Occurrence of Methicillin-Resistant Staphylococcus aureus with Reduced Susceptibility to Vancomycin in Srinagarind Hospital

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1 Original Article Occurrence of Methicillin-Resistant Staphylococcus aureus with Reduced Susceptibility to Vancomycin in Srinagarind Hospital Aroonlug Lulitanond, M.Sc. 1,3 Aroonwadee Chanawong, Ph.D. 1,3 Pipat Sribenjalux, M.Sc. 1,3 Wanlop Kaewkes, M.D. 2 Nicha Charoensri, Ph.D. 1,3 Danaisak Leumsai, B.Sc. 1 Pensri Monpou, B.Sc. 1 ABSTRACT We investigated the prevalence of heterogeneous vancomycin-intermediate Staphylococcus aureus (hvisa) in methicillin-resistant Staphylococcus aureus (MRSA) from patients in Srinagarind Hospital, Khon Kaen, Thailand by screening with a one-point population analysis. The suspected isolates were then confirmed by a population analysis profile. Of the 246 MRSA isolated from patients between June and December 2003, 20 isolates were positive when screened by the one-point population analysis. However, only 7 of the 20 isolates showed an hvisa population analysis profile curve. It was also observed that the 7 isolates were susceptible to vancomycin when tested by either the disk diffusion or agar dilution test: minimal inhibitory concentrations ranged between 2 and 3 µg/ml. These findings suggest a surveillance program for hvisa and vancomycin-resistant S. aureus in hospitals where the prevalence of MRSA is high. (J Infect Dis Antimicrob Agents 2005;22:9-14.) INTRODUCTION these organisms. However, the first S. aureus with an For more than four decades, methicillin-resistant intermediate-level resistance to vancomycin (VISA) Staphylococcus aureus (MRSA) has caused serious was reported in Japan in Since then, VISA has hospital infections worldwide. 1,2 Vancomycin has long been been reserved as the drug of choice for infection with reported around the globe. 4-8 These isolates display 1 Department of Clinical Microbiology, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. 2 Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. 3 Center for Research and Development in Medical Diagnostic Laboratory, Khon Kaen University, Khon Kaen 40002, Thailand. Received for publication: January 17, Reprint request: Aroonlug Lulitanond, M.Sc., Department of Clinical Microbiology, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. Keywords: MRSA, h-visa, Staphylococcus aureus, vancomycin, antimicrobial resistance, surveillance 9

2 10 J INFECT DIS ANTIMICROB AGENTS Jan.-Apr a heterogeneous phenotype (hvisa). Although their overall minimal inhibitory concentrations (MICs) are < 4 mg/ml, subpopulations resistant to higher vancomycin concentrations occurred at a minimum frequency These strains were of clinical importance since failure treatment with vancomycin has been reported. 8,11,12 The first VISAs in Thailand were reported in 2001 in three patients who did not respond to vancomycin treatment at Siriraj Hospital. 13 The hvisa isolates could not be detected using routine clinical microbiological laboratory methods, such as disk diffusion or MIC determination. Since the MRSA infection rate at Srinagarind Hospital is around percent of all coagulase-positive Staphylococcus infections, vancomycin therapy cannot be avoided. Surveillance of hvisa in this hospital must therefore be performed for early detection and control planning. We screened 246 MRSA isolates for hvisa using a one-point population analysis (OPA), then confirmed by a population analysis profile (PAP) as described by Hiramatsu et al. 11 MATERIALS AND METHODS Bacterial isolates The bacterial isolates used were 246 clinically significant MRSA recovered from patients attended at Srinagarind Hospital, Khon Kaen, Thailand between June and December S. aureus ATCC was used as the control strain for MIC determination. The reference strains of S. aureus Mu50 (VISA) and S. aureus Mu3 (hvisa) were provided by Professor Malai Vorachit and Ms. Piriyaporn Chongtrakool at Ramathibodi Hospital, Bangkok. All of the studied isolates were identified by conventional biochemical tests, and stored in skim milk with glycerol at -70 C. Culture media were prepared according to the recommendations of the manufacturer, including Brain Heart Infusion (BHI) broth, Brain Heart Infusion agar (BHIA) and Mueller Hinton agar (MHA) (BBL, USA). Vancomycin powder was purchased from Sigma (Switzerland). OPA The screening for hvisa by OPA required a 100-µL aliquot of bacterial suspension, adjusted to 0.5 McFarland standard (absorbance at 625 nm is ~ ), was spread on a BHIA plate with 4 µg/ml of vancomycin. The plates were then incubated at 37 C and the 48-h-growth was recorded. Any isolate yielding growth within 24 h was considered VISA while any that grew during the h incubation, hvisa. All of the isolates that grew were subjected to a further confirmation test. PAP The population analysis profile (PAP) required a 100-µL aliquot of each 10-fold dilution of the bacterial suspension, adjusted to 0.5 McFarland standard. This was spread on each plate of BHIA supplemented with 0, 1, 2, 3, 4, 5, 6, 7 and 8 µg/ml of vancomycin. The plates were incubated at 37 C for 48 h. The colony-forming unit (CFU) per ml of each isolate was calculated and plotted on a semi-logarithmic graph. Agar dilution The MIC of vancomycin for all isolates with a positive result from the OPA, and the other 13 isolates with a negative result, were determined by the agar dilution method according to the National Committee for Clinical Laboratory Standards (NCCLS) of the United States. 14 RESULTS Of the 246 MRSA isolates, twenty gave positive results by OPA. The growth of 12 isolates was detected after a 24-h incubation and the other eight, after 48 h. Only seven of the 20 isolates had hvisa curves from the PAP vs. the reference strains (Figure 1) whereas the

3 Vol. 22 No. 1 Occurrence of hvisa in Srinagarind Hospital:- Lulitanond A, et al. other 13 showed VSSA curves (Figure 2). The results of OPA, PAP and the types of specimens are presented in Table 1. Based on the NCCLS criteria (MIC < 4 µg/ml), the MIC results revealed that all of the isolates were susceptible to vancomycin. The MIC 50 and MIC 90 of vancomycin for the seven hvisa isolates was 3 and 3 µg/ml, respectively, whereas those of the VSSA isolates (N = 26) were 1 and 2 µg/ml, respectively (Table 2). DISCUSSION The OPA is a screening method for hvisa. In our study, 13 isolates yielded false positives (5.3%), slightly less than that (10.3%) reported by Walsh et al. 15 Therefore, the positive isolates from the OPA test Figure 1. Population analysis profile of the three control and seven MRSA strains which showed a hvisa pattern. Figure 2. Population analysis profile of six MRSA isolates compared with that of S. aureus ATCC All isolates show the VSSA pattern.

4 12 J INFECT DIS ANTIMICROB AGENTS Jan.-Apr Table 1. Types of specimens for MRSA isolates and results of one-point population analysis and population analysis profile. Specimens No. of MRSA OPA* test positive after PAP* test positive after isolates 24 h 48 h 24 h 48 h Blood Urine Pus Body fluid Sputum Total OPA: one-point population analysis, PAP: population analysis profile Table 2. MICs of vancomycin for hvisa and VSSA. Organisms (n) Numbers of isolates yielding various MICs (µg/ml) MIC 50 MIC 90 hvisa (7) VSSA (26) should always be confirmed by a PAP test. The PAP has been proposed as a reference method for confirmation of hvisa but implementation in clinical microbiology laboratories is problematic. Therefore, other tests for assessing hvisa have recently been proposed such as the E-test MIC of vancomycin, compared to that of teicoplanin We found seven of the 246 MRSA isolates were hvisa and susceptible to vancomycin by both disk diffusion (data not shown) and agar dilution methods. All of the seven isolates were from six hospitalized patients (three females and three males); three isolates were from two children and four from adults. Four of the isolates were from two Medical Wards, two from the same patient in the Neonatal Intensive Care Unit, and one from the Rehabilitation Ward. Three of the seven isolates were from patients treated with vancomycin. This is the first warning for Srinagarind Hospital that MRSA, which is an important hospital pathogen, will become a problem vis-à-vis antimicrobial therapy. In future, patients infected with these organisms may not be successfully treated with vancomycin. Most strains of hvisa were isolated after longterm treatment with vancomycin. 3,13,19 However, Whitener et al 20 reported a VRSA strain isolated from a patient not exposed to vancomycin for five years, implying that VRSA is becoming prevalent even without vancomycin selective pressure. 21 We should therefore be prepared to encounter hvisa and VRSA in MRSAinfected patients. ACKNOWLEDGEMENT This study was supported by Research Funds from Khon Kaen University. We thank the Faculty of Associated Medical Sciences and CMDL, Khon Kaen University, for support, and the staff of the Clinical Microbiology Laboratory at Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, for collecting the clinical isolates. We thank Mr. Bryan Roderick Hamman for assistance with the English-language presentation.

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