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Antimicrobial Agents and Chemotherapy, May 2000, p. 1349-1351, Vol. 44, No. 5
Medical Service, Department of Veterans
Affairs Medical Center,3 and
Adult1 and
Pediatric2 Infectious Diseases
Divisions, Case Western Reserve University School of Medicine,
Cleveland, Ohio
Received 12 July 1999/Returned for modification 30 August
1999/Accepted 27 January 2000
In several clonally unrelated VanB-type vancomycin-resistant
Enterococcus faecium strains, we demonstrated a common
physical relationship between pbp5 and Tn5382
as well as common mutations within pbp5. The majority of
these strains transferred vancomycin and ampicillin resistance to
E. faecium in vitro, suggesting the dissemination of
similar transferable pbp5-vanB-containing mobile elements
throughout the United States.
Enterococcus faecium
strains are resistant to penicillin through the expression of
low-affinity penicillin-binding protein PBP5 (9) and are
resistant to vancomycin most often through the action of resistance
operons encoding VanA and VanB types of resistance (1-3,
8). PBP5-mediated low-level ampicillin resistance has been
thought to be intrinsic to the enterococci (and nontransferable),
whereas the vancomycin resistance determinants are acquired and transferable.
We recently described the cotransfer of ampicillin and vancomycin
resistance within a large, chromosomally located element that possessed
the vanB-containing transposon Tn5382 and
pbp5 (5). The present study was undertaken to
determine whether similar large chromosomal elements were present in
enterococci from diverse geographic regions.
Ten clinical E. faecium strains from northeast Ohio, 1 strain from Hawaii, and 12 VanB strains from diverse geographical
locations (kindly provided by Fred Tenover of the Centers for Disease
Control and Prevention) were used in these studies. Bacterial strains were grown overnight in brain heart infusion (BHI) broth at 37°C. MICs were determined using dilution on BHI agar for ampicillin and
vancomycin in concentrations ranging from 0.5 to 512 µg/ml. One or
two concentrations were tested for the following antibiotics: tetracycline (10 µg/ml), erythromycin (10 µg/ml), ciprofloxacin (10 µg/ml), chloramphenicol (10 µg/ml), streptomycin (2,000 µg/ml), and gentamicin (500 and 2,000 µg/ml).
PCR experiments were carried out using 10 µl of an overnight culture
diluted 1:10 in sterile water and heated to 95°C for 10 min. The
sample was diluted 1:10 in a previously prepared PCR mixture
(Perkin-Elmer) and amplified using a Perkin-Elmer 9600 thermal cycler
with Taq DNA polymerase for 25 cycles of denaturation at
95°C (10 s), annealing at 66°C (10 s), and extension at 74°C (1 min 30 s). Primers used to amplify a 1,076-bp product spanning the
downstream end of pbp5 and the left end of Tn5382
were 1492 (5'-TCAGCCGATTTGCGACAGGTTATG-3') and 3206 (5'-TGGGGTGGCGGGTATTAGCAGTAT-3'). DNA sequencing reactions
were performed on PCR product templates with the ALF automated
sequencing kit and Cy5 indodicarbocyanine dye-labeled primer (Pharmacia
LKB). The sequence was determined with the ALFExpress automated
sequencer (Pharmacia LKB) and analyzed using the MacDNAsis, version 2.0 (Hitachi, Ltd.), sequence analysis program.
Filter matings were performed using PCR-positive clinical strains as
donors and E. faecium GE-1 (Aps Fusr
Rifr) (5) as the recipient as previously
described (5). Six of the 10 clinical strains from northeast
Ohio and all other PCR-positive strains were used as donors.
Transconjugants were selected on BHI agar plates containing vancomycin
(6 µg/ml), fusidic acid (25 µg/ml), and rifampin (100 µg/ml).
Pulsed-field gel electrophoresis (PFGE) was performed on genomic DNA
extracted from overnight cultures using the GenePath Group 1 reagent
kit (Bio-Rad, Hercules, Calif.), digested with SmaI as
previously described (7), and separated on 1% agarose gels
using the enterococcal program on the Bio-Rad GenePath electrophoresis system. Isolates were considered unrelated if they differed by more
than five bands. DNA bands separated by PFGE were transferred to nylon
filters using the technique of Southern (10). The filter was
baked at 80°C for 1 to 2 h, hybridized overnight at 68°C with a digoxigenin-labeled internal vanB probe, and washed under
conditions of high stringency according to the specifications of the
manufacturer (Boehringer Mannheim, Indianapolis, Ind.). Hybridized
fragments were detected using an antidigoxigenin antibody and a
chromogenic enzyme substrate (Boehringer Mannheim).
The 1,076-bp product was amplifiable from 10 of 10 clinical strains
from northeast Ohio and 6 of 13 strains from diverse geographic locations. Six of the 10 strains from northeast Ohio (chosen because they represented different clonal groups) and the 6 other PCR-positive strains were selected for MIC determination and conjugation
experiments. All 12 strains exhibited resistance to high levels of
ampicillin and vancomycin and to 10 µg of ciprofloxacin per ml and
were susceptible to chloramphenicol (Table
1). Vancomycin resistance was transferred at a low frequency (<10
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Geographic Distribution of a Large Mobile Element
That Transfers Ampicillin and Vancomycin Resistance between
Enterococcus faecium Strains
ABSTRACT
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9/donor CFU) from 7 of the 12 donor strains. All transconjugants expressed resistance to ampicillin
and vancomycin at a MIC that was the same as or lower than that for the
donor strains (Table 1), a discordance that has been noted previously
with transfer of a similar element from E. faecium C68
(5). Transfer of erythromycin, streptomycin, and
tetracycline resistance was variable. Neither ciprofloxacin nor
gentamicin resistance was transferred.
TABLE 1.
MICs for isolates
Sequence analysis from all PCR-positive strains revealed five mutations previously identified in the pbp5 gene from E. faecium C68 (GenBank accession number AF117609). Using the numbering system for the pbp5 gene described for E. faecium strain D63r in reference 14, these changes include an additional serine at amino acid position 466 and four additional nucleotide changes (T-1918-C, T-1912-G, C-1899-T, and A-1884-C).
PFGE analysis of the seven strains from which transconjugants were
obtained confirmed that the strains were not clonally related (Fig.
1). Southern transfers of the PFGE
separation revealed hybridization of vanB probes to large
fragments in each of the donors and transconjugants (Fig. 1).
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Among the more interesting, and as yet unexplained, attributes of the ongoing outbreaks of vancomycin-resistant enterococci (VRE) in the United States is the almost exclusive occurrence of this phenotype in E. faecium strains expressing high levels of ampicillin resistance. The coexpression of these resistance phenotypes has significant clinical implications, most prominently the potential for selection of resistant strains with different antibiotics. It has been noted that restriction of vancomycin usage does not consistently affect the overall prevalence of VRE within institutions. This observation is understandable if antibiotic selective pressure persists despite reductions in vancomycin use. Extended-spectrum cephalosporins were observed to select for colonization by ampicillin-resistant E. faecium even before the VRE outbreak (6) and have been associated in several studies with infection or colonization by VRE (4, 7, 11, 12).
The data presented in this paper suggest that the close association of the vancomycin and ampicillin resistance phenotypes, at least in VanB-type VRE, is explainable by their inclusion within large, transferable genetic elements. The precise relationship between pbp5 and Tn5382 demonstrated in different strains by these studies and the presence of the same point mutations within the pbp5 genes suggest that all of these elements evolved from a single genetic event. The nature of this event remains obscure, but it most likely involved the insertion of Tn5382 into a larger, mobile element that encodes PBP5-mediated ampicillin resistance. Although pbp5 has previously been described as transferable on a plasmid (13), evidence for the geographic dispersion of a chromosomally located element possessing Tn5382 and pbp5 has been lacking. It is likely that enterococci only recently acquired this gene or that prior attempts to demonstrate transfer were thwarted by the variable expression of ampicillin resistance in the transconjugants. In fact, we were unable to select directly for transfer of ampicillin resistance. Rather, we noted the variable expression of ampicillin resistance (despite the presence of the pbp5 gene) in transconjugants selected for the transfer of resistance to vancomycin. Further characterization of these large, transferable elements promises to provide significant insight into the evolution of multiresistance in nosocomial enterococci.
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ACKNOWLEDGMENTS |
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This work was supported by the Office of Research and Development, Medical Research Service of the Department of Veterans Affairs (L.B.R.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Medical Service 111(W), VA Medical Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800, ext. 4801. Fax: (216) 231-3289. E-mail: louis.rice{at}med.va.gov.
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Antimicrobial Agents and Chemotherapy, May 2000, p. 1349-1351, Vol. 44, No. 5
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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