JAC Advance Access originally published online on May 13, 2008
Journal of Antimicrobial Chemotherapy 2008 62(3):514-517; doi:10.1093/jac/dkn208
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Original research |
Longitudinal analysis of chlorhexidine susceptibilities of nosocomial methicillin-resistant Staphylococcus aureus isolates at a teaching hospital in Taiwan
1 Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan 2 Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan 3 Graduate Institute of Clinical Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
* Correspondence address. Department of Internal Medicine, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei 100, Taiwan. Tel: +886-2-23123456 ext. 5401; Fax: +886-2-23958721; E-mail: changsc{at}ntu.edu.tw
Received 10 October 2007; returned 18 January 2008; revised 8 April 2008; accepted 23 April 2008
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Abstract |
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Background: Chlorhexidine has been widely used for hand hygiene to prevent transmission of nosocomial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). However, data on longitudinal surveillance of the susceptibility of MRSA isolates to chlorhexidine are limited.
Methods: A total of 240 nosocomial MRSA isolates obtained in 1990, 1995, 2000 and 2005 at National Taiwan University Hospital (NTUH), a hospital where chlorhexidine gluconate was used for hand hygiene for more than 20 years, were included in the study. Chlorhexidine susceptibility, molecular typing using multilocus sequence typing and distribution of the qacA/B gene of these MRSA isolates were studied.
Results: The proportion of tested MRSA with a high MIC of chlorhexidine (
4 mg/L) was 1.7% in 1990, 50% in 1995, 40% in 2000 and 46.7% in 2005. Among these 83 isolates with high chlorhexidine MICs, 55.4% carried the qacA/B gene. MRSA isolates carrying the qacA/B gene were first detected in 1995 and belonged to a single clone at that time. However, the qacA/B gene was detected in MRSA isolates belonging to seven different clones in 2005.
Conclusions: The proportion of tested MRSA isolates with high chlorhexidine MICs at NTUH increased from 1990 to 1995 and remained steady thereafter. The presence of the qacA/B gene may contribute to the spread of specific MRSA clones.
Keywords: MRSA , multilocus sequence typing , qacA/B , molecular typing
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Introduction |
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The nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infection rate at National Taiwan University Hospital (NTUH) increased rapidly during the 1990s, and the prevalence of methicillin resistance among S. aureus isolates causing nosocomial infections at NTUH has remained at 60% to 80% during the past 10 years.1 One of the major mechanisms that spreads MRSA rapidly across the hospital is transfer of MRSA among patients via the contaminated hands of healthcare workers (HCWs).2 To prevent this, Hibscrub (Zeneca Ltd Co., Macclesfield Cheshire, England, UK), containing chlorhexidine gluconate (4% w/v), has been used for hand wash for more than 20 years at NTUH. Chlorhexidine resistance can emerge among S. aureus isolates, especially MRSA.3
Resistance of S. aureus to chlorhexidine is conferred by two gene families, qacA/B and smr.4 The qacA/B gene confers high-level resistance to antiseptics, whereas the smr gene confers low-level resistance. The current study aimed to understand the changes in susceptibility to chlorhexidine as well as the proportion of MRSA isolates carrying the qacA/B gene at NTUH, where a high prevalence of MRSA nosocomial infections and long-term chlorhexidine use were present.
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Materials and methods |
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Six isolates in 1990 and 60 randomly selected isolates each in 1995, 2000 and 2005 from MRSA isolates causing nosocomial bloodstream infections at NTUH, a 2500 bed hospital in Taiwan, were enrolled first (only six nosocomial bloodstream infections in total in 1990). Because of the limited number of blood isolates in 1990, 54 nosocomial MRSA isolates from other clinical specimens in 1990 were also included (only 63 nosocomial MRSA isolates in total in 1990). The total number of nosocomial blood S. aureus isolates in 1990, 1995, 2000 and 2005 at NTUH was 596. The total number of nosocomial blood MRSA isolates in 1990, 1995, 2000 and 2005 at NTUH was 388.
The chlorhexidine MIC was determined using the agar dilution method.5 The chlorhexidine MIC for MRSA isolates carrying the qacA/B gene is typically 4 mg/L according to previous reports.6 Therefore, an isolate with a high chlorhexidine MIC was defined in this study as having a chlorhexidine MIC 4 mg/L.
The detection of qacA/B gene was performed by the PCR method.4 Because of the high sequence similarity between qacA and qacB genes, the PCR products were further sequenced.
All 240 MRSA isolates underwent molecular typing, using multilocus sequence typing and susceptibility tests to erythromycin, clindamycin, gentamicin, minocycline, trimethoprim/sulfamethoxazole and vancomycin, using disc diffusion methods.7
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Results and discussion |
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The chlorhexidine MIC ranges of MRSA isolates collected in 1990, 1995, 2000 and 2005 were 1–4, 0.5–8, 1–8 and 1–16 mg/L, respectively (for the six blood isolates in 1990, the MIC range was 0.5–2 mg/L) and the MIC90s were 2, 4, 8 and 8 mg/L, respectively. The proportion of tested MRSA isolates with high chlorhexidine MICs (
4 mg/L) increased markedly from 1.7% in 1990 to 50% in 1995. After 1995, the proportion stabilized (40% in 2000 and 46.7% in 2005) (testing for heterogeneity of frequencies: with all four time points, P = 0.003; with only 1995–2005, P = 0.54). A total of 83 isolates (34.6%) expressed high chlorhexidine MICs. The distribution of the sequence types of the tested isolates is listed in Table 1. Proportionately, ST254 was the predominant clone at NTUH in 1990 (accounting for 45%), but decreased in 1995 and thereafter. ST30 MRSA isolates accounted for a significant portion (36.7%) in 1990, but were absent after 1995. Of six blood isolates in 1990, four belonged to ST254 and two belonged to ST239. MRSA isolates of ST241 were predominant (55%) only in 1995. ST239 MRSA isolates were persistently noted during the study period and predominated (55%) in 2000. ST59 MRSA isolates were noted in 1995, increased in proportion thereafter and reached predominance (48.3%) in 2005. In total, 33.8% of the 240 isolates belonged to ST239, 20.4% belonged to ST59, 16.3% belonged to ST241, 15.4% belonged to ST254 and 9.2% belonged to ST30. The others belonged to six other minor sequence types.
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The qacA/B gene was not detected in 1990, but was detected in 16 isolates (26.7%) from 1995, 21 isolates (35%) from 2000 and 20 isolates (33.3%) from 2005. Nucleotide sequence determinations revealed that all 16 isolates from 1995, 20 isolates from 2000 and 18 isolates from 2005 had the qacA gene, while 1 from 2000 and 2 from 2005 had the qacB gene.
The relationships between sequence types and chlorhexidine susceptibility, sequence types and qacA/B gene distribution, as well as qacA/B gene and chlorhexidine MIC are also shown in Table 1. In 1990, only one ST239 MRSA isolate expressed high chlorhexidine MIC. In 1995, 1 ST59 isolate and 29 ST241 isolates expressed high chlorhexidine MICs. In 2000, 2 ST5, 3 ST59, 15 ST239, 2 ST241 and 2 ST254 isolates had high chlorhexidine MICs. In 2005, 1 ST1, 3 ST5, 1 ST8, 6 ST59, 16 ST239 and 1 ST594 isolates had high chlorhexidine MICs.
Among the 54 MRSA isolates carrying the qacA gene, 31 belonged to ST239, 19 belonged to ST241, 2 belonged to ST59, 1 belonged to ST1 and 1 belonged to ST594. In 1995, all MRSA isolates carrying qacA/B genes belonged to ST241. ST239 MRSA isolates did not carry the qacA/B gene until 2000, despite the fact that they had been noted to circulate at NTUH since 1990. ST59 isolates had circulated at NTUH since 1995, but carried the qacA/B gene only after 2000. Among the three isolates carrying the qacB gene, two belonged to ST5 and the other one belonged to ST8.
All MRSA isolates carrying the qacA/B gene had chlorhexidine MICs 2 mg/L and 80.7% (46/57) expressed the defined ‘high’ MIC. For those MRSA isolates devoid of qacA/B genes. only 20.2% (37/183) had high MICs (P < 0.0001, 
2 test).
Antibiotic susceptibilities are listed in Table 2. Isolates from 1990 were more susceptible to minocycline, trimethoprim/sulfamethoxazole and ciprofloxacin than those from 1995, 2000 and 2005. Isolates of ST254 were more susceptible than those of ST239 and ST241 to trimethoprim/sulfamethoxazole and ciprofloxacin. Isolates of ST59 were more susceptible than those of ST239, ST241 and ST254 to all tested antibiotics except erythromycin and clindamycin.
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Previous reports demonstrated that MRSA isolates carrying the qacA/B gene usually expressed chlorhexidine MICs
4 mg/L.6 Consistent with this, we observed that 80.7% of the MRSA isolates carrying the qacA/B gene demonstrated chlorhexidine MICs matching or above the 4 mg/L benchmark. However, among MRSA isolates that exhibited high chlorhexidine MICs, only 55.4% carried the qacA/B gene. In the current study, the smr gene was not screened. The proportions of MRSA isolates carrying smr genes without the qacA/B gene were quite low among Asian countries except India.8 The study by Noguchi et al.8 also demonstrated that about one-third of the MRSA isolates expressing high chlorhexidine MICs did not harbour the qacA/B gene suggesting other mechanisms can contribute to chlorhexidine resistance. We found that ST241 isolates were the first clone to carry the qacA/B gene in MRSA isolates causing nosocomial bloodstream infections at NTUH during the study period. ST241 isolates were also more resistant to trimethoprim/sulfamethoxazole and ciprofloxacin than ST254 isolates. During this time period, the amount of prescribed fluoroquinolones at NTUH increased 3-fold.7 These factors may have led to ST241 replacing the predominance of ST254. In addition, MRSA isolates belonging to ST239 were found since 1990 and predominated in 2000. However, they did not carry qacA/B genes till 2000. After acquiring the qacA/B gene, most ST239 isolates expressed high chlorhexidine MICs. The resistance to other antibiotics was similar among ST239 and ST241 isolates. The resistance to chlorhexidine may again facilitate the spread of ST239 isolates at NTUH and lead to its predominance in 2000.
ST59 MRSA isolates have been found to be present in our hospital since 1995 and predominated in 2005. ST59 MRSA isolates usually were not multidrug-resistant, did not harbour the qacA/B gene and did not express high chlorhexidine MICs. However, they were identified as the predominant clone causing community-associated MRSA (CA-MRSA) infections in Taiwan.9 Their predominance in 2005 may represent the emergence of CA-MRSA isolates in the hospital environment and the purported superior fitness (other than resistance to antibacterials) of CA-MRSA over hospital-associated MRSA.
During the study period, increasing numbers of MRSA clones carrying the qacA/B gene were noted. This change is also worthy of continuous monitoring.
In conclusion, the present study demonstrated that the proportion of MRSA isolates with high chlorhexidine MICs at NTUH increased from 1990 to 1995 and remained steady thereafter. More than half (55.4%) of the isolates with high chlorhexidine MICs harboured the qacA/B gene, and it is presumable that the presence of these genes may contribute to the spread of specific MRSA clones.
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Funding |
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This study was funded by the National Science Council, Taiwan (NSC-94-2314-B-002-163).
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None to declare.
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References |
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6 Alam MM, Kobayashi N, Uehara N, et al. Analysis on distribution and genomic diversity of high-level antiseptic resistance genes qacA and qacB in human clinical isolates of Staphylococcus aureus. Microb Drug Resist (2003) 9:109–21.[CrossRef][Web of Science][Medline]
7 Wang JT, Fang CT, Chen YC, et al. Staphyloccocal cassette chromosome mec in MRSA, Taiwan. Emerg Infect Dis (2007) 13:494–7.[Web of Science][Medline]
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Noguchi N, Suwa J, Narui K, et al. Susceptibilities to antiseptic agents and distribution of antiseptic resistance genes qacA/B and smr of methicillin-resistant Staphylococcus aureus isolated in Asia during 1998 and 1999. J Med Microbiol (2005) 54:557–65.
9 Huang YH, Tseng SP, Hu JM, et al. Clonal spread of SCCmec type IV methicillin-resistant Staphylococcus aureus between community and hospital. Clin Microbiol Infect (2007) 13:717–24.[CrossRef][Medline]
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