Molecular characterization of gentamicin-resistant Enterococci in the United States: evidence of spread from animals to humans through food.

We evaluated the molecular mechanism for resistance of 360 enterococci for which the gentamicin MICs were >/=128 micro g/ml. The aac(6')-Ie-aph(2")-Ia, aph(2")-Ic, and aph(2")-Id genes were identified by PCR in isolates from animals, food, and humans. The aph(2")-Ib gene was not identified in any of the isolates. Two Enterococcus faecalis isolates (MICs > 1,024 micro g/ml) from animals failed to generate a PCR product for any of the genes tested and likely contain a new unidentified aminoglycoside resistance gene. Pulsed-field gel electrophoresis (PFGE) analysis showed a diversity of strains. However, 1 human and 18 pork E. faecalis isolates from Michigan with the aac(6')-Ie-aph(2")-Ia gene had related PFGE patterns and 2 E. faecalis isolates from Oregon (1 human and 1 grocery store chicken isolate) had indistinguishable PFGE patterns. We found that when a gentamicin-resistant gene was present in resistant enterococci from animals, that gene was also present in enterococci isolated from food products of the same animal species. Although these data indicate much diversity among gentamicin-resistant enterococci, the data also suggest similarities in gentamicin resistance among enterococci isolated from humans, retail food, and farm animals from geographically diverse areas and provide evidence of the spread of gentamicin-resistant enterococci from animals to humans through the food supply.

PCR fragment length polymorphism analysis of vancomycin-resistant Enterococcus faecium.

In this study, the glycopeptide resistance element, Tn1546, in 124 VanA Enterococcus faecium clinical isolates from 13 Michigan hospitals was evaluated using PCR fragment length polymorphism. There were 26 pulsed-field gel electrophoresis (PFGE) types, which consisted of epidemiologically related and unrelated isolates from separate patients (1992 to 1996). Previously published oligonucleotides specific for regions in the vanA gene cluster of Tn1546 were used to amplify vanRS, vanSH, vanHAX, vanXY, and vanYZ. The glycopeptide resistance element, Tn1546, of E. faecium 228 was used as the basis of comparison for all the isolates in this study. Five PCR fragment length patterns were found, as follows. (i) PCR amplicons were the same size as those of EF228 for all genes in the vanA cluster in 19.4% of isolates. (ii) The PCR amplicon for vanSH was larger than that of EF228 (3.7 versus 2.3 kb) due to an insertion between the vanS and vanH genes (79.2% of isolates). (iii) One isolate in a unique PFGE group had a vanSH amplicon larger than that of EF228 (5.7 versus 2.3 kb) due to an insertion in the vanS gene and an insertion between the vanS and vanH genes. (iv) One isolate did not produce a vanSH amplicon, but when vanS and vanH were amplified separately, both amplicons were the same size as those as EF228. (v) One isolate had a vanYZ PCR product larger than that of EF228 (2.8 versus 1.6 kb). This study shows that in a majority of the VanA E. faecium isolates, Tn1546 is altered compared to that of EF228. A total of 79.2% of the study isolates had the same-size insertion between the vanS and vanH genes. The results of this study show dissemination of an altered Tn1546 in heterologous VanA E. faecium in Michigan hospitals.

Comparative in vitro and bactericidal activity of oxazolidinone antibiotics against multidrug-resistant enterococci.

Increasing resistance among enterococci poses a considerable therapeutic problem. In this study, we evaluated the comparative in vitro activity of two investigational oxazolidinone antibiotics, eperezolid and linezolid, versus clinical isolates of multidrug-resistant enterococci. One hundred isolates (16 Enterococcus faecalis, 69 E. faecium, 10 E. gallinarum, 2 E. casseliflavus, 1 E. avium, 1 E. hirae, and 1 E. raffinosus) evaluated were collected from diverse geographic areas in North America and Europe from 1991 to 1995. Eperezolid MIC50 and MIC90 were 1.0 microgram/mL and 2.0 micrograms/mL (1.0-2.0 micrograms/mL range). Linezolid MIC50 and MIC90 were 2.0 micrograms/mL and 2.0 micrograms/mL (0.5-2.0 micrograms/mL range), respectively. MICs were the same at 10(3) CFU/mL and 10(8) CFU/mL initial inoculum. In time-kill experiments using 10 strains and concentrations of 4 micrograms/mL, 8 micrograms/mL, and 16 micrograms/mL (achievable serum concentrations) of eperezolid and linezolid there was a 2 log10 reduction of growth for 2 of 10 isolates tested using eperezolid and a 1 log10 reduction for 50% of isolates with both agents. There was indifferent bactericidal killing when either oxazolidinone was combined with gentamicin, ampicillin, or streptomycin for isolates lacking these resistances. This study demonstrates these oxazolidinone agents to have excellent in vitro activity versus multidrug-resistant enterococci.

Epidemiologic evaluation of antimicrobial resistance in community-acquired enterococci.

Fecal samples from 200 consecutive patients admitted to a community hospital yielded 107 enterococci. High-level gentamicin resistance occurred in 10 (14%) of the Enterococcus faecalis isolates. Ampicillin resistance occurred in two (3%) of the E. faecalis isolates and six (23%) of the Enterococcus faecium isolates. There were no vancomycin-resistant enterococci. Risk factors for enterococci with high-level aminoglycoside (gentamicin) or ampicillin resistance included prior hospitalization and previous antibiotic use.

Antimicrobial resistance in enterococci isolated from Turkey flocks fed virginiamycin.

From 125 separate cloacal cultures from three turkey flocks fed virginiamycin, 104 Enterococcus faecium and 186 Enterococcus faecalis isolates were obtained. As the turkeys aged, there was a higher percentage of quinupristin-dalfopristin-resistant E. faecium isolates, with isolates from the oldest flock being 100% resistant. There were no vancomycin-resistant enterococci. Results of pulsed-field gel electrophoresis (PFGE) indicated there were 11 PFGE types of E. faecalis and 7 PFGE types of E. faecium that were in more than one group of flock cultures.

The effect of Tn916 insertions on contour-clamped homogeneous electrophoresis patterns of Enterococcus faecalis.

Contour-clamped homogeneous gel electrophoresis has increasingly been used and has generally been considered the method of choice for the delineation of enterococcial strains. It has been suggested that the contour-clamped homogeneous electric field (CHEF) electrophoresis digestion patterns of genomic DNA indicate the relatedness of strains when SmaI patterns differ by six or fewer bands. To evaluate the potential reasons for the diversity of bands among clonally related isolates, we studied plasmid-free Enterococcus faecalis FA2-2 and derivatives with transposon Tn916 (encoding for tetracycline resistance) insertions on the chromosome. We obtained derivatives with seven different Tn916 insertion sites and up to two copies of Tn916 on the chromosome, resulting in differences of one to seven bands by CHEF electrophoresis; eight different patterns were observed. With Tn916 insertions, there was either (i) loss of a band(s) with the generation of a new band(s), (ii) the generation of a new band(s) only, or (iii) the loss of a band(s) with no new visible band(s). These results indicate that strains can have up to seven different SmaI bands by CHEF electrophoresis and still be closely related.

A novel gentamicin resistance gene in Enterococcus.

Enterococcus gallinarum SF9117 is a veterinary isolate for which the MIC of gentamicin is 256 micrograms/ml. Time-kill studies with a combination of ampicillin plus gentamicin failed to show synergism against SF9117. A probe representing aac(6')-aph(2") did not hybridize to DNA from SF9117. A 3.2-kb fragment from plasmid pYN134 of SF9117 was cloned and conferred resistance to gentamicin in Escherichia coli DH5 alpha. Nucleotide sequence analysis revealed the presence of a 918-bp open reading frame whose deduced amino acid sequence had a region with homology to the C-terminal domain of the bifunctional enzyme AAC(6')-APH(2"). The gene is designated aph(2")-Ic, and its observed phosphotransferase activity is provisionally designated APH(2")-Ic. An intragenic probe hybridized to the genomic DNA from an Enterococcus faecium isolate from the peritoneal fluid of one patient and to the plasmid DNA of an Enterococcus faecalis isolate from the blood of another patient. An enterococcal isolate containing this novel resistance gene might not be readily detected in clinical laboratories that use gentamicin at 500 or 2,000 micrograms/ml for screening for high-level resistance.

Characterization of antimicrobial resistance in enterococci of animal origin.

Among 97 enterococci cultured from animals, gentamicin MICs were > or = 2,000 micrograms/ml for 9 isolates and between 250 and 1,024 micrograms/ml for 6 isolates. For two isolates tested (gentamicin MICs, 256 and 512 micrograms/ml, respectively), there was no in vitro synergy with penicillin plus gentamicin, resistance was transferable, and there was no hybridization with a probe specific for 6'-aminoglycoside acetyltransferase-2"-aminoglycoside phosphotransferase. The results of the study indicate the presence of a unique gentamicin resistance genotype in enterococci of animal origin.

Tn924, a chromosome-borne transposon encoding high-level gentamicin resistance in Enterococcus faecalis.

Enterococcus faecalis SF350 is a clinical isolate from Winnipeg, Canada, with high-level (MIC > 2,000 micrograms/ml) gentamicin resistance. The genetic determinant for gentamicin resistance was located on the chromosome of SF350 and could be mobilized by a coresident conjugative plasmid, pYN120. Genetic and physical analyses showed that the gentamicin resistance determinant was located on a 27-kb transposable element which was designated Tn924.

Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocarditis.

A rabbit endocarditis model was utilized to evaluate the virulence conferred by the conjugative plasmid pAD1 with the following strains: Enterococcus faecalis plasmid-free FA2-2 and FA2-2 containing plasmids pAD1 (hemolysin and aggregation substance positive), pAM9058 (insertional inactivation of hemolysin), and pAM944 or pAM947 (insertional inactivation of aggregation substance). All isolates were similar in ability to produce endocarditis. Mean vegetation weight was greater in animals inoculated with strains that produced aggregation substance (P < 0.01). Mortality was significantly increased in animals given FA2-2 containing pAD1 compared with those given all other strains (P < 0.01). These results suggest that the combination of hemolysin and aggregation substance is associated with increased mortality and that vegetation weight is associated with production of aggregation substance in experimental E. faecalis endocarditis.

Sparfloxacin and clinafloxacin alone or in combination with gentamicin for therapy of experimental ampicillin-resistant enterococcal endocarditis in rabbits.

Sparfloxacin and clinafloxacin alone and in combination with gentamicin, were evaluated for the treatment of experimental endocarditis in rabbits caused by ampicillin-resistant enterococci. Clinafloxacin was tested against Enterococcus faecalis strain WH245, a beta-lactamase producer lacking high-level gentamicin resistance (MIC 12.5 mg/L). Sparfloxacin was tested against Enterococcus faecium strain SF2149 a non-producer of beta-lactamase (ampicillin MIC 400 mg/L, gentamicin MIC 12.5 mg/L). For strain WH245, clinafloxacin alone significantly reduced enterococcal counts in vegetations (7.7 log10 cfu/g) and for strain SF2149, sparfloxacin significantly reduced counts (7.0 log10 cfu/g) compared with untreated controls (WH245, 8.8 log10 cfu/g and SF2149, 9.3 log10 cfu) or treatment with ampicillin plus gentamicin (WH245, 9.7 log10 cfu/g). The addition of gentamicin resulted in no further reduction of bacterial counts in vegetations but resulted in an increase in sterilization of blood for strain SF2149. These results suggest that sparfloxacin and clinafloxacin and may prove useful in the therapy of infections due to ampicillin-resistant enterococci.

Mobilization of the penicillinase gene in Enterococcus faecalis.

Enterococcus faecalis SF4855 is a beta-lactamase-producing isolate resistant to high levels of gentamicin, with determinants for these resistances on the chromosome. SF4855 transferred both determinants into E. faecalis FA2-2 and UV202 at a frequency of 10(-9) in the presence of the MLS plasmid pYN120. beta-Lactamase and gentamicin resistance probes hybridized to three locations on the chromosome of FA2-2 transconjugants on contour-clamped homogeneous electric field electrophoresis. The study results suggest mobilization of the beta-lactamase determinant.

Molecular characterization of highly gentamicin-resistant Enterococcus faecalis isolates lacking high-level streptomycin resistance.

Antimicrobial susceptibilities and DNA contents were analyzed for six clinical isolates of Enterococcus faecalis that had high-level resistance to gentamicin (MIC > 2,000 micrograms/ml) but not streptomycin and were obtained from patients in diverse geographic areas. Contour-clamped homogeneous electric field electrophoresis of genomic DNA showed all isolates to be different strains. Gentamicin resistance was transferred from four isolates to plasmid-free enterococcal recipients in filter matings. Restriction enzyme analysis of transconjugants showed distinct gentamicin resistance plasmids. A probe specific for the gentamicin resistance determinant hybridized to the plasmids of four isolates and to the chromosomes of two isolates. These findings suggest that clonal dissemination is not responsible for the spread of these resistant strains, that resistance determinants occur on different plasmids as well as on the chromosome of E. faecalis, and that the genetic determinants of resistance are related.