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Antimicrobial Agents and Chemotherapy, December 2004, p. 4762-4765, Vol. 48, No. 12
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.12.4762-4765.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Derivatives of a Vancomycin-Resistant Staphylococcus aureus Strain Isolated at Hershey Medical Center

Bülent Bozdogan, Lois Ednie, Kim Credito, Klaudia Kosowska, and Peter C. Appelbaum^*

Department of Pathology, Hershey Medical Center, Hershey, Pennsylvania

Received 9 June 2004/ Returned for modification 1 August 2004/ Accepted 29 August 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antimicrobial susceptibilities and genetic relatedness of the vancomycin-resistant Staphylococcus aureus strain (VRSA) isolated at Hershey, Pa. (VRSA Hershey), and its vancomycin-susceptible and high-level-resistant derivatives were studied and compared to 32 methicillin-resistant S. aureus strains (MRSA) isolated from patients and medical staff in contact with the VRSA patient. Derivatives of VRSA were obtained by subculturing six VRSA colonies from the original culture with or without vancomycin. Ten days of drug-free subculture caused the loss of vanA in two vancomycin-susceptible derivatives for which vancomycin MICs were 1 to 4 µg/ml. Multistep selection of three VRSA clones with vancomycin for 10 days increased vancomycin MICs from 32 to 1,024 to 2,048 µg/ml. MICs of teicoplanin, dalbavancin, and oritavancin were also increased from 4, 0.5, and 0.12 to 64, 1, and 32 µg/ml, respectively. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing analysis indicated that VRSA Hershey was the vanA-acquired variety of a common MRSA clone in our hospital with sequence type 5 (ST5). Three of five vancomycin-intermediate S. aureus strains tested from geographically different areas were also ST5, and the Michigan VRSA was ST371, a one-allele variant of ST5. Derivatives of VRSA Hershey had differences in PFGE profiles and the size of SmaI fragment that carries the vanA gene cluster, indicating instability of this cluster in VRSA Hershey. However induction with vancomycin increased glycopeptide MICs and stabilized the resistance.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Staphylococcus aureus is one of the most common causes of nosocomial infections, especially pneumonia, surgical site infections, and bloodstream infections (13, 17). This bacterium has the ability to rapidly acquire antimicrobial resistance. Most S. aureus strains (>90%) are resistant to penicillin (12), and since the 1980s, methicillin-resistant S. aureus (MRSA) strains have become endemic in hospitals worldwide (20). One decade later during the1990s, S. aureus isolates with diminished susceptibility to vancomycin (vancomycin-intermediate S. aureus [VISA]) were reported (8, 9, 15). Recent reports of three S. aureus clinical strains with the vanA gene open a new era in staphylococcal antibacterial resistance (2-4). This latter development limits further potentially therapeutic options against these strains.

Two of the three vancomycin-resistant S. aureus (VRSA) strains were isolated from foot ulcers of patients with circulation disorders in the lower limbs concomitant with amputation histories (5, 19). Both isolates had vanA, but the level of vancomycin resistance was high (MIC, 1,024 µg/ml) in VRS1, isolated from a patient treated with vancomycin in Michigan, and was lower in VRS2 (32 µg/ml), isolated in the absence of vancomycin treatment (prior or current) in Hershey, Pa. (5, 19). The vancomycin MICs for the third VRSA strain (isolated from urine) were also lower, similar to those of the Hershey strain (4).

The aims of this study were to (i) analyze glycopeptide-susceptible derivatives of the VRSA Hershey strain, (ii) analyze the capability of vancomycin to induce raised glycopeptide MICs for the parent Hershey strain, (iii) determine clonality by comparing the latter strain with S. aureus strains isolated from contacts of our index case, and (iv) identify its relationship with the major internationally spread MRSA clones as well as with five VISA strains and the Michigan VRSA strain by multilocus sequence typing (MLST).

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Bacteria. VRSA strain Hershey (VRS2) was isolated from the foot ulcer of a 70-year-old patient (1, 2, 19). Two hundred thirty-nine patients and medical staff from Hershey Medical Center in contact with the VRSA index patient were studied for MRSA and VRSA carriage by nasal culture and culture of open wounds where present (19). Prior Institutional Review Board approval was sought and obtained in this study for each person cultured in the search for possible carriers.

Strains VRS2.1 to VRS2.6 were randomly taken colonies from the original culture of the VRSA patient. VRSA Michigan (VRS1) and five VISA strains (NRS1, NRS12, NRS17, NRS56, and NRS63), obtained from The Network on Antimicrobial Resistance in Staphylococcus aureus through Focus Technologies, Herndon, Va., were also included in this study.

Selection of derivative strains and antimicrobial susceptibility testing. VRSA clones VRS2.1, VRS2.2, and VRS2.3, isolated from the original VRS2 culture, were used for multistep selection studies. For multistep studies, MICs were determined by the standard broth microdilution method with cation-adjusted Mueller-Hinton broth (BBL Microbiology Systems, Cockeysville, Md.) as recommended by the National Committee for Clinical Laboratory Standards (10). A final inoculum of 5 x 10⁵ CFU/ml was used to obtain mutants. Daily passages were then performed for 10 days by taking an inoculum from the tube nearest the MIC (usually 1 to 2 dilutions below) that had the same turbidity as the antibiotic-free controls. Subculturing was stopped when the vancomycin MICs for the mutants were 1,024 µg/ml. Cultures were incubated for 24 h at 37°C.

VRSA clone VRS2.3 was subcultured for 10 days on a blood agar plate, without antibiotic. After 10 days, vancomycin susceptibilities of the colonies were tested by macrobroth MIC (11) and vancomycin-susceptible colonies were selected for further analysis.

Glycopeptide MICs for the high-level resistant and vancomycin-susceptible derivatives of VRS2 were tested by broth macrodilution with cation-adjusted Mueller-Hinton broth (11).

MIC testing of S. aureus strains isolated from contacts. MICs for the strains collected from contacts were determined by agar dilution as recommended by the National Committee for Clinical Laboratory Standards (11). Standard quality control strains were included in each run. Cultures were incubated for 16 to 20 h in ambient air. Vancomycin plates were incubated for 24 h (11). Drugs were obtained from their respective manufacturers.

PFGE and Southern blot. Pulsed-field gel electrophoresis (PFGE) was performed with a CHEF DR III apparatus (Bio-Rad, Hercules, Calif.) as described previously (10). The SmaI-digested DNA fragments were separated in 1% agarose gel for an initial time of 5 s and a final time of 40 s; the electrophoresis time was 21 h at 6.6 V with an angle of 120°. Gels were treated with depurination and denaturation buffers. After depurination (HCl, 0.25 M) and denaturation (1.5 M Tris HCl, 0.5 N NaOH), DNA was transferred to a nylon membrane with a Vacublotter (Bio-Rad, Hercules, Calif.) with transfer buffer (1.5 M Tris HCl, 0.25 N NaOH). An internal fragment obtained after amplification by PCR was labeled with the ECL enhanced chemiluminescence nucleic acid labeling and detection system (Amersham Bioscience Buckinghamshire, England) and used as a probe.

Determination of resistance mechanisms. Resistance mechanisms for methicillin, macrolides, aminoglycosides, and tetracycline were tested as described previously (1). A double-disk test was used to determine inducibility of clindamycin resistance, using erythromycin (15 µg/ml) and clindamycin (2 µg/ml) disks.

MLST. The seven housekeeping genes were amplified with primers and the PCR method described by Enright et al. for MLST (7). After amplification, PCR products were purified from excess primers and nucleotides with a QIAquick PCR purification kit (QIAGEN, Valencia, Calif.) and were sequenced directly with a CEQ8000 genetic analysis system (Beckman Coulter, Fullerton, Calif.). Analysis of the sequences was done with software of the Multilocus Sequence Typing website (http://www.mlst.net).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Epidemiology of VRSA Hershey. A total of 32 MRSA strains were isolated from 20 of 239 contacts. Multiple strains isolated from the same individual were identical by PFGE profile, and only one strain from each contact was studied further. All 20 strains were methicillin resistant and carried mecA. Among 20 strains, 18 were resistant to erythromycin and carried the erm(A) gene. Five of 18 strains with erm(A) were susceptible to clindamycin, and resistance was inducible by the double-disk test. The MICs of erythromycin and clindamycin for inducible strains were 16 and <0.125 µg/ml, respectively. Thirteen of 18 erythromycin-resistant strains were constitutively resistant, and their MICs were >64 for both clindamycin and erythromycin. Erythromycin-resistant strains were also resistant to ciprofloxacin. The MIC ranges for ciprofloxacin, vancomycin, kanamycin, gentamicin, and tetracycline were 0.5 to >64, 0.5 to 1, 2 to >64, 0.5 to 16, and 0.25 to 32 µg/ml, respectively. Only one strain was resistant to tetracycline and carried the tetK gene. All MRSA strains were susceptible to gentamicin and vancomycin.

Seventeen of 20 S. aureus isolates had zero to three band differences in PFGE profile compared to strain VRS2.3 and were genetically related. Among those, five isolates were identical to VRS2.3 (Fig. 1). VRS2.3 and two strains with zero and one band differences were studied by MLST. Allelic profiles of the strains are shown in Table 1. All three strains had the same allelic profile, which corresponds to ST5.

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FIG. 1. PFGE patterns after digestion by SmaI of MRSA strains isolated from contacts. Seventeen of 20 strains were clonally related to the VRSA strain and had zero to three band differences. Strains studied for MLST (V3, VRS2.3; 12, S12; and 11, S11) are underlined. S. aureus NCTC 8325 was used as a molecular size marker (M).

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TABLE 1. Allelic profiles and MLST types of two VRSA and five VISA strains as well as two MRSA strains from contacts

The possibility of a common origin among vancomycin-nonsusceptible strains was tested by including five VISA strains (NRS1, NRS12, NRS17, NRS56, and NRS63) isolated from geographically distinct parts of the world (Japan, United States, France, Brazil, and Oman) and the VRS1 strain (VRSA Michigan). Three of five VISA strains isolated from Japan, United States, and France displayed an ST5 profile by MLST (Table 1). The VISA strain NRS56 from Brazil was ST239. The NRS63 isolated in Oman was a single-locus variant of ST241 and had 1-base difference from allele 4 (C257T) in tpi locus. This new tpi allele was numbered 73 and the new ST was designated ST372. VRS1 represents a single-locus variant of the MLST profile of the VRS2 and had 1 nucleotide substitution; thymine at position 378 was replaced with adenine in the glpF locus. This new glpF allele was named allele 53, and the variant of ST5 was designated ST371 (Table 1).

Derivatives of VRSA Hershey. Selection with vancomycin increased the vancomycin MICs at days 4 to 6 from 32 µg/ml to 1,024 to 2,048 µg/ml in derivatives of VRS2. Increase in vancomycin MICs was associated with increase in MICs of teicoplanin (16- to 128-fold) and dalbavancin (16- to 64-fold). Oritavancin MICs increased only two- to fourfold. Some high-level-resistant derivatives lost gentamicin and tetracycline resistance (Table 2). Drug-free subculture for 10 days caused loss of the vanA cluster and concomitant vancomycin resistance in two derivatives. MICs of the susceptible derivatives for oritavancin and teicoplanin did not change but were 8-fold lower for dalbavancin and 8- to 64-fold lower for vancomycin. Vancomycin resistance was more stable in the selected high-level-resistant derivatives, which did not lose resistance after 10 days of drug-free subculture.

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TABLE 2. Glycopeptide MICs of the derivatives of VRSA Hershey

Pulsed-field profiles of derivatives of VRS2 strain were different (Fig. 2). VRS1 and VRS2.1 to -3 and VRS2.6 had three band differences. The second band in VRS2.1 was absent in VRS1, in which two different bands appeared: the larger of these two bands (360 kb) on first inspection appeared to carry the vanA gene. However, careful analysis of hybridization results (Fig. 2) showed that the vanA-positive fragments of VRS1 as well as VRS2.6 and VRS2.31 represented different forms of a nondigested plasmid. The absence of SmaI restriction sites in the sequence of the plasmid pLW043 from VRSA Michigan that carry the vanA gene support this hypothesis (18). The molecular sizes of the SmaI fragments that carry the vanA gene also varied between 90 and 135 kb among variants of VRS2. In two variants, VRS2.S1 and VRS2.S2, absence of vanA was associated with vancomycin susceptibility (Fig. 2).

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FIG. 2. Analysis of genomic DNA from VRSA strains isolated at Hershey and from Michigan by Southern blot hybridization. Panel A shows total DNA separated by PFGE after digestion by SmaI, and panel B shows hybridization of DNA transferred to a nylon sheet with biotin-labeled vanA probe. Some of the derivatives of VRSA Hershey show different PFGE profiles. Hybridization with vanA probe indicates that the size of the SmaI fragment, which carries vanA, may vary within the derivatives of the same strain. Strains VRS1 (lane MI), VRS2.1 (lane V1), VRS2.2 (lane V2), VRS2.3 (lane V3), VRS2.4 (lane V4), and VRS2.6 (lane V6) are derivatives of VRSA Hershey. Strains VRS2.11 (lane 11), VRS2.12 (lane 12), VRS2.21 (lane 21), VRS2.31 (lane 31), and VRS2.32 (lane 32) are vancomycin-induced derivatives of VRSA strains. Strains VRS2.S1 (lane S1) and VRS2.S2 (lane S2) are vancomycin-susceptible derivatives of VRSA Hershey.

Top Abstract Introduction Materials and Methods Results Discussion References

 
VRS2.3 was genetically related to 85% of MRSA strains isolated from carriers in contact with the index VRSA patient. MLST indicated that VRS2 belongs to ST5, one of the widespread MRSA clones. Crisostomo et al. have shown that the New York clone, which is widely disseminated in the northeastern part of the United States, also displayed ST5 (6). Sixty percent of VISA strains that were tested were also ST5. Other glycopeptide-intermediate S. aureus strains have also been reported to belong to ST5 (14). The VRS1 is a one-allele variant of ST5. Further analyses are necessary to determine if the vancomycin resistance has a possible association with ST5.

In the absence of vancomycin pressure, vancomycin resistance was found to be unstable and expressed at a low level. This low-level expression of vancomycin resistance in S. aureus may be the reason why these strains are hard to detect clinically. It has been shown that automated susceptibility testing systems fail to detect low-level vancomycin-resistant strains (4, 16). Use of disk diffusion, E-test, and vancomycin screening plates with 6-µg/ml vancomycin will increase the detectability of these strains (4, 16). The incidence of gentamicin-resistant strains in carriers was lower than that seen from clinical isolates in our hospital. However, the majority of S. aureus infections in our institution are acquired nosocomially in the presence of aminoglycoside selective pressure.

We conclude that VRSA Hershey is the vanA-acquired variant of a very common major epidemic MRSA clone, which has the facility to acquire antimicrobial resistance, including to the glycopeptides, and to spread and become endemic. Different clones of VRSA Hershey with different locations of the vanA gene indicate the instability of the genetic element that carries the vanA gene. Vancomycin overuse in the community may play a role in maintenance of an unstable vanA determinant in VRSA clones.

 
We thank NARSA and NIAID for supplying the Michigan VRSA strain (VRS1) and the five VISA strains.

 
* Corresponding author. Mailing address: Hershey Medical Center, Department of Pathology, 500 University Dr., Hershey, PA 17033. Phone: (717) 531-5113. Fax: (717) 531-7953. E-mail: pappelbaum{at}psu.edu.

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Antimicrobial Agents and Chemotherapy, December 2004, p. 4762-4765, Vol. 48, No. 12
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.12.4762-4765.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bozdogan, B. Articles by Appelbaum, P. C. Search for Related Content PubMed PubMed Citation Articles by Bozdogan, B. Articles by Appelbaum, P. C.