Departments of 1 Pathology and 2 Medicine, Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA
Received 14 August 2003; returned 22 August 2003; revised 26 August 2003; accepted 28 August 2003
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Abstract |
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Keywords: S. aureus, VRSA, antimicrobial susceptibilities, mechanisms of resistance
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Introduction |
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We tested antimicrobial susceptibilities and mechanisms of resistance of the VRSA strain isolated at the Hershey Medical Center in September 2002, as well as of a cured vancomycin-susceptible derivative of this VRSA, using a variety of clinically used and experimental antibacterials.
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Materials and methods |
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Vancomycin-resistant S. aureus strain HMC3 was isolated at the Hershey Medical Center from a heel wound in a 70-year-old male patient.2 A spontaneously cured vancomycin-susceptible derivative of this VRSA, S. aureus strain HMC3-1, was obtained by serial passage of the VRSA in drug-free medium.
Antimicrobials
Oritavancin (LY 333328) was from Lilly Research Laboratories (Indianapolis, IN, USA); dalbavancin from Vicuron Pharmaceuticals, Inc. (King of Prussia, PA, USA); ramoplanin from the Genome Therapeutics Corp. (Waltam, MA, USA); BAL9141 from Basilea Pharmaceutica AG (Basle, Switzerland); RWJ-54428 from Johnson & Johnson (Raritan, NJ, USA); daptomycin from Cubist Pharmaceuticals, Inc. (Lexington, MA, USA); tigecycline from Wyeth-Ayerst Laboratories (Pearl River, NY, USA); ranbezolid from Ranbaxy Laboratories (New Delhi, India); WCK 771 A, WCK 919, and the latters constituent enantiomers (WCK 1152 and WCK 1153) from Wockhardt Research Center (Aurangabad, India); DK-507k and sitafloxacin from Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan); iclaprim from Arpida AG (Münchenstein, Switzerland); NVP-PDF713 from the Novartis Pharmaceutical Corp. (Summit, NJ, USA); and GS02-02, GS02-12, GS2-10547 and GS02-104 from GeneSoft Pharmaceuticals Inc. (South San Francisco, CA, USA). Other drugs were obtained from their manufacturers.
MIC determinations and timekill studies
MICs were determined using the macrobroth dilution method.3 Timekill studies were carried out as described previously.4
Determination of resistance mechanisms and DNA manipulation
The presence of antibiotic resistance genes in strains HMC3 and HMC3-1 was examined by PCR.57 Alterations in quinolone resistance determining regions (QRDRs) in gyrA, gyrB, grlA and grlB genes were studied as described previously.8 After amplification, PCR products were purified from excess primers and nucleotides using a QIAquick PCR Purification Kit (Qiagen, Inc., Valencia, CA, USA) and sequenced directly using a CEQ 8000 Genetic Analysis System (Beckman Coulter, Inc., Fullerton, CA, USA). Pulsed-field gel electrophoresis of total DNA was carried out as previously described.9
To locate the vanA gene, an internal PCR fragment of vanA, labelled using the ECL nucleic acid labelling and detection system, was used as a probe for hybridization to digested and separated genomic DNA, which had been transferred to nylon membranes under a vacuum.
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Results |
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VRSA strain HMC3 contained the vanA gene on a 135 kb SmaI genomic DNA fragment (data not shown); it also contained mecA, erm(A), erm(B), tet(K) and aac(6')-aph(2''). A spontaneous vancomycin-susceptible derivative HMC3-1 lacked vanA, erm(B) and aac(6')-aph(2''). Variations in the size of the SmaI fragment carrying the vanA gene were observed among seven separate VRSA isolates from the same patient, suggesting instability of the resistance element (data not shown).
For both HMC3 and HMC3-1, analysis of QRDRs of gyrA, gyrB, grlA and grlB showed substitution of serine by leucine at position 84 in GyrA and substitution of glutamic acid by lysine at position 471 in GrlB, but no alterations in either GyrB or GrlA.
Antimicrobial susceptibility
The vancomycin MIC for strain HMC3 was 32 mg/L, though it remained susceptible to teicoplanin (4 mg/L). MICs of the experimental glycopeptides oritavancin and dalbavancin and the glycolipodepsipeptide ramoplanin were low (0.1250.5 mg/L). Teicoplanin, dalbavancin, oritavancin and ramoplanin were bactericidal against HMC3 after 612 h in the 12 x MIC range. Regrowth was observed at the MIC after 24 h with dalbavancin (Table 1).
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In comparison with HMC3, derivative HMC3-1 was susceptible to vancomycin (4 mg/L) and showed a lower dalbavancin MIC (0.06 mg/L); however, activities of teicoplanin, oritavancin and ramoplanin were unchanged. HMC3-1 also became susceptible to gentamicin (0.5 mg/L). Activities of other drugs tested against HMC3 and HMC3-1 were identical.
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Discussion |
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The level of expression of vanA in VRSA strain HMC3 is currently under investigation in our laboratory. However, this study indicates that the vanA gene cluster is not a stable genetic element in strain HMC3; serial passage of the strain in the absence of vancomycin led to loss of the vanA cluster and, consequently to restoration of vancomycin susceptibility. A decrease in dalbavancin MIC was also noted in strain HMC3-1, though MICs of teicoplanin, oritavancin and ramoplanin were unaffected. Vancomycin susceptibility was accompanied by emergence of gentamicin susceptibility, attributable to loss of the aac(6')-aph(2'') gene; erm(B) was also lost. Loss of these resistance genes may result from the instability of transposon Tn1546, which is often carried by conjugative plasmids with a broad host range in Gram-positive bacteria.
Emergence of a truly vancomycin-resistant S. aureus seriously threatens the most important treatment option available to clinicians for infections resulting from methicillin-resistant S. aureus. VRSA strain HMC3 is resistant to most available bactericidal drugs; it was bacteriostatically inhibited by linezolid, tigecycline, iclaprim and the PDF inhibitors. Though quinupristin/dalfopristin is typically rapidly bactericidal against Gram-positive cocci, it was bacteriostatic against strain HMC3, perhaps because of the presence of constitutively expressed erm genes. Antibiotics bactericidal against strain HMC3 included cephalosporins BAL9141 and RWJ-54428; quinolones WCK 771 A, WCK 1153, sitafloxacin and DK-507k; the DNA nanobinder GS02-02; glycopeptides oritavancin and dalbavancin; glycolipodepsipeptide ramoplanin; and lipopeptide daptomycin.
Acknowledgements
This work was supported in part by grants from Cubist Laboratories (Lexington, MA, USA) and Johnson & Johnson Research Laboratories (Raritan, NJ, USA).
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Footnotes |
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Corresponding author. Tel: +1-717-531-5113; Fax: +1-717-531-7953; E-mail: pappelbaum{at}psu.edu
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References |
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