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PubMed2004

Vancomycin-resistant Enterococcus faecium isolates causing hospital outbreaks in northern Italy belong to the multilocus sequence typing C1 lineage.

Multilocus sequence typing (MLST) was used to obtain insights into the genetic relationships between 14 vancomycin-resistant Enterococcus faecium (VREF) isolates from humans (hospitalized patients, 5 strains) and nonhuman sources (meat and poultry, 9 strains) in northern Italy over the period 1993-2001. The typing scheme (Homan et al., 2002, J. Clin. Microb., 40:1963-1971) based on seven housekeeping genes--adk (adenylate kinase), atpA (ATP synthase, alpha subunit), ddl (D-alanine-D-alanine ligase), gyd (glyceraldehyde-3-phosphate dehydrogenase), gdh (glucose-6-phosphate dehydrogenase), purK (phosphoribosylaminoimidazole carboxylase ATPase subunit), and pstS (phosphate ATP-binding cassette transporter)--was used. In the 14 VREF analyzed, the number of unique alleles ranged from 1 (gyd) to 8 (atpA). Isolates from hospitalized patients were defined by the unique allele purK 1. Nine sequence types (STs) were identified. All of the epidemic strains isolated over the period 2000-2001 showed identical or closely related pulsed-field gel electrophoresis (PFGE) patterns and clustered in the same ST78. These strains shared six of the seven alleles with the strain CA20 representative of the 1993-1999 outbreaks, which PFGE indicated as being unrelated to those of the recent outbreaks. MLST confirmed the unrelatedness of human and nonhuman strains already detected by PFGE. All isolates clustered in three main genetic lineages: group A comprised two of the three isolates from meat; group C the human strains of all outbreaks and one poultry strain; and group B four of the five poultry strains and one meat strain. All human strains carried the esp gene and clustered in the C1 sublineage that has been described as having emerged recently worldwide.

Alleles↗
PubMed1995

Serious infections caused by multiply-resistant Enterococcus faecium.

Three liver transplant patients developed serious intraabdominal infections and recurrent bacteremias due to strains of Enterococcus faecium with high-level resistance to vancomycin. The enterococci were also resistant to all other antibacterials except pristinamycin, which, given orally, proved ineffective. One strain was sensitive to tetracycline. Increasingly, clinicians are likely to encounter infections caused by multiply-resistant enterococci, and these cases illustrate the seriousness of such infections in compromised patients.

Abdomen↗
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↗
PubMed1995

Enterococcus faecium strains with vanA-mediated high-level glycopeptide resistance isolated from animal foodstuffs and fecal samples of humans in the community.

The occurrence and the further spread of high-level glycopeptide-resistant, vanA-positive Enterococcus faecium strains outside of hospitals have been investigated. We could isolate such bacteria directly from thawing liquids of commercially produced frozen poultry (chickens, turkeys; no further data on previous feeding with avoparcin were available). In 5 of 13 samples of raw minced meat of pigs originating from 13 different butcher's shops, glycopeptide-resistant E. faecium (VanA type) could be detected after overnight broth cultivation of these samples. No glycopeptide-resistant enterococci could be isolated from meat samples of chickens that were fed without avoparcin. VanA type E. faecium strains were also identified in 12 fecal samples recovered from 100 nonhospitalized humans in the rural area of Saxony-Anhalt federal county. These results suggest a possible role of the food chain in the spread of glycopeptide-resistant E. faecium. Molecular typing (macrorestriction and multilocus enzyme analysis) reveal a wide dissemination of the vanA gene among strains of different ecological origins.

Animals↗
PubMed1995

In vitro activity of RP 59500 (quinupristin/dalfopristin) and ramoplanin against vancomycin-resistant Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (VREF) collected between July 1991 and February 1994 were tested in vitro against RP 59500 and ramoplanin using agar dilution and standard macro broth dilution procedures. Colony counts were determined at 0, 4, and 24 h. RP 59500 had an MIC range of < or = 0.5-8 micrograms/ml with an MIC90 of 2 micrograms/ml and a MBC range of < or = 0.5-16 micrograms/ml with an MBC90 of 16 micrograms/ml. Ramoplanin had an MIC range of < or = 0.125-1 microgram/ml with an MBC range of < or = 0.125-4 micrograms/ml. The MIC90 for ramoplanin was 1 microgram/ml and the MBC90 was 4 micrograms/ml for the tested isolates. Against these isolates of E. faecium, RP 59500 was bactericidal at 8x MIC, a potentially achievable level using a high drug dosage. Ramoplanin was bactericidal at 2x MIC.

Anti-Bacterial Agents↗
PubMed1996

The gene encoding for penicillin-binding protein 5 of Enterococcus faecalis is useful for development of a species-specific DNA probe.

Recently, in Escherichia coli was cloned a Sau3AI 3.4-kb fragment containing the gene encoding for penicillin-binding protein 5 (PBP5) of Enterococcus faecalis. The structural gene for the PBP of E. faecalis and the flanking regions were entirely sequenced (C. Signoretto, M. Boaretti, and P. Canepari, FEMS Microbiol. Lett. 123:99-106, 1994). When the entire cloned E. faecalis DNA insert, labeled with digoxigenin, was used as a probe to detect a homology gene in enterococci, it was observed that only DNAs of all the E. faecalis strains reacted to the probe. The same results were obtained when a HindIII fragment of 0.35 kb from the entire insert of 3.4 kb was used. In this study we tested a total of 62 clinically isolated enterococcal strains, belonging to the species E. faecalis (36 strains), E. faecium (13), E. gallinarum (6), E. bovis (2) E. avium (3), E. hirae (1), and E. casseliflavus (1). The results indicate that both the entire segment and the HindIII fragment may be useful for preparing a species-specific probe for rapid identification of E. faecalis species.

Bacterial Proteins↗
PubMed1996

Peptidoglycan composition of vancomycin-resistant Enterococcus faecium.

Muropeptide composition of peptidoglycan isolated from isogenic vancomycin-resistant and sensitive Enterococcus faecium strains was analyzed by reverse-phase high-performance liquid chromatography combined with amino acid and fast atom bombardment mass spectrometric analyses. Peptidoglycan of the sensitive and resistant strains was the same and was composed of tri- and tetrapeptides stem peptide subunits with or without aspartate or asparagine substitutions on the epsilon-amino group of the lysine residue. Thus, the synthesis of lactate-terminating peptidoglycan precursors in vancomycin-resistant E. faecium did not affect the chemical composition of peptidoglycan.

Amino Acids↗
PubMed1996

Genotypic characterization of a nosocomial outbreak of VanA Enterococcus faecalis.

Despite growing concern about vancomycin-resistant enterococci (VRE) as nosocomial pathogens, especially in the United States, in Italy VRE still represent an uncommon and occasional experience for most diagnostic laboratories. We report a genotypic characterization of the first reported nosocomial outbreak of VRE in Italy. Some experiments, including plasmid analysis and pulsed-field gel electrophoresis (PFGE) assays, aimed at investigating the genetic relatedness of the VRE isolates. Other experiments, based on hybridization and polymerase chain reaction (PCR) assays, aimed at characterizing the vancomycin resistance determinants. Over a 6-month period, 21 VRE, all identified as Enterococcus faecalis, were isolated from eight patients (all treated earlier with glycopeptide antibiotics) in a neurosurgical intensive care unit. All isolates had the same biochemical profile and antibiotic susceptibility pattern, including high-level resistance to aminoglycosides and vancomycin and teicoplanin MICs of 256 and 128 micrograms/ml, respectively. Three plasmids, one strongly hybridizing with a vanA probe, were detected in all but the last of the 21 VRE isolates. The last isolate of the cluster lacked the smallest of the three plasmids. Similar restriction profiles were obtained after plasmid DNA digestion with several endonucleases, with minor differences appreciated only in the first and last isolates. Analysis of genomic DNA restriction fragment patterns by PFGE confirmed that the reported cluster of VRE isolations was due to a single nosocomial strain of E. faecalis, despite some modifications in plasmid DNA at the beginning and at the end of the outbreak. Completely different PFGE patterns were yielded by vancomycin-susceptible E. faecalis strains isolated during the same period from inpatients in the same intensive care unit. Hybridization experiments with vanA and vanS-vanH probes and DNA amplification assays using 14 PCR primer pairs specific for vanA cluster genes (vanR, vanS, vanH, vanA, and vanY), orf1, orf2, vanB, and vanC showed identical organization of resistance determinants in all epidemic VRE isolates. This organization appeared to be the same as that described for Tn1546 in VanA prototype strain E. faecium BM4147.

Anti-Bacterial Agents↗
PubMed1996

Testing the efficacy of a molecular surveillance network: methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF) genotypes in six hospitals in the metropolitan New York City area. The BARG Initiative Pilot Study Group. Bacterial Antibiotic Resistance Group.

Molecular fingerprinting techniques are rapidly becoming indispensable tools for hospital epidemiology. On the other hand, the relative complexity and unfamiliarity of these techniques to most hospital diagnostic laboratories limit their usefulness. In an attempt to provide a solution for this dilemma, we tested the feasibility and efficacy of a cooperative venture in which molecular typing of isolates recovered from patients in six hospitals was performed at two microbiology research laboratories with expertise in these techniques. In a small preliminary study, 30 methicillin-resistant Staphylococcus aureus (MRSA) and 30 vancomycin-resistant Enterococcus faecium (VREF) isolates were collected over a 3-week period from six hospitals in the metropolitan New York area and transported to the Laboratory of Microbiology at The Rockefeller University during the summer months of 1994. Nineteen of the 27 confirmed MRSA isolates were closely related strains carrying the same mecA and the same Tn554 polymorphs in a pulsed-field gel electrophoresis (PFGE) background represented by closely related subtypes of a single pattern, indicating the wide distribution of this MRSA clone among the participating hospitals. Typing of the same 27 MRSA isolates was also performed at the Tuberculosis Center of the Public Health Research Institute and identical results were obtained. The 29 confirmed VREF isolates were highly heterogeneous and belonged to as many as 23 distinct clonal types as defined by PFGE patterns and probing with vanA. Characterization of the 60 isolates by these methods was completed in one month of full-time effort by a single experienced laboratory assistant guided by a doctoral-level expert in molecular fingerprinting techniques. The collection of samples for both MRSA and VREF was not intended to address epidemiological questions but to determine the feasibility of a multicenter study. On the basis of our preliminary findings we are encouraged that a larger cooperative effort is possible and with the correct sampling method we believe that epidemiological and surveillance studies could be accomplished that would provide a tracking system to assist hospitals, clinics, and chronic care facilities in controlling the spread of multidrug-resistant pathogens.

Anti-Bacterial Agents↗
PubMed1996

Treatment of vancomycin-resistant Enterococcus faecium infections with an investigational streptogramin antibiotic (quinupristin/dalfopristin): a report of fifteen cases.

New therapies for vancomycin-resistant Enterococcus faecium (VREF) infections are urgently needed. We describe the treatment of 15 patients with VREF infection with quinupristin/dalfopristin (RP 59500), a new injectable streptogramin antibiotic. Primary infections treated were bacteremia (4), urinary tract (4), intraabdominal (5), otitis externa (1), and meningitis (1). Minimum inhibitory concentrations for quinupristin/dalfopristin ranged from 0.5 microgram/ml or less to 2 micrograms/ml, and minimum bactericidal concentrations were greater than 64 micrograms/ml for all VREF isolates tested. Peak serum inhibitory titers following infusion of quinupristin/dalfopristin ranged from 1:8 to 1:64; all bactericidal titers were less than 1:2. Development of resistance to quinupristin/dalfopristin during therapy was not observed. The only drug-related adverse effect noted was phlebitis in 4 patients; all had received quinupristin/dalfopristin by peripheral venous infusion. Three patients had clinical and bacteriologic cures. Relapses occurred in 5 patients with recovery of VREF from infected sites in post-treatment cultures. Ten patients died of severe underlying disease; VREF was believed to contribute directly to the death of only 1 patient. While evaluation of clinical efficacy was complicated by the severity of underlying disease in patients with VREF infection, our experience suggests that quinupristin/dalfopristin is a safe and potentially useful agent for the treatment of VREF infections.

Adult↗
PubMed1996

Persistence of vancomycin-resistant Enterococcus faecium gastrointestinal tract colonization in antibiotic-treated mice.

Colonization with vancomycin-resistant Enterococcus faecium (VREF) is strongly associated with previous antimicrobial therapy. The gastrointestinal (GI) tract appears to be the major reservoir for this organism. We used antibiotic-treated Swiss Webster mice to study GI tract colonization with a characterized strain of VREF (E. faecium 228). Mice were pretreated with antibiotics in their daily drinking water and inoculated with 10(9) colony-forming units (CFU) of E. faecium 228 by oral gavage. We were able to establish persistent colonization with high concentrations of E. faecium 228 (> 8.0 log10 CFU/g of feces) in animals treated with 5 mg/ml of streptomycin plus 1 mg/ml of cefotetan. RP 59500, a streptogramin antibiotic with good in vitro activity against VREF, was administered orally in mice (n = 8) colonized with E. faecium 228. After 14 days of treatment VREF was undetectable in feces of all treated mice (< 3.0 CFU/g). Seven days after discontinuation of RP 59500, VREF was present in the feces of all animals. VREF isolates recovered after treatment remained susceptible to RP 59500. Attempts to eradicate E. faecium 228 colonization by oral administration of a vancomycin-sensitive E. faecium strain (SF68) or Lactobacillus spp. were unsuccessful as long as animals continued to receive streptomycin and cefotetan. Recovery of E. faecium 228 from cultures of livers and gallbladders in some animals with persistent GI tract colonization suggests that the organisms may also colonize the hepatobiliary system.

Animals↗
PubMed1997

Studies on the distribution of high-level gentamicin-resistant Enterococcus faecalis using ribotyping and restriction analysis of the 16S-23S rRNA intergenic spacer sequences.

Twenty-five high-level gentamicin resistant (HLGR) Enterococcus faecalis strains were isolated from three different University laboratories in Italy. The resistant strains were variously distributed in the three centers with percentages of prevalence ranging from about 3% up to 14%. Almost all strains shared high-level resistance to streptomycin (23 out of 25). Ribotyping and restriction analysis of the 16S-23S rRNA intergenic spacer sequences were used to genetically differentiate the various strains and to study their spreading in the university hospitals serviced by the three laboratories. At least three ribotypes were identified, which showed a peculiar distribution in the various centers. Only the ribotype B was isolated from the University of Padua. In Cagliari, most strains belonged to ribotype A (4/6), whereas in Genoa there was an equal distribution of the ribotypes A and B. A clonal spreading of some HLGR strains is suggested by these findings. The restriction analysis of the 16S-23S rRNA intergenic-spacer sequences gave comparable results with classical ribotyping and, in addition, was quicker and easier to perform than the latter.

Drug Resistance, Microbial↗
PubMed1999

The use of avoparcin as a growth promoter and the occurrence of vancomycin-resistant Enterococcus species in Norwegian poultry and swine production.

This study documents a strong and statistically significant association between the use of the glycopeptide avoparcin as a growth promoter in Norwegian poultry production and the occurrence of vancomycin-resistant Enterococcus species (VRE). Avoparcin was approved as a feed additive for broilers and turkeys in Norway in 1986 and was banned from June 1, 1995. In a survey conducted in Norway between June, 1995 and March, 1997, VRE were isolated from fecal samples from 106 out of 109 poultry houses previously exposed to avoparcin (97%) and from six out of 33 poultry houses never exposed to avoparcin (18%) (RR = 5.35). Samples from previously exposed poultry houses were collected in three time periods. The proportion of positive samples remained high (96-98%), in all three time periods indicating a persistence of vancomycin resistance among enterococci for more than a year and a half after the withdrawal of avoparcin. VRE were also isolated from six out of 10 poultry farmers living on farms previously exposed to avoparcin, and from none of 16 farmers living on farms never exposed to avoparcin. Moreover, VRE were isolated from 68 out of the 225 broiler carcasses investigated (30%). The resistance to vancomycin was a high-level type (MIC > or = 256 microg/ml) mediated by the vanA gene. For comparison, VRE could only be isolated from two out of 147 fecal samples from Norwegian flocks of swine (1%). Because avoparcin never has been used in Norwegian swine production, this observation strengthens the association between the use of avoparcin in animal husbandry and the occurrence of VRE.

Animal Husbandry↗
PubMed1999

First confirmed case of a vancomycin-resistant Enterococcus faecium with vanA phenotype from Brazil: isolation from a meningitis case in São Paulo.

The importance of enterococci as a nosocomial etiologic agent is well documented; however, enterococci are also capable of causing a variety of community-acquired infections. Vancomycin resistance in a clinical Enterococcus isolate was first reported in 1986, and since then vancomycin-resistant enterococci (VRE) have been reported world-wide. This report describes a case of E. faecium with the VanA phenotype, isolated from meningitis in Sao Paulo, Brazil. Two E. faecium strains were isolated. One strain showed VanA phenotype, and the molecular characterization of the VanA gene was confirmed by polymerase chain reaction. The other strain was susceptible to vancomycin and teicoplanin. The authors would like to call the attention of the scientific community to this first identification of a VRE case in Sao Paulo, Brazil.

Anti-Bacterial Agents↗
PubMed1999

Glycopeptide resistance in Enterococcus faecium from broilers and pigs following discontinued use of avoparcin.

The use of the glycopeptide growth promoter avoparcin was discontinued in Denmark in 1995 following concerns that vancomycin-resistant Enterococcus faecium occurring as a result of its use could be transferred to humans via food. The present study is an analysis of results obtained by the continuous surveillance of an antimicrobial resistance in Denmark (DANMAP) with the aim of determining the effect of the ban on the occurrence of glycopeptide resistance among E. faecium isolated from broilers and pigs. Among isolates from broilers, the proportion that were resistant to glycopeptides has shown a statistically high significant decline between the end of 1995 and the first half of 1998, whereas in pigs the ban appears to have no such effect. One possible explanation is that the broiler industry generally uses all in-all out production compared with continuous production in pig herds. Alternatively, the results indicate that the different outcomes may result from different co-selection patterns in pigs and broilers. In pigs, the antimicrobials most commonly used favored co-selection of glycopeptide resistant strains of E. faecium while in broilers the antimicrobials most widely used selected for glycopeptide-susceptible strains. The results show that intervention to reduce antimicrobial resistance may not always be effective and preventing resistance problems therefore becomes essential.

Animals↗
PubMed2000

Associations between the use of antimicrobial agents for growth promotion and the occurrence of resistance among Enterococcus faecium from broilers and pigs in Denmark, Finland, and Norway.

This study compares the susceptibility of Enterococcus faecium isolated from pigs and poultry in Denmark, Finland, and Norway to antimicrobial agents used for growth promotion. E. faecium was isolated from 211 broilers and 55 pigs in Denmark in 1997, from Norwegian 55 poultry farms (turkey and broiler farms) and 4 swine farms between 1995 and 1997, and Finnish poultry (52) and swine (43) in 1996 and examined for susceptibility to avilamycin, avoparcin, bacitracin, flavomycin, monensin, salinomycin, spiramycin, tylosin, and virginiamycin. Only a limited number of isolates were categorized as resistant to monensin or salinomycin. In general, an association between the usage of antimicrobial agents in the respective countries and the occurrence of associated resistance was observed. Resistance to avilamycin was frequently observed among isolates from broilers in Denmark, where avilamycin has been used, whereas all isolates from Finland and Norway, where these drugs have not been used, were susceptible. The same phenomenon could be observed for avoparcin, bacitracin, tylosin, and virginiamycin; resistance was frequently observed among isolates from where these antimicrobials have been widely used, but rarely among isolates from where the use has been limited. Also for avoparcin and bacitracin, an association between usage and occurrence of resistance was observed. All isolates categorized as avoparcin resistant contained the vanX gene; isolates from broilers had the T variant in position 8,234 and isolates from pigs the G variant. Three (1%) of the 222 isolates resistant to tylosin contained the ermA gene and 196 (88%) ermB. Sixteen (11%) of the 146 virginiamycin-resistant isolates from broilers and two (7%) of the 27 virginiamycin-resistant isolates from pigs in Denmark contained the satA gene, whereas satA was not observed among any of the virginiamycin-resistant isolates from Finland. A total of 72% of the virginiamycin-resistant E. faecium from broilers in Denmark and all nine virginiamycin-resistant E. faecium from Finland contained satG. This gene was also observed among two (7%) of the virginiamycin-resistant isolates from pigs in Denmark. This study indicates that the use of antimicrobial agents for growth promotion in Denmark, Finland, and Norway have selected for resistance to most of these drugs among E. faecium in food animals.

Animals↗
PubMed2000

Association between the use of avilamycin for growth promotion and the occurrence of resistance among Enterococcus faecium from broilers: epidemiological study and changes over time.

This study describes the changes in the occurrence of resistance to avilamycin among Enterococcus faecium from broilers in Denmark and the epidemiological association between usage of avilamycin for growth promotion and the occurrence of avilamycin-resistant E. faecium on broiler farms. The consumption of avilamycin for growth promotion increased from 10 kg in 1990 to 2,740 kg 1996 and decreased in the following years to only 7 kg in 1998. Most of this has been used for broilers. As part of the nationwide monitoring program for antimicrobial resistance, a total of 473 E. faecium isolates from broilers and 290 isolates from pigs have been tested for their susceptibility to avilamycin from 1995 to 1998. A very limited number of isolates from pigs were resistant to avilamycin, whereas the occurrence of resistance among isolates from broilers increased from 63.6% at the end of 1995 to a maximum of 80.7% during the last half of 1996. Since then, the occurrence of resistance has decreased to 23.3% in the last half of 1998. The epidemiological association between consumption of avilamycin and occurrence of resistant E. faecium fecal droppings were examined on 10 poultry farms that had not used avilamycin for growth promotion during 1996 or 1997 and eight farms that had used avilamycin during 1997. We tested a total of E. faecium isolates from the exposed farms and 104 from the nonexposed farms for their susceptibility to avilamycin. Resistant isolates were found on all eight exposed farms, and on seven of 10 nonexposed farms. Sixty-four isolates (72%) from the exposed farms were resistant, compared with 24 (23%) of the isolates from nonexposed farms. The adjusted chi-square p value equaled 0.01065, and showed a significant association between use of avilamycin and occurrence of resistance. The national monitoring program showed a decrease in the occurrence of resistance following a decreased use of avilamycin in Denmark, and the epidemiological study showed a statistically significant association between the use of avilamycin for growth promotion and the occurrence of avilamycin-resistant E. faecium on broiler farms.

Animals↗
PubMed2005

Clonality among Enterococcus faecium clinical isolates.

In the course of a survey to determine the epidemiology of enterococcal infections in Italy, a sudden increment, in a 1-year time, was noted in the number of glycopeptide resistant Enterococcus faecium isolated from different wards of the University Hospital in Rome, Italy. The isolates were characterized for clonal relatedness by comparing SmaI gel electropherotypes, presence of vancomycin-resistance genes, and expression of virulence factors. PFGE identified in a single pulsed type all the glycopeptide-resistant isolates but one. Resistance to high levels of aminoglycosides was expressed by these same isolates, which also included a majority of non biofilm-forming strains. Two esp gene-carrying strains were also identified in different PFGE types. Data indicates that a specific clone acquired, in the clinical setting, the genetic determinant for glycopeptide resistance, thus improving environmental adaptation and favoring its persistence and spread.

Drug Resistance, Multiple, Bacterial↗