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Comparison of inoculation methods for testing enterococci by using vancomycin screening agar.

One hundred four recent clinical isolates of Enterococcus species were screened for vancomycin resistance by using inocula of 10(5) or 10(6) CFU dispensed by pipet and by use of a cotton swab dipped in a 0.5 McFarland standard organism suspension applied to the surface of brain heart infusion agar containing 6 micrograms of vancomycin per ml. The three inoculation methods were equivalent in the detection of nonsusceptible isolates. The use of swab inoculation was convenient and less costly than the use of micropipets.

Agar↗

Random amplified polymorphic DNA typing versus pulsed-field gel electrophoresis for epidemiological typing of vancomycin-resistant enterococci.

Sixty vancomycin-resistant vanA mutant Enterococcus faecium (VRE) isolates, collected during a 40-month period from 48 patients hospitalized in a French Cancer Referral Center, were typed by using random amplified polymorphic DNA (RAPD), and the results were compared with those previously obtained by typing with SmaI pulsed-field gel electrophoresis (PFGE), which is currently recognized as the "gold standard." The discriminating power of RAPD typing, with seven primers and 11 combinations of primers, was tested on 18 strains, and only the most discriminating combination was further tested on the whole collection. We compared the epidemiological usefulness of RAPD typing of 60 clinical VRE isolates with that of SmaI PFGE typing. With primers AP4 and ERIC1R, RAPD generated 30 patterns versus the 36 patterns generated by SmaI PFGE. However, this did not hamper the epidemiologically correct clustering of 15 related strains and the detection of multiple colonization in nine patients. We conclude that this simple RAPD technique is well suited to the epidemiological typing of VRE and the monitoring of its nosocomial spread.

Bacterial Typing Techniques↗

Determination of 16S rRNA sequences of enterococci and application to species identification of nonmotile Enterococcus gallinarum isolates.

The 16S rRNA sequences of enterococcal species E. faecium, E. faecalis, E. gallinarum, E. casseliflavus/flavescens, E. dispar, E. pseudoavium, E. sulfureus, E. malodoratus, E. raffinosus, E. cecorum, E. hirae, E. saccharolyticus, E. seriolicida, E. mundtii, E. avium, E. durans, E. columbae, and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified as E. faecium. The implications of this finding are discussed.

Anti-Bacterial Agents↗

Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci.

Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 438-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 19 enterococcal type strains (Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus seriolicida, Enterococcus solitarius, and Enterococcus sulfureus). Sequence analysis of the sodA(int) fragments enabled reliable identification of 18 enterococcal species, including E. casseliflavus-E. flavescens and E. gallinarum. The sodA(int) fragments of E. casseliflavus and E. flavescens were almost identical (99.5% sequence identity), which suggests that they should be associated in a single species. Our results confirm that the sodA gene constitutes a more discriminative target sequence than 16S rRNA gene in differentiating closely related bacterial species.

Bacterial Proteins↗

Outbreak of vancomycin-resistant enterococci in a hospital in Gdask, Poland, due to horizontal transfer of different Tn1546-like transposon variants and clonal spread of several strains.

Twenty-two vancomycin-resistant enterococcal (VRE) isolates of the VanA phenotype (21 Enterococcus faecium isolates and 1 E. faecalis isolate), representative of a large outbreak that occurred in a hospital in Gdańsk, Poland, were studied. All of the isolates demonstrated resistance to a wide variety of other antimicrobial agents in addition to glycopeptides. Several lines of evidence suggested that the outbreak most probably consisted of two epidemics that followed the independent introduction of VanA determinants into two separate hematological wards of the hospital. This hypothesis is supported by the fact that isolates recovered in these wards possessed two different polymorphs of the highly conserved DNA region encompassing the vanRSHAX genes and two distinct polymorph types of Tn1546-like transposons, which contain these genes. According to pulsed-field gel electrophoresis data, the outbreak in the adult hematology ward (HW) was highly polyclonal, which suggested a major role for the horizontal transmission of Tn1546-like elements among nonrelated strains of E. faecium and E. faecalis in this environment. On the other hand, the outbreak in the pediatric hematology ward (PHW) was most probably due to the clonal spread of two epidemic E. faecium strains, which had exchanged a plasmid carrying the Tn1546-like transposon. Restriction fragment length polymorphism studies of transposons and their insertion loci in plasmid DNA have suggested that numerous isolates from both HW and PHW contained two or more copies of Tn1546-like elements that underwent diversification due to various genetic modifications. The reported data demonstrated a very complex epidemiology of the first, and up to now the only, VanA VRE outbreak characterized in Poland.

Adult↗

Antimicrobial growth promoter ban and resistance to macrolides and vancomycin in enterococci from pigs.

Ninety-six enterococcus isolates from fecal samples of pigs receiving tylosin as an antimicrobial growth promoter and 59 isolates obtained in the same farms 5 to 6 months after the ban of antimicrobial growth promoters in Switzerland were tested for susceptibility to nine antimicrobial agents. A clear decrease in resistance to macrolides, lincosamides, and tetracycline was visible after the ban. Vancomycin-resistant Enterococcus faecium belonged to the same clonal lineage as vancomycin-resistant isolates previously isolated from Danish pigs.

Animals↗

Characterization of Tn1546 in vancomycin-resistant Enterococcus faecium isolated from canine urinary tract infections: evidence of gene exchange between human and animal enterococci.

Thirty-five enterococcal isolates were recovered from dogs diagnosed with urinary tract infections at the Michigan State University Veterinary Teaching Hospital over a 2-year period (1996 to 1998). Isolated species included Enterococcus faecium (n = 13), Enterococcus faecalis (n = 7), Enterococcus gallinarum (n = 11), and Enterococcus casseliflavus (n = 4). Antimicrobial susceptibility testing revealed several different resistance phenotypes, with the majority of the enterococcal isolates exhibiting resistance to three or more antibiotics. One E. faecium isolate, CVM1869, displayed high-level resistance to vancomycin (MIC > 32 micro g/ml) and gentamicin (MIC > 2,048 micro g/ml). Molecular analysis of this isolate revealed the presence of Tn1546 (vanA), responsible for high-level vancomycin resistance, and Tn5281 carrying aac6'-aph2", conferring high-level aminoglycoside resistance. Pulsed-field gel electrophoresis analysis revealed that CVM1869 was a canine E. faecium clone that had acquired Tn1546, perhaps from a human vancomycin-resistant E. faecium. Transposons Tn5281 and Tn1546 were located on two different conjugative plasmids. Sequence analysis revealed that in Tn1546, ORF1 had an 889-bp deletion and an IS1216V insertion at the 5' end and an IS1251 insertion between vanS and vanH. To date, this particular form of Tn1546 has only been described in human clinical vancomycin-resistant enterococcus isolates unique to the United States. Additionally, this is the first report of a vancomycin-resistant E. faecium isolated from a companion animal in the United States.

Animals↗