JAC Advance Access originally published online on October 16, 2003
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Journal of Antimicrobial Chemotherapy (2003) 52, 864-868
© 2003 The British Society for Antimicrobial Chemotherapy
Antibacterial susceptibility of a vancomycin-resistant Staphylococcus aureus strain isolated at the Hershey Medical Center
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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Staphylococcus aureus strain HMC3 isolated at the Hershey Medical Center, was resistant to vancomycin (VRSA) through the presence of the vanA resistance gene; it also contained mecA, erm(A), erm(B), tet(K) and aac(6')-aph(2''), conferring resistance to licensed ß-lactams, macrolides, tetracycline and aminoglycosides. HMC3 also had alterations in GyrA and GrlB and was resistant to available quinolones. Experimental drugs with low MICs (<2 mg/L) for VRSA HMC3 included cephalosporins BAL9141 and RWJ-54428; glycopeptides oritavancin and dalbavancin; the lipopeptide daptomycin; the glycolipodepsipeptide ramoplanin; new fluoroquinolones WCK 771 A, WCK 1153, DK-507k and sitafloxacin; and the DNA nanobinder GS02-02. These agents were all bactericidal as were trimethoprim/sulfamethoxazole and teicoplanin (MIC 4 mg/L). Oxazolidinones linezolid and ranbezolid; the injectable streptogramin quinupristin/dalfopristin; DNA nanobinders GS2-10547 and GS02-104; peptide deformylase inhibitors NVP-PDF713 and GS02-12; tetracycline derivative tigecycline; the antifolate iclaprim; mupirocin and fusidic acid were all active in vitro but bacteriostatic.
Keywords: S. aureus, VRSA, antimicrobial susceptibilities, mechanisms of resistance
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Introduction |
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Fifteen years after the initial report of Enterococcus faecium with a transferable vanA gene and 6 years after the first description of vancomycin-intermediate Staphylococcus aureus, two vancomycin-resistant S. aureus (VRSA) strains containing the vanA gene cluster were isolated in the US between June and September 2002.1,2
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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Bacterial strains
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 latter’s 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 time–kill studies
MICs were determined using the macrobroth dilution method.3 Time–kill 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.5–7 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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Mechanism of resistance
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 (0.125–0.5 mg/L). Teicoplanin, dalbavancin, oritavancin and ramoplanin were bactericidal against HMC3 after 6–12 h in the 1–2 x MIC range. Regrowth was observed at the MIC after 24 h with dalbavancin (Table 1).
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Two experimental anti-MRSA cephalosporins, BAL914110 and RWJ-54428,11 each had low MICs for HMC3 and were bactericidal at 2 x MIC after 6–12 h (Table 1). The strain was highly resistant to macrolides, telithromycin and clindamycin (>64 mg/L), but susceptible to quinupristin/dalfopristin, although this was only bacteriostatic. The MICs of oxazolidinones linezolid and ranbezolid were 1 mg/L and they were bacteriostatic. Though marketed fluoroquinolones were not active, experimental quinolones sitafloxacin, WCK 771 A,12 WCK 115312 and DK-507k,13 each had MICs in the 0.5–1 mg/L range and were bactericidal in the 1–2 x MIC range after 3–6 h (Table 1). DNA nanobinders are a new class of antibiotics that bind to the minor groove of DNA and inhibit DNA function and transcription;14 DNA nanobinders GS02-02, GS2-10547 and GS02-104 all had low MICs for VRSA strain HMC3, and GS02-02 was bactericidal (Table 1). Peptide deformylase (PDF) inhibitors interfere with removal of the formyl moiety of nascent polypeptides by PDF.15 PDF inhibitors NVP-PDF713 and GS02-12 had low MICs for VRSA strain HMC3, and were bacteriostatic. The VRSA was resistant to aminoglycosides and tetracycline, intermediate to chloramphenicol, and susceptible to tigecycline, iclaprim, trimethoprim/sulfamethoxazole, mupirocin and rifampicin (<0.06 mg/L), with a fusidic acid MIC of 4 mg/L. Tigecycline, mupirocin, fusidic acid and iclaprim were all bacteriostatic. Trimethoprim/sulfamethoxazole had a low MIC (0.25 mg/L) for the VRSA and was bactericidal at a concentration of 4 x MIC after 12 h (Table 1).
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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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Within a short period of time, two distinct, non-clonal strains of S. aureus resistant to vancomycin were isolated in the USA, first in Detroit, MI and shortly afterwards in Hershey, PA.1,2 Although both isolates possessed the vanA gene cluster, they have different glycopeptide resistance phenotypes; the Detroit strain of VRSA being highly resistant to both vancomycin and teicoplanin, and with an oritavancin MIC of 4 mg/L, whereas the Hershey VRSA strain HMC3 is susceptible to teicoplanin, and has low oritavancin and dalbavancin MICs.
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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* Present address. Erciyes Üniversitesi T
p Fakültesi, Mikrobiyoloji BD, Kayseri, Turkey. 
Corresponding author. Tel: +1-717-531-5113; Fax: +1-717-531-7953; E-mail: pappelbaum{at}psu.edu
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References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
1 . Centers for Disease Control and Prevention. (2002). Staphylococcus aureus resistant to vancomycin—United States. Morbidity and Mortality Weekly Report 51, 565–7.
2 . Centers for Disease Control and Prevention. Public Health Dispatch. (2002). Vancomycin-resistant Staphylococcus aureus—Pennsylvania, 2002. Morbidity and Mortality Weekly Report 51, 902–3.
3 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
4 . Pankuch, G. A., Jacobs, M. R. & Appelbaum, P. C. (1984). Study of comparative antipneumococcal activities of penicillin G, RP 59500, erythromycin, sparfloxacin, ciprofloxacin, and vancomycin by using time–kill methodology. Antimicrobial Agents and Chemotherapy 38, 2065–72.
5 . Sutcliffe, J., Grebe, T., Tait-Kamradt, A. et al. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 2562–6.[Abstract]
6
.
Kao, S. J., You, I., Clewell, D. B. et al. (2000). Detection of the high-level aminoglycoside resistance gene aph(2'')-Ib in Enterococcus faecium. Antimicrobial Agents and Chemotherapy 44, 2876–9.
7 . Warsa, U. C., Nonoyama, M., Ida, T. et al. (1996). Detection of tet(K) and tet(M) in Staphylococcus aureus of Asian countries by the polymerase chain reaction. Journal of Antibiotics (Tokyo) 49, 1127–32.
8
.
Discotto, L. F., Lawrence, L. E., Denbleyker, K. L. et al. (2001). Staphylococcus aureus mutants selected by BMS-284756. Antimicrobial Agents and Chemotherapy 45, 3273–5.
9
.
Murray, B. E., Singh, K. V., Heath, J. D. et al. (1990). Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. Journal of Clinical Microbiology 28, 2059–63.
10
.
Entenza, J. M., Hohl, P., Heinze-Krauss, I. et al. (2002). BAL9141, a novel extended-spectrum cephalosporin active against methicillin-resistant Staphylococcus aureus in treatment of experimental endocarditis. Antimicrobial Agents and Chemotherapy 46, 171–7.
11
.
Chamberland, S., Blais, J., Hoang, C. M. et al. (2001). In vitro activities of RWJ-54428 (MC-02,479) against multiresistant gram-positive bacteria. Antimicrobial Agents and Chemotherapy 45, 1422–30.
12 . Bozdogan, B. & Appelbaum, P. C. (2003). Bactericidal activities of new quinolones WCK 771 A, WCK 919, and its two isomers WCK 1152 and WCK 1153 against vancomycin resistant Staphylococcus aureus (VRSA). In Abstracts of the forty-third Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2003. Abstract F-438, p. 228. American Society for Microbiology, Washington, DC, USA.
13 . Otani, T., Tanaka, M., Akasaka, T. et al. (2001). DK-507k, a new 8-methoxyquinolone: in vitro and in vivo antibacterial activities. In Abstracts of the forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2001. Abstract F-547, p. 218. American Society for Microbiology, Washington, DC, USA.
14 . Burli, R. W., Ge, Y., White, S. et al. (2002). DNA binding ligands with excellent antibiotic potency against drug-resistant gram-positive bacteria. Bioorganic and Medicinal Chemistry Letters 12, 2591–4.
15 . Hao, B., Gong, W., Rajagopalan, P. T. et al. (1999). Structural basis for the design of antibiotics targeting peptide deformylase. Biochemistry 38, 4712–9.[CrossRef][Medline]
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