity genes esp(Efm) and hyl(Efm), and identification of purK alleles were studied. A group of isolates was also analyzed by amplified fragment length polymorphism (AFLP) and multilocus sequence typing. Medical charts (30 variables collected) were reviewed for 60/84 patients. ASEF showed high clonal diversity (32 PFGE types, 11 purK alleles, 4 AFLP genogroups), did not harbor putative virulence genes, and had no specific association with hospital acquisition. AREF isolates belonged to a clonal complex (CC) of genetically related strains (purK-1, AFLP genogroup C), occasionally harboring putative virulence traits, and were from patients with particular risk factors. Within this CC, previously associated with vancomycin-resistant E. faecium isolates causing outbreaks worldwide (W. L. Homan et al., J. Clin. Microbiol. 40:1963-1971, 2002), a great genetic diversity of antibiotic resistance and virulence/epidemicity profiles was found. Associations between esp and a >7-day hospital stay and between purK-1, hospital location, and nosocomial acquisition were noted (P < 0.001). These findings reflect the importance of local environmental differences in the evolution of this CC, suggesting that the emergence of vancomycin resistance among AREF strains in Spain may be a question of time.

Ampicillin↗

PubMed1999

Intestinal colonization of a human subject by vancomycin-resistant Enterococcus faecium.

OBJECTIVE: To study the ability of two strains of vancomycin-resistant Enterococcus faecium to colonize the human intestine. METHODS: A single human subject ingested separately two strains of vancomycin-resistant E. faecium isolated from a pig and a chicken. The feces were cultured on selective medium. Prior to ingestion no vancomycin-resistant cocci were present in the feces. Ingestion of 104-105 CFU resulted in either no colonization or isolation only after enrichment. Ingestion of 107 CFU of one strain resulted in colonization for a period of nearly 3 weeks, with fecal counts at times in excess of 106 CFU/g. Ingestion of similar numbers of the other strain and reingestion of the first strain resulted in excretion in the feces for much shorter periods. When the fecal count of the ingested strains was greater than 104-105 CFU/g, the strains were isolated from swabs taken from perianal skin. CONCLUSIONS: Vancomycin-resistant E. faecium strains from pigs and poultry are able to colonize the human gut and the perianal skin.

Journal Article↗