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PubMed2003

Antimicrobial resistance in recent fecal enterococci from healthy volunteers and food handlers in Spain: genes and phenotypes.

Susceptibility patterns to 15 different antibiotics and the presence of resistance genes were evaluated in recent fecal Enterococcus isolates recovered from 42 healthy volunteers (HV) and 43 food-handlers (FH). A total of 142 Enterococcus faecalis, 74 Enterococcus faecium, and 23 Enterococcus spp. with different antibiotic susceptibility patterns were studied. A higher percentage of resistance for moxifloxacin, erythromycin, glycopeptides and high-level resistance (HLR) to gentamicin were observed in the FH group. Ampicillin- or linezolid-resistant isolates were not recovered in any of the groups. The tet(M) gene was found in 96% and in 85% of tetracycline-resistant isolates from HV and FH, respectively. HLR-kanamycin was mediated by aph(3')-IIIa, or aac(6')-aph(2"), or both genes in all isolates from HV group and in 86% from FH group. The aac(6')-aph(2") gene was found in all HLR-gentamicin isolates. Ninety-one percent of HV and 71% of FH erythromycin-resistant isolates harbored the erm(B) gene (erythromycin MIC range of 8-128 microg/ml), whereas erm(A), erm(C), or mef(A) genes were not detected. Coexistence of erm(B), aph(3')-IIIa, and tet(M) genes was observed in 17% of the isolates of both groups. The HLR-gentamicin isolates presented unrelated PFGE patterns while 2 out of 3 vanA E. faecium isolates showed an indistinguishable SmaI-pulsed-field gel electrophoresis (PFGE) pattern. This study shows that despite 4 years of official banning of antibiotic growth promoters in animals, enterococci isolated from FH are more resistant than those from HV. This suggests the permanence of resistant clones or transferable resistance elements in farms and a possible exchange between food products and humans, or eventually the long-term permanence of certain clones in the FH intestinal tract.

Anti-Bacterial Agents↗
PubMed2000

Coverage of enterococci in community acquired secondary peritonitis: results of a randomized trial.

INTRODUCTION: The increasing number of enterococcal infections in hospitals and reports about the development of resistance of these bacteria make it necessary to review their importance as co-pathogens in secondary peritonitis. MATERIALS AND METHODS: A prospective randomized controlled trial on primary therapy of secondary peritonitis was carried out in six centers comparing cephalosporin-based antibiotic therapy to acylaminopenicillin-based therapy. RESULTS: Enterococci were only cultured in 6 of 110 cases from the abdomen and were found in only 5 cases of postoperative complications. No differences were found between penicillin-based vs. cephalosporin-based therapy. CONCLUSION: The study supports the view that these bacteria continue to play a minor role in secondary peritonitis. The point has to be emphasized, however, that the patients under study were in relatively good condition (APACHE II median 9 for cephalosporins and 10 for penicillins) and that postoperative cases of peritonitis were excluded.

Adult↗
PubMed1995

Molecular epidemiology of enterococci with high-level resistance to aminoglycosides.

DNA-based methodologies are considerably more powerful than other phenotype-based typing systems, providing a finer level of epidemiological discrimination, differentiating both closely and distantly related independent isolates that otherwise may appear as identical. In this study, plasmid analysis and pulsed-field gel electrophoresis were used to compare 28 isolates of Enterococci (respectively 13 strains of Enterococcus faecalis and 15 strains of Enterococcus faecium) with high-level resistance to aminoglycosides, isolated in Catania (Italy). Plasmid profile analysis resolved 20 different patterns among 24 plasmid harboring strains; many isolates showed one or two plasmids of the same size, but different plasmid content. Analysis of the PFGE-based RFLP patterns after SmaI digestion of genomic DNA resolved 26 different clones from 28 isolates: particularly, it resolved two different clones from three isolates showing identical plasmid profiles, and it identified as a single clone two isolates exhibiting different plasmid profiles. Thus, on the basis of our PFGE-based RFLP analysis data, we concluded that all the strains included in the study were genetically unrelated with two exceptions.

Aminoglycosides↗
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↗
PubMed1998

Transmission of VanA-type vancomycin-resistant enterococci and vanA resistance elements between chicken and humans at avoparcin-exposed farms.

The genetical relatedness between epidemiologically linked fecal VRE strains from poultry farmers (n = 5) and their broilers (n = 7) at five avoparcin-exposed Norwegian farms was examined. Pulsed-field gel electrophoresis (PFGE) of bacterial chromosomal digests and structural analysis of vanA resistance elements was performed. Animal and human Enterococcus faecium strains at one farm were genetically closely related with indistinguishable vanA elements and a single band position difference in PFGE analysis. Examination of the vanA elements in genetically unrelated strains by restriction enzyme digestion of Tn1546 long-PCR amplicons and ORF2-vanR intergenic sequencing revealed a pool of at least two distinct vanA gene cluster groups in the two reservoirs. The results indicate that transmission of VanA glycopeptide resistance in enterococci between human and animal at avoparcin-exposed farms can occur by direct transfer of VRE strains as well as horizontal spread of resistance genes between strains.

Animals↗
PubMed1999

Comparison of direct and enrichment methods for the selective isolation of vancomycin-resistant enterococci from feces of pigs and poultry.

Isolation results of vancomycin-resistant enterococci (VRE) of fecal samples from pigs and broiler and layer chickens obtained with two vancomycin-supplemented enrichment media, kanamycin aesculin azide (KAA) broth and Enterococcosel (ECC) broth, and three isolation media, KAA agar, ECC agar, and Slanetz and Bartley (SL) agar, were compared. Direct isolation on vancomycin-containing agar plates was not efficient in swine and layer chickens, which had only low numbers of VRE. In broilers chickens, the VRE content of the samples was high, and SL as well as ECC were found to perform better than KAA agar. The same three agar media were used as selective plating media after 1 and 2 days incubation of the samples in KAA and ECC enrichment broths. Sensitivities of the 12 different enrichment-plate combinations tested ranged from 0 to 81% in layer chickens and from 5 to 44% in samples from pigs. In the high prevalence type of samples from broilers, sensitivities still varied substantially from 52 to 78%. Incubating vancomycin-containing enrichment broths for 2 days compared with 1 day was favorable for the isolation of vancomycin-resistant Enterococcus faecalis, E. gallinarum, and E. casseliflavus but not for E. faecium and E. hirae/E. durans. ECC broth and ECC plates yielded the highest number of E. gallinarum and E. casseliflavus. In layer as well as in broiler chickens, ECC broth incubated for 2 days and plated on ECC agar was the most sensitive method. In pigs, however, KAA broth incubated for 2 days and plated on ECC medium yielded the highest number of VRE.

Animals↗
PubMed1999

Decreased incidence of VanA-type vancomycin-resistant enterococci isolated from poultry meat and from fecal samples of humans in the community after discontinuation of avoparcin usage in animal husbandry.

The use of the glycopeptide antibiotic avoparcin (AVO) as a feed additive in animal husbandry of many European countries led in 1994-1995 to frequent isolation of VanA-type vancomycin-resistant enterococci (VRE) from commercially produced animal foodstuffs as well as from fecal samples of nonhospitalized persons in Germany (Saxony-Anhalt state). However, at the end of 1997, a decreasing number of such VRE was detected in frozen and fresh poultry meat (chickens and turkeys) from German producers. At this point in time, AVO had been discontinued in animal husbandry for more than 2 and one-half years in Denmark/Norway, nearly 2 years in Germany, and about 8-9 months in all countries of the European Community and Switzerland, respectively. VRE were then only detected in very low concentrations in one-quarter of the poultry meat samples (eight of 31, originating from 18 distinct German producers and bought in 12 different supermarkets). A decline of VRE prevalence was also observed in the gut flora of healthy persons (VRE carriers) in the same region (Saxony-Anhalt state, Germany), having fallen from 12% (12/100) in 1994 when AVO was being used to 6% (6/100) in 1996 and 3% (13/400) in 1997 after it was discontinued. These results likely indicate the importance of antibiotic selective pressure by glycopeptides such as AVO for the presence of VRE in animal meat products from commercial animal husbandry. Additionally, it underlines the role of animal products for the spread of resistant bacteria and transferable resistance genes to humans in the community.

Animal Husbandry↗
PubMed2000

Detection and characterization of vancomycin-resistant enterococci in farm animals and raw meat products in Italy.

The emergence of vancomycin-resistant enterococci (VRE) in Europe has been ascribed to the long-time use of the glycopeptide antibiotic avoparcin as feed additive in food animals, until its ban in 1997 in EU. The pres- ence of VRE in food of animal origin is believed to represent a potential risk for the consumer. We studied the fecal carriage of VRE in broiler chickens and slaughter pigs in Italy before the avoparcin ban and eval- uated the impact of avoparcin withdrawal on the presence of VRE in raw meat products. Broilers and pigs were both found to be frequently colonized by VRE, as 36% and 24.6% of the flocks or the herds, respec- tively, were positive. Molecular typing of VRE strains by PFGE showed that animals housed in different pens within the same farm were colonized by clonally related strains. After the avoparcin ban, a decrease in the rate of VRE contamination in meat products was observed. Such a decrease was statistically significant in poultry (from 18.8% to 9.6%) but not in pork products (from 9.7% to 6.9%). The majority of VRE from all sources carried the vanA resistance gene and included Enterococcus faecium, E. faecalis, E. hirae, E. durans, and E. gallinarum. None of the strains carried the vanB gene, whereas constitutively resistant vanC-positive strains were frequently found. Our results show that avoparcin withdrawal has been successful in reducing VRE contamination in meat products. However, this measure needs to be complemented by a prudent use of glycopeptide antibiotics in human medicine.

Animals↗
PubMed2000

Typeability of Tn1546-like elements in vancomycin-resistant enterococci using long-range PCRs and specific analysis of polymorphic regions.

Molecular subtyping of the VanA-type resistance element Tn1546 in an international collection of 81 genomically diverse vancomycin-resistant enterococci (VRE) from human, animal, and environmental reservoirs was evaluated by restriction analysis of long-range PCR amplicons (PCR-RFLP), single gene PCRs, Southern blot analysis of genomic digests, and partial DNA sequencing. A dominant Tn1546-RFLP in accordance with Enterococcus faecium BM4147 was detected in 43 of the 49 long-range PCR positive strains from ecologically diverse sources in several European countries and the US. Tn1546-like elements from the 32 (40%) long-range PCR negative strains were typed into 17 different groups by single-gene PCRs and Southern blot analysis of the ORF1, ORF2, vanS-vanH, vanX-vanY, and vanZ regions. All these isolates showed deletions in the ORF1 and/or vanZ primer binding regions explaining the failure of long-range PCR amplification. Enlarged vanS-vanH or vanX-vanY fragments were detected in 7 (22%) and 16 (50%) of the long-range PCR negative strains, respectively. The enlarged vanS-vanH regions of five clinical isolates from the US (n = 2), Ireland (n = 2), and Norway (n = 1) contained identical IS1251-like insertions indicating intercontinental spread of the vanA gene cluster. Intergenic vanS-vanH IS1251 insertions have so far not been reported in European studies. Structural rearrangements of Tn1546-like elements may represent single recombination events that can serve as fingerprints in the molecular examination of vanA gene cluster evolution and transmission. The optimal strategy for such analysis has yet to be determined. Two alternative long-range PCRs with subsequent RFLP analysis were successfully used to type the majority of vanA gene clusters in an ecologically and geographically heterogeneous VRE strain collection, but failed to detect and type a group of variant Tn1546-like elements truncated in the left-end ORF1/ORF2 region. Further subtyping of such variants should specifically target the polymorphic vanS-vanH and vanX-vanY regions.

Base Sequence↗
PubMed2005

Detection of a novel aph(2") allele (aph[2"]-Ie) conferring high-level gentamicin resistance and a spectinomycin resistance gene ant(9)-Ia (aad 9) in clinical isolates of enterococci.

Aminoglycoside-modifying enzymes (AMEs) are major factors that confer aminoglycoside resistance to enterococci. In an epidemiologic study on distribution of 12 AME genes in 534 recent clinical strains isolated from a Japanese hospital, two uncommon AME genes, ant(9)-Ia and a novel aph(2") allele, aph(2")-Ie, were detected. ant(9)-Ia had been reported only in Staphylococcus aureus and encodes spectinomycin adenylyltransferase ANT(9)-I, which confers resistance to spectinomycin. The ant(9)-Ia gene was detected in three strains, a single strain each of Enterococcus faecalis, E. faecium, and E. avium. Nucleotide sequences of ant(9)-Ia from these three enterococcal species were identical to that reported for S. aureus and considered to be located on Tn 554. The new aph(2") allele, designated aph(2")-Ie, was identified in three E. faecium strains. The aph(2")-Ie allele was genetically close to aph(2")-Id reported in E. casseliflavus (93.7% amino acid sequence identity; 96.3% similarity), while distant from aph(2")-Ia, aph(2")-Ib, or aph(2")-Ic (26.3-29.5% amino acid sequence identity). Sequence divergence between APH(2")-Id and APH(2")-Ie was mostly located in amino-terminal half. In contrast, sequences corresponding to the three motifs required for aminoglycoside phosphotransferase were conserved except for a single amino acid. Three E. faecium strains having aph(2")-Ie showed high-level resistance to gentamicin and streptomycin, but not to kanamycin, dibekacin, and tobramycin, unlike enzyme specificity described for aph(2")-Id in E. casseliflavus. Such a difference in resistance phenotype was suggested to be related to amino acid sequence divergence between APH(2")-Id and APH(2")-Ie.

Alleles↗
PubMed1997

Skin colonization with vancomycin-resistant enterococci among hospitalized patients with bacteremia.

To assess the prevalence of skin and rectal colonization by vancomycin-resistant enterococci (VRE) in hospitalized bacteremic patients and to determine the relation between colonization and bacteremia, we compared 14 case patients who had bacteremia due to VRE with 30 control patients who had bacteremia due to other pathogens. Rectal colonization and skin (inguinal area and/or antecubital fossa) colonization with VRE were common among both case patients (100% had rectal colonization, and 86% had skin colonization) and control patients (37% had rectal colonization and 23% had skin colonization). Among patients with rectal colonization, skin colonization was more common when diarrhea or fecal incontinence was present. The bloodstream cleared without appropriate antimicrobial therapy in nine of the 14 patients with bacteremia due to VRE. The high prevalence of skin colonization with VRE may increase the risk of catheter-related sepsis, cross-infection, or blood culture contamination (which may explain the frequent spontaneous resolution of bacteremia due to VRE).

Anti-Bacterial Agents↗
PubMed1995

High-level resistance to aminoglycosides: comparison of community and nosocomial fecal isolates of enterococci.

Fecal carriage of enterococci highly resistant to streptomycin, gentamicin, and kanamycin was examined in 64 healthy volunteers with no exposure to hospitals and in 53 hospitalized individuals. High-level resistance to streptomycin and gentamicin was found in fecal specimens from 3% and 0, respectively, of the healthy volunteers and in fecal specimens from 41% and 15%, respectively, of the hospitalized individuals. We found that high-level resistance to kanamycin was also more common among hospitalized individuals than among healthy volunteers (36% vs. 17%). The frequent occurrence of high-level resistance to kanamycin in fecal isolates confirms that amikacin is a poor choice when attempting to achieve synergistic therapy for enterococcal infections, in particular for those infections that are nosocomially acquired.

Base Sequence↗
PubMed1995

Stool carriage, clinical isolation, and mortality during an outbreak of vancomycin-resistant enterococci in hospitalized medical and/or surgical patients.

During a nosocomial outbreak of infection due to vancomycin-resistant enterococci (VRE), rectal swabs that were collected weekly were used to identify and isolate VRE carriers. Over 6 months, 1,458 stool specimens from 724 high-risk patients were cultured, and 187 VRE isolates were recovered from 61 patients; 96% of the isolates were Enterococcus faecium. VRE tended to be isolated from clinical specimens from patients identified as VRE carriers by stool surveillance (P < .01). However, isolation of VRE from surveillance cultures preceded clinical isolation for only approximately 50% of the patients from whom a clinical VRE isolate was recovered. Mortality was greater (P < .05) among patients from whom a clinical VRE isolate was recovered than among patients from whom VRE was isolated only by stool surveillance. The mortality (1[17%] of 6) among patients for whom VRE was isolated from blood was similar to that (10 [27%] of 37) among patients for whom vancomycin-susceptible enterococcus was isolated from blood (P = .97). Despite prompt initiation of contact precautions for VRE carriers, the incidence of fecal carriage of VRE remained approximately 8% among this patient population for the 6-month period of the study.

Adult↗
PubMed1996

Manipulation of a hospital antimicrobial formulary to control an outbreak of vancomycin-resistant enterococci.

Infection control practices are not uniformly successful in limiting outbreaks of vancomycin-resistant enterococci (VRE). Despite the implementation of barrier precautions for VRE-infected patients, nearly one-half of the inpatients at our center were found to have gastrointestinal colonization by VRE. In an attempt to control the outbreak, we altered the antibiotic formulary by restricting the use of cefotaxime and vancomycin and adding beta-lactamase inhibitors to replace third-generation cephalosporins. The use of clindamycin was also restricted because of a concomitant outbreak of Clostridium difficile colitis. After 6 months, the average monthly use of cefotaxime, ceftazidime, vancomycin, and clindamycin had decreased by 84%, 55%, 34%, and 80%, respectively (P < .02). The point prevalence of fecal colonization with VRE decreased from 47% to 15% (P < .001), and the number of patients whose clinical specimens were culture positive also gradually decreased. A change in antibiotic use appears to have significantly affected our VRE outbreak when previous measures failed.

Ampicillin↗
PubMed1989

Pathogenicity of enterococci in a rat model of fecal peritonitis.

The pathogenicity of enterococci in intraabdominal sepsis has not been clarified. Therefore, fecal-type peritonitis was induced in rats by intraperitoneal injection of barium sulfate along with a bacterial inoculum consisting of Escherichia coli, Bacteroides fragilis, and Clostridium perfringens with or without Streptococcus faecalis. Mortality at 19 d and characteristics of intraabdominal abscesses in survivors at 19 d were analyzed. The presence of S. faecalis in the original inoculum was significantly associated with death or large (greater than 20 mm) abscess formation when these two end points were examined together. S. faecalis may synergize with other bacteria in intraabdominal sepsis to augment morbidity and possibly mortality.

Abscess↗
PubMed1993

The vanB gene confers various levels of self-transferable resistance to vancomycin in enterococci.

Thirty-nine strains of Enterococcus faecium and Enterococcus faecalis resistant to vancomycin and susceptible to teicoplanin on disk susceptibility testing (phenotypic class B) were isolated in 15 hospitals in Europe and the United States. The MICs of vancomycin for these strains ranged from 4 to 1024 micrograms/mL. Part of the vancomycin resistance gene vanB from E. faecalis V583 hybridized with a single but variably sized HindIII-KpnI fragment of total DNA from all 39 strains. This indicates that a single class of resistance determinants accounts for the VanB phenotype. No hybridization was detected with DNA from intrinsically resistant Enterococcus gallinarum or Enterococcus casseliflavus. Hybridization with DNA from enterococcal strains susceptible to or with acquired resistance to vancomycin and teicoplanin was not observed. The genes conferring resistance to vancomycin were self-transferable to other Enterococcus strains in 14 of the 39 strains. It thus appears that vanB confers various levels of conjugative vancomycin resistance in enterococci.

Bacterial Proteins↗
PubMed1995

Enterococcus faecalis cytolysin without effect on the intestinal growth of susceptible enterococci in mice.

A murine model was developed to determine whether the Enterococcus faecalis cytolysin, through its bacteriolytic action on gram-positive bacteria, could promote intestinal overgrowth of cytolytic strains. Sets of E. faecalis strains with varying cytolytic production and susceptibility to cytolytic activity were mixed 1:1 and allowed to compete in vitro in broth or in vivo after orogastric administration in mice pretreated with antibiotics. In general, cytolytic strains outgrew, by as much as 2000-fold, competing cytolysin-susceptible or -hypersusceptible strains in vitro. In contrast, no growth advantage was observed in vivo, despite similar transient colonization of the murine intestinal tract by both cytolytic and cytolysin-susceptible strains. These data suggest that cytolysin plays little role in promoting intestinal overgrowth of enterococci through bacteriolytic activity.

Animals↗