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Treatment failure due to methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin

Peter B Ward, Elizabeth A Grabsch and Barrie C Mayall
Med J Aust 2001; 175 (9): 480-483.
Published online: 5 November 2001

We report the first instance in Australia of treatment failure due to a strain of methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin — heteroresistant vancomycin-intermediate S. aureus (hVISA). The infection occurred in a 41-year-old man with multiple risk factors. No transmission of the organism to other patients or the environment was detected. This case may herald the beginning of a new phase of staphylococcal resistance in Australia.

Peter B Ward, Paul D R Johnson, Elizabeth A Grabsch,
Barrie C Mayall and M Lindsay Grayson

MJA 2001; 175: 480-483
  

Clinical record - Assessment of patient isolates - Results - Discussion - Competing interests - Acknowledgements - References - Author's details -
- - More articles on Infectious diseases and parasitology

The glycopeptides, vancomycin and, to a lesser extent, teicoplanin, are the mainstay of therapy for infections caused by methicillin-resistant Staphylococcus aureus (MRSA),1,2 and currently up to half of all S. aureus strains isolated in hospitals in Australia are MRSA.3,4 Despite substantial glycopeptide use over many years, the emergence of MRSA strains with reduced susceptibility to vancomycin and teicoplanin has been reported only recently.5-7 Subsequently, MRSA strains have been reported that contain limited subpopulations with intermediate resistance to glycopeptides, while most of the population remains glycopeptide-susceptible.8-14 These are termed heteroresistant vancomycin-intermediate S. aureus (hVISA) (see glossary in Box 1). We report here the first Australian case of infection due to hVISA.

Clinical record

 A 41-year-old male smoker with long-standing type 1 diabetes, haemodialysis-dependent end-stage renal failure, hepatitis C and peripheral vascular disease was admitted to hospital in late August 2000 with bilateral lower-limb ischaemia refractory to prostacyclin therapy. Despite hyperbaric oxygen and multiple courses of antibiotics (including cephalexin, flucloxacillin, gentamicin and clindamycin), the patient developed increasing lower-limb gangrene, necessitating a right below-knee amputation on Day 41 of hospital admission. On Day 47, ulcers on his left foot were found to be infected with MRSA and Enterobacter spp.

Therapy with vancomycin (1 g) and meropenem (500 mg) postdialysis (ie, three times a week) was commenced. Intravenous teicoplanin (400 mg every third day) was later substituted for vancomycin, as the patient developed a rash after the initial dose of vancomycin. However, his condition worsened, necessitating a left below-knee amputation on Day 50. Therapy was continued with teicoplanin, intravenous gentamicin (160 mg daily) and metronidazole (500 mg twice daily), but both amputation wounds broke down and repeated cultures grew MRSA and E. coli, finally necessitating a left above-knee amputation on Day 105.

Despite therapeutic serum levels of teicoplanin (troughs of 6.5-15.9 mg/L, measured on 10 occasions over eight weeks), both amputation sites remained actively infected with MRSA and E. coli. On Day 120 (after 73 days' glycopeptide therapy), teicoplanin was ceased, and a new oxazolidinone, linezolid (600 mg intravenously, twice daily), was commenced in combination with oral ciprofloxacin and metronidazole. Over the next five days, dramatic improvement was noted in all infected wounds. After 11 days, intravenous linezolid was changed to oral linezolid (600 mg twice daily). MRSA was isolated from the amputation sites nine days after linezolid was begun, but was not detected again in any subsequent cultures.

The patient continued to receive oral linezolid for 81 days. His condition improved steadily, and he was transferred to a rehabilitation unit on Day 179. Over the subsequent six months, he remained reasonably well, with no evidence of MRSA infection.


Assessment of patient isolates

After attending a presentation during which new laboratory methods for the accurate identification of VISA and hVISA were presented (Annual Conference of the Australian Society for Antimicrobials, Melbourne, April, 2001), we decided to further investigate stored MRSA isolates from the patient. Two MRSA strains obtained from the right below-knee amputation stump were retrieved from storage at -70ºC. These strains (AR1 and AR2) were isolated on hospital Day 104 (after 57 days of teicoplanin therapy) and Day 129 (nine days after changing from teicoplanin to linezolid therapy), respectively.

The two strains were assessed for in-vitro antibiotic susceptibility using routine methods (agar dilution and broth microdilution).15 They were also assessed for glycopeptide-resistant subpopulations using methods described previously.5-7 These comprised:

Colony morphology: Each isolate was examined macroscopically for the heterogeneous colony morphology typical of hVISA; pure cultures have been reported to produce a mix of both large and small colonies when cultured on Columbia agar with 5% horse blood (Oxoid, Basingstoke, UK) and other media.6,7 Colonies suspected of glycopeptide resistance were further assessed for vancomycin and teicoplanin resistance.

Vancomycin gradient plates: Vancomycin resistance was assessed using vancomycin gradient plates prepared as described previously.6 Thirty mL of brain-heart infusion (BHI) agar (Oxoid, Basingstoke, UK) containing vancomycin (4 mg/L) was poured into a 10 cm square petri dish raised on one edge by 6 mm. After setting, the resultant wedge was overlaid with a 30 mL layer of BHI agar without antibiotic and allowed to set horizontally. Plates were stored for 24 hours at 4ºC to allow diffusion of vancomycin into the upper agar layer. Twenty-four-hour cultures of organisms in brain-heart infusion (BHI) broth were adjusted to a 0.5 McFarland standard, and 20 µL aliquots were spread on the gradient plates in an even line along the increasing antibiotic gradient. Plates were assessed after 48 hours' aerobic incubation at 37ºC.6

E test minimum inhibitory concentration: Both vancomycin and teicoplanin resistance was assessed by E test (AB Biodisk, Solna, Sweden), using methods and interpretations recommended in the United States7 and Europe.11 US methods use Mueller-Hinton agar (Oxoid, Basingstoke, UK) and an inoculum equivalent to the 0.5 McFarland standard, and define intermediate vancomycin resistance as MIC, 8-16 mg/L. European methods use BHI agar and a heavier inoculum (2 McFarland standard) and define intermediate vancomycin resistance as MIC ≥8 mg/L. Teicoplanin intermediate resistance is defined as MIC > 8 mg/L (US) or > 6 mg/L (Europe). Inoculated media were incubated aerobically for 24 hours at 37ºC.7,11

Population analysis profile: The proportion of cells in the population of each isolate that was resistant to a range of vancomycin and teicoplanin concentrations was assessed, using methods described previously.6,9

Assessment of nosocomial transmission

As the presence of hVISA was first recognised five months after the patient's infection was cured empirically, screening was undertaken to ascertain whether hVISA had been transmitted nosocomially:

  • Nose, groin, hand and wound specimens from the index patient were cultured to assess current MRSA and hVISA infection or colonisation.

  • Nose and groin specimens were collected from all patients attending the in-centre haemodialysis units also attended by the index patient. These were assessed, along with multiple environmental samples from the units, for the presence of MRSA and hVISA.

  • All patients in the Nephrology Department who had been diagnosed with MRSA infection or colonisation between October 2000 and May 2001 were identified from the hospital's microbiology database. Their clinical course was reviewed to identify those whose condition did not respond to vancomycin. Stored MRSA isolates from these patients were assessed for glycopeptide resistance, as described above.

  • All MRSA isolates obtained at our institution since May 2001 were assessed prospectively for hVISA using a screening plate of BHI agar with vancomycin (4 mg/L).
    • 12

    Results

    Antibiotic susceptibility of index isolate

    Routine antibiotic sensitivity testing: The two patient isolates, AR1 and AR2, were confirmed to be MRSA and to have identical susceptibility profiles. Both tested resistant to penicillin, methicillin, erythromycin, trimethoprim, cotrimoxazole, clindamycin and ciprofloxacin, but susceptible to tetracycline, chloramphenicol, mupirocin, fusidic acid, vancomycin (MIC, 2 mg/L) and teicoplanin (MIC, ≤ 8 mg/L) using agar dilution methods,15 and to linezolid (MIC, 1.5 mg/L) using the E test.15

    Glycopeptide-resistant subpopulations: Both AR1 and AR2 displayed small and large colony variants, consistent with previous reports of hVISA, VISA and VRSA.6,7 By E test, both isolates had a vancomycin MIC of 6-8 mg/L (US method) and 8 mg/L (European method), and a teicoplanin MIC of 24 mg/L (US method) and 16-24 mg/L (European method).

    Repeated analysis of AR2 using vancomycin gradient plates demonstrated growth across the entire 4 mg/L vancomycin gradient, confirming the MIC to be > 4 mg/L, and the isolate to be hVISA. Population analysis profiles were also consistent with both isolates' being hVISA. In particular, detailed analyses of AR2 demonstrated that colony subpopulations were able to grow on 3 mg/L and 4-6 mg/L vancomycin plates at frequencies of 1 in 10 and 1 in 105-106, respectively (Box 2). In comparison, control organisms generated resistant colonies at a rate of < 1 in 108 at these concentrations. Similarly, population analysis of AR2 using 8 mg/L and 16 mg/L teicoplanin demonstrated presence of resistant subpopulations at frequencies of 1 in 103 and 1 in 105-106, respectively (Box 2). These findings are consistent with those for hVISA reported by Hiramatsu.5,6,9

    Nosocomial transmission of hVISA

    MRSA, hVISA and VISA were not detected in cultures obtained from the index patient after completion of linezolid therapy. Similarly, hVISA was not detected from nose or groin cultures of 85 patients who were either current renal ward inpatients or undergoing in-centre haemodialysis. Also, hVISA was not detected in cultures of 28 environmental sites in the ward and haemodialysis units, suggesting that routine cleaning was effective in limiting significant hVISA colonisation and contamination.

    Twenty-six renal patients were identified from the microbiology database with MRSA infection during the eight months between October 2000 and May 2001. Six of these patients were considered by the Nephrology Department to have had a slow clinical response to anti-MRSA treatment. MRSA isolates were retrieved from frozen storage for all six patients, and the most recently obtained MRSA isolate was assessed for all (except one patient in whom the second most recent isolate was assessed). None of these MRSA strains were hVISA.

    Prospective screening of all MRSA isolates at our institution for hVISA began in July 2001. Of 315 isolates obtained from 128 patients, none were hVISA.

    Discussion

    		 This is the first report of clinical treatment failure caused by MRSA with reduced susceptibility to glycopeptides in Australia. Similar cases have been described in Europe, North America and South-East Asia,7,16 and a single strain of vancomycin-resistant S. aureus has been reported in Japan.17

    Slow clinical response, and even treatment failures, associated with glycopeptide therapy for MRSA infections have been described previously.1,18-20 This has generally been attributed to the characteristics of glycopeptides: their penetration into sites of established sepsis, which is generally inferior to that of other agents, such as β-lactams, and their slow bactericidal activity.20-22 Empirically, we attributed the clinical failure of teicoplanin in our patient to multiple factors, including his advanced vascular disease, and poor drug delivery (despite adequate serum teicoplanin levels), as well as the inherent characteristics of the drug.2 However, the patient's very rapid clinical improvement and the ultimate clearance of MRSA soon after commencement of linezolid is consistent with our later identification of the infecting strain as hVISA.

    Our detailed search did not detect hVISA contamination of the haemodialyis environment, or colonisation or infection of other haemodialysis patients, or any subsequent patients with MRSA at our hospital. Thus, we believe that hVISA is not widespread in our hospital, and that our case is unusual.

    Now that the microbiological methods to identify hVISA have been clearly described,6,7,14 it is likely that strains will be identified in Australia. However, it is a challenge to establish a laboratory screening protocol for hVISA and to determine what resources should be allocated to screening for hVISA. The Centers for Disease Control and Prevention in Atlanta recommend that routine screening of all MRSA isolates for vancomycin resistance is currently unnecessary and probably wasteful. Instead, attention should be focused on patients in whom glycopeptide therapy is failing, or those at increased risk of MRSA carriage and infection, such as patients undergoing haemodialysis or chronic ambulatory peritoneal dialysis.7,12

    Our identification of hVISA may be the beginning of a new phase in the emergence of antibiotic resistance in Australia, when the glycopeptides vancomycin and teicoplanin will no longer be effective in some cases of MRSA infection.18-20,23 This raises challenges for clinical management, laboratory detection and infection control. Furthermore, while two recently available agents, linezolid and quinupristin-dalfopristin, appear active against hVISA, VISA, MRSA and vancomycin-resistant enterococci, resistance to linezolid has already been reported among strains of both MRSA and Enterococcus faecium.21,22,24 Thus, we may be approaching an era when there are no effective therapies for some strains of MRSA.

    Competing interests

    None declared.

    Acknowledgements

    We wish to acknowledge the invaluable assistance of the infection control practitioners and staff of the Microbiology and Nephrology departments.

    References
    1. Kucers A. Vancomycin. In: Kucers A, Crowe S, Grayson ML, Hoy J. The use of antibiotics. 5th ed. Oxford: Butterworth Heinemann, 1997: 763-790.
    2. Fekety R. Vancomycin, teicoplanin, and the streptogramins: quinupristin and dalfopristin. In: Mandell GL, Bennett JE, Dolin R, editors. Principles and practice of infectious diseases. 5th ed. Philadelphia: Churchill Livingstone, 2000: 382-392.
    3. Turnidge JD, Bell JM. Methicillin-resistant Staphylococcus aureus evolution in Australia over 35 years. Microb Drug Resist 2000; 6: 223-229.
    4. Gottlieb T, Mitchell D. The independent evolution of resistance to ciprofloxacin, rifampicin and fusidic acid in methicillin-resistant Staphylococcus aureus in Australian teaching hospitals (1990-1995). Australian Group for Antimicrobial Resistance (AGAR). J Antimicrob Chemother 1998; 42: 67-73.
    5. Hiramatsu K, Hanaki H, Ino T, et al. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J Antimicrob Chemother 1997; 40: 135-136.
    6. Hiramatsu K. The emergence of Staphylococcus aureus with reduced susceptibility to vancomycin in Japan. Am J Med 1998; 104 Suppl 5A: 7S-10S.
    7. Tenover FC, Biddle JW, Lancaster MV. Increasing resistance to vancomycin and other glycopeptides in Staphylococcus aureus. Emerg Infect Dis 2001; 7: 327-332.
    8. Howe RA, Wootton M, Walsh TR, et al. Heterogeneous resistance to vancomycin in Staphylococcus aureus. J Antimicrob Chemother 2000; l45: 130-132.
    9. Trakulsomboon S, Danchaivijitr S, Rongrungruang Y, et al. First report of methicillin-resistant Staphylococcus aureus with reduced susceptibility to vancomycin in Thailand. J Clin Microbiol 2001; 39: 591-595.
    10. Wong SS, Ho PL, Woo PC, Yuen KY. Bacteremia caused by staphylococci with inducible vancomycin heteroresistance. Clin Infect Dis 1999; 29: 760-767.
    11. Walsh TR, Bolmstrom A, Qwarnstrom A, et al. Evaluation of current methods for detection of staphylococci with reduced susceptibility to glycopeptides. J Clin Microbiol 2001; 39: 2439-2444.
    12. Fridkin SK. Vancomycin-intermediate and -resistant Staphylococcus aureus: what the infectious disease specialist needs to know. Clin Infect Dis 2001; 32: 108-115.
    13. Wootton M, Howe RA, Hillman R, et al. A modified population analysis profile (PAP) method to detect hetero-resistance to vancomycin in Staphylococcus aureus in a UK hospital. J Antimicrob Chemother 2001; 47: 399-403.
    14. Centers for Disease Control and Prevention. Staphylococcus aureus with reduced susceptibility to vancomycin-Illinois, 1999. MMWR Morb Mortal Wkly Rep 2000; 48: 1165-1167.
    15. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Supplement M100 S11. Wayne, Pa: The Committee, 2001.
    16. Geisel R, Schmitz FJ, Thomas L, et al. Emergence of heterogeneous intermediate vancomycin resistance in Staphylococcus aureus isolates in the Dusseldorf area. J Antimicrob Chemother 1999; 43: 846-848.
    17. Hiramatsu K, Aritaka N, Hanaki H, et al. Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 1997; 350: 1670-1673.
    18. Levine DP, Fromm BS, Reddy BR. Slow response to vancomycin or vancomycin plus rifampin in methicillin-resistant Staphylococcus aureus endocarditis. Ann Intern Med 1991; 115: 674-680.
    19. Wood CA, Wisniewski RM. Beta-lactams versus glycopeptides in treatment of subcutaneous abscesses infected with Staphylococcus aureus. Antimicrob Agents Chemother 1994; 38: 1023-1026.
    20. Small PM, Chambers HF. Vancomycin for Staphylococcus aureus endocarditis in intravenous drug users. Antimicrob Agents Chemother 1990; 34: 1227-1231.
    21. Drew RH, Perfect JR, Srinath L, et al. Treatment of methicillin-resistant Staphylococcus aureus infections with quinupristin-dalfopristin in patients intolerant of or failing prior therapy. J Antimicrob Chemother 2000; 46: 775-784.
    22. Prystowsky J, Siddiqui F, Chosay J, et al. Resistance to linezolid: characterization of mutations in rRNA and comparison of their occurrences in vancomycin-resistant enterococci. Antimicrob Agents Chemother 2001; 45: 2154-2156.
    23. Hanaki H, Kuwahara-Arai K, Boyle-Vavra S, et al. Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50. J Antimicrob Chemother 1998; 42: 199-209.
    24. Tsiodras S, Gold HS, Sakoulas G, et al. Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet 2001; 358: 207-208.

    (Received 10 Sep, accepted 26 Sep, 2001)

    Authors' details

    Austin and Repatriation Medical Centre, Melbourne, VIC.
    Peter B Ward, BAppSc, PhD, Senior Scientist, Microbiology Department;
    Paul D R Johnson, FRACP, PhD, Deputy Director, Infectious Diseases Department,
    and Associate Professor, Department of Medicine, University of Melbourne, VIC;
    Elizabeth A Grabsch, BSc, MPH, Infection Control Scientist, Microbiology Department;
    Barrie C Mayall, FRACP, FRCPA, Medical Microbiologist, Microbiology Department;
    M Lindsay Grayson, FRACP, FAFPHM, MD, Director, and Professor,
    Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, VIC,
    and Department of Medicine, University of Melbourne, Melbourne, VIC.

    Reprints: Dr P B Ward, Microbiology Department,
    Austin and Repatriation Medical Centre, Studley Road, Heidelberg, VIC 3084.
    Peter.WardATarmc.org.au


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    1: Glossary (adapted from references 7 and 12)

    MRSA: Methicillin-resistant Staphylococcus aureus.
    An isolate of S. aureus, resistant to methicillin, with minimum inhibitory concentration (MIC) to vancomcyin ≤2mg/L. MRSA does not produce vancomycin-resistant subpopulations during routine laboratory susceptibility tests.

    hVISA: Heteroresistant vancomycin-intermediate S. aureus.
    An isolate of MRSA which produces subpopulations with vancomycin MICs ≥4mg/L, typically at a rate of 1:105 to 1:106 resistant:sensitive colonies. Antibiotic-resistance detection methods that use large inocula, such as E test, are needed to screen for hVISA.

    VISA: Vancomycin-intermediate S. aureus.
    An isolate of MRSA which produces colonies with vancomycin MICs of 8-16mg/L at high frequency, and is detectable as "intermediate resistant" using standard low-inocula susceptibility tests.

    VRSA: Vancomycin-resistant S. aureus.
    An isolate of MRSA which produces populations of colonies with vancomycin MICs >32mg/L at high frequency.
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    2: Number of colonies resistant to defined concentrations of vancomycin and teicoplanin among subpopulations of a Staphylococcus aureus strain, AR2, isolated from the index patient. A range of inocula (103-109) were used to measure viable subpopulations at each antibiotic concentration.

    Table 2
    table 2

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    • Peter B Ward
    • Elizabeth A Grabsch
    • Barrie C Mayall



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