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J Med Microbiol 54 (2005), 557-565; DOI: 10.1099/jmm.0.45902-0
© 2005 Society for General Microbiology
ISSN 0022-2615

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

Norihisa Noguchi1, Junichi Suwa1, Koji Narui1, Masanori Sasatsu1, Teruyo Ito2, Keiichi Hiramatsu2 and Jae-Hoon Song3

1Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan 2Department of Bacteriology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku Tokyo 113-8421, Japan 3Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University, 50 Ilwon-dong, Kangnam-ku Seoul 135-710, Korea

Correspondence Norihisa Noguchi noguchin{at}ps.toyaku.ac.jp

Received September 21, 2004
Accepted February 17, 2005

Many antiseptic agents are used in hygienic handwashes in the prevention of nosocomial infections by methicillin-resistant Staphylococcus aureus (MRSA). The plasmid-borne genes qacA/B and smr confer resistance to cationic antiseptic agents in S. aureus. In this study, the susceptibilities for dyes and antiseptic agents (e.g. acriflavine, acrinol, benzalkonium chloride, benzethonium chloride, chlorhexidine digluconate and alkyldiaminoethylglycine hydrochloride) of 894 isolates of MRSA collected from 11 Asian countries (South Korea, China, the Philippines, Singapore, Vietnam, Thailand, Indonesia, India, Sri Lanka, Saudi Arabia and Japan) between 1998 and 1999 were examined. In addition, the distributions of the antiseptic-resistance genes qacA/B and smr were studied by PCR. Among the Asian MRSA isolates 57.7 % (516/894) were acriflavine-resistant. The smr gene was detected in 31.6 % (12/38) of MRSA isolates from India but only in 1.9 % (16/856) of all the isolates from other Asian countries. MRSA with qacA/B comprised 41.6 % (372/894) of the isolates across Asia. In addition, PFGE was performed to type the MRSA and grouped the tested 30 MRSA isolates with qacA/B into 21 PFGE types. The results indicated that qacA/B is functionally the most important gene mediating antiseptic resistance in the MRSA strains of Asia and that a specific MRSA with qacA/B was not prevalent in Asia but qacA/B were widely spread among MRSA of Asia, while the geographical distribution of smr is more limited.

Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; QAC, quaternary ammonium compounds.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen. Hand hygiene has been considered to be the important tool in control of nosocomial infection (Nystrom, 1994). Many antiseptic agents are used in hygienic handwashes (Boyce & Pittet, 2002). Antiseptic agents include various compounds with different chemical structures such as dyes, alcohols and surfactants (McDonnell & Russell, 1999). MRSA with decreased antiseptic susceptibility have been isolated from clinical samples and settings (Noguchi et al., 1999; Suller & Russell, 1999). However, it is difficult to clearly evaluate the antiseptic resistance of MRSA because the difference in the MICs for antiseptic agents between a resistant strain and a susceptible strain is small, and the MIC of antiseptic agents for antiseptic-resistant strains is less than the user concentration of the agents. Decreased susceptibility to cationic antiseptic agents, i.e. an increase in MIC, has been attributed mainly to the excretion of drugs into the extracellular space by proton motive force-dependent multidrug-transport systems (Grinius et al., 1992; Kaatz et al., 1993; Putman et al., 2000). In S. aureus, qacA (Rouch et al., 1990), qacB (Paulsen et al., 1996), smr (Grinius et al., 1992) and norA (Neyfakh et al., 1993), which encode multidrug-transporter proteins, have been identified as antiseptic-resistance genes which confer resistance to cationic antiseptic agents including dyes such as acriflavine, acrinol and ethidium bromide, quaternary ammonium compounds (QACs) such as benzethonium chloride and benzalkonium chloride, and biguanides such as chlorhexidine digluconate (Littlejohn et al., 1992; Putman et al., 2000). Therefore, an MRSA strain with decreased susceptibility to antiseptic agents mediated by an antiseptic-resistance gene is interpreted as an antiseptic-resistant strain (Lyon & Skurry, 1987).

The norA gene is located on the chromosome of S. aureus (Ng et al., 1994; Yoshida et al., 1990). Since the resistance of norA seems to be due to mutation in the transcriptional regulatory region that results in overexpression of norA mRNA (Kaatz et al., 1993; Noguchi et al., 2002), it is difficult to clearly identify norA-mediated antiseptic resistance in S. aureus by detection of the resistance gene. In comparison, the qacA, qacB and smr genes have been found mainly on plasmids (Littlejohn et al., 1991; Lyon & Skurry, 1987; Tennent et al., 1985). Except for 7 bp, the sequence of qacB is identical to that of qacA, although their resistance profiles differ slightly (Littlejohn et al., 1991; Paulsen et al., 1996). Thus, the qacA and qacB genes can not be separately amplified by simple PCR. The smr gene is identical to qacC, qacD and ebr (Grinius et al., 1992; Littlejohn et al., 1991; Sasatsu et al., 1989). The proteins encoded by qacA/B and smr belong to a major facilitator superfamily and a small multidrug-resistance family, respectively (Putman et al., 2000). Therefore, these plasmid-borne antiseptic-resistance genes can be classified structurally into two families, qacA/B and smr.

Epidemiological information on antiseptic susceptibility and the distribution of resistance genes is useful for nosocomial infection control. Although multidrug-efflux proteins have been studied extensively and have emerged as a major medical problem (Anthonisen et al., 2002; Okamoto et al., 2002; Putman et al., 2000; Russell, 2002), there is little information about antiseptic-resistant MRSA (Alam et al., 2003; Mayer et al., 2001; Noguchi et al., 1999). In the present study, we examined the sensitivities to antiseptic agents of MRSA isolates collected from 11 Asian countries during 1998 and 1999. The antiseptic-resistance genes qacA/B and smr were detected by PCR. The results were analysed with regard to geographical distribution.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Strain, antimicrobial compounds and medium.

A total of 894 MRSA isolates were collected from Japan, South Korea, China, the Philippines, Singapore, Vietnam, Thailand, Indonesia, India, Sri Lanka and Saudi Arabia between 1998 and 1999 by the Asian Network for Surveillance of Resistant Pathogens (Oh et al., 2004) (Table 1). MRSA isolates in Japan were collected by the provision of approximately 30 isolates from each of 14 hospitals. S. aureus JCM 2874 (= ATCC 29213), JCM 2413 (= ATCC 25923), JCM 2151 (= 209P) and RN2677 were used as reference strains and for quality control during susceptibility testing. TS77 and L20 strains were used as the positive controls with qacA/B and with smr, respectively, in PCR experiments (Noguchi et al., 1999). Acriflavine, acrinol, benzethonium chloride, benzalkonium chloride, chlorhexidine digluconate and alkyldiaminoethylglycine hydrochloride were purchased from Sigma-Aldrich and Wako-Pure Chemical Industries. Trypto-soy medium (Eiken Chemical) and Mueller–Hinton (MH) medium (Difco) were used for growth of S. aureus and for susceptibility testing, respectively.


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  		Table 1. Antimicrobial susceptibility of MRSA isolated from Asia between 1998 and 1999
 

Antimicrobial susceptibility testing.

MICs of antiseptic agents were determined by the agar doubling dilution method according to NCCLS guidelines (NCCLS, 2000). The range of the MIC was as follows: acriflavine and acrinol, 1–1024 µg ml–1; QACs and chlorhexidine digluconate, 1–32 µg ml–1; alkyldiaminoethylglycine hydrochloride, 2–128 µg ml–1 (Noguchi et al., 2004).

PCR.

A search for qacA/B and smr genes was performed by multiple PCR with the following sets of primers: 5'-GCAGAAAGTGCAGAGTTCG-3' and 5'-CCAGTCCAATCATGCCTG-3' for qacA/B (product size 361 bp) (Rouch et al., 1990) and 5'-GCCATAAGTACTGAAGTTATTGGA-3' and 5'-GACTACGGTTGTTAAGACTAAACCT-3' for smr (product size 195 bp) (Sasatsu et al., 1989). PCR assays were performed using the modified colony direct method of Tsuchizaki et al. (2000). Briefly, the point of a toothpick was gently placed into contact with one colony on an agar plate and then transferred into 100 µl H2O. The small sample of cells was suspended and 1 µl of the cell suspension was added directly into 25 µl of the PCR mixture containing two sets of primers and 12.5 µl PCR master mix (Promega). PCR was performed using an initial denaturation step of 96 °C for 3 min, 25 cycles of 94 °C for 20 s, 53 °C for 20 s and 72 °C for 20 s, and a final extension step at 72 °C for 5 min. PCR products were analysed by agarose gel electrophoresis. All results were confirmed by at least two independent experiments.

PFGE typing.

PFGE of SmaI-digested chromosomal DNA was performed with a CHEF Mapper system according to the instructions provided by the manufacturer (Bio-Rad). Lambda and S. aureus N315 were used as a DNA reference standard because the genome of N315 has been completely determined (Kuroda et al., 2001). The DNA patterns obtained by PFGE were analysed with BIONUMERICS software (Applied Maths), using the Dice coefficient, and by the unweighted pair group method (UPGMA) with 1.5 % tolerance and 1.5 % optimization (Murchan et al., 2003; Tenover et al., 1995).


   RESULTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The MICs of dyes and antiseptic agents for MRSA isolated from Asia between 1998 and 1999 were determined, and the MIC range and the MICs at which 50 and 90 % of isolates were inhibited (MIC50 and MIC90, respectively) by antimicrobial agents for MRSA are shown in Table 1. The dye acriflavine had the broadest MIC range while the other antiseptic agents showed a narrow MIC range. The MIC50 and MIC90 of acriflavine, QACs and chlorhexidine for MRSA isolated from Asia were higher than the MICs for the reference strains.

The distributions of MIC and the antiseptic-resistance genes qacA/B and smr were analysed in Asian isolates (Fig. 1). In the strains with qacA/B, 98.3 % (366/372) had an acriflavine MIC of >=64 µg ml–1 and the distribution of the strains with qacA/B was at the higher MICs of acriflavine, QACs and chlorhexidine. No difference in the MIC of acrinol between strains with and without qacA/B was observed. Based on the results of the distributions and the MICs of antiseptic agents for the reference strains used (Table 1), the criteria for ‘susceptible to’ and ‘intermediate to’ acriflavine in S. aureus were defined as MIC <=16 µg ml–1 and 32 µg ml–1 respectively. Among Asian MRSA isolates, 57.7 % were resistant to acriflavine. Table 2 summarizes the distribution of antiseptic-resistance genes in acriflavine-resistant strains. qacA/B alone or smr alone was detected in 38.5 % (344/894) and 3.1 % (29/894) of the Asian isolates, respectively. MRSA with both qacA/B and smr comprised an additional 3.1 % (28/894) of the isolates. When the strains with both qacA/B and smr and those with only qacA/B were considered together, the overall incidence of qacA/B increased to 41.6 % (372/894) of the Asian isolates. Among acriflavine-resistant MRSA isolates, qacA/B were found in 70.9 % (366/516), while smr alone was detected in only 2.5 % (13/516). The remaining acriflavine-resistant isolates (26.6 %) had neither qacA/B nor smr. Therefore, qacA/B were the major genes that conferred resistance to cationic antiseptic agents in MRSA isolated from Asia.



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  		Fig. 1. Distribution of antiseptic MICs for acriflavine-resistant MRSA with antiseptic-resistance genes. Acriflavine-resistant MRSA are grouped as follows: acriflavine-resistant MRSA with only qacA/B (qacA/B), both qacA/B and smr (qacA/B + smr), only smr (smr); acriflavine-resistant MRSA without qacA/B and smr (AF-R; acriflavine MIC >=64 µg ml–1); and acriflavine-susceptible and -intermediate MRSA (AF-S/I; MIC <=32 µg ml–1).

 

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  		Table 2. Distribution of acriflavine susceptibility and antiseptic-resistance genes in MRSA in Asia Values are the number (%) of strains carrying antiseptic-resistance genes.
 

The distributions of antiseptic-resistance genes in strains in each Asian country and in acriflavine-resistant MRSA are shown in Table 3. No acriflavine-resistant MRSA strains were found in Indonesia or Saudi Arabia. In agreement with this finding, neither qacA/B nor smr was found in isolates from Indonesia or Saudi Arabia. One acriflavine-resistant strain (1/15) was isolated from the Philippines and this strain carried qacA/B. The MIC90 of acriflavine in MRSA from India was 64 µg ml–1. The qacA/B genes were detected in only one strain from India while 63.6 % (7/11) of the acriflavine-resistant strains from India carried smr but not qacA/B. The remaining three acriflavine-resistant strains had neither qacA/B nor smr. Twenty-nine additional MRSA strains with only smr were isolated in Asia beyond India. These were found in Japan, Vietnam and Singapore at the following rates: 3.4 % (14/413), 2.6 % (1/15) and 1.1 % (1/87), respectively. Thus, although smr is prevalent in India, there were few MRSA isolates with smr among other Asian countries.


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  		Table 3. Distribution of antiseptic-resistance genes in MRSA isolated from Asia
 

The MIC90 of acriflavine in isolates from Japan, Korea, Thailand, Sri Lanka, Vietnam and Singapore was 1024 µg ml–1. The percentages of acriflavine-resistant MRSA with qacA/B in each country were as follows: Japan, 67.5 % (193/283); Korea, 49.3 % (34/69); Thailand, 87.9 % (29/33); Sri Lanka, 84.2 % (16/19); Vietnam, 94.1 % (16/17); and Singapore, 96.1 % (74/77). Furthermore, a total of 65 isolates were chosen from 11 Asian countries and PFGE typing was performed (Fig. 2). These 65 isolates tested were classified into 42 PFGE types. The PFGE type of antiseptic-resistant strains was different from that of antiseptic-susceptible strains in each country, while there were similarities in PFGE banding patterns between MRSA without antiseptic-resistance genes in India, Saudi Arabia, Indonesia, Vietnam, Thailand and China. Although the identical PFGE type was found in MRSA with qacA/B isolated from Singapore, China, Sri Lanka and Thailand, at least 70 % (21/30) of the tested MRSA with qacA/B were of different PFGE types, and the PFGE types of MRSA with only smr were also different.



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  		Fig. 2. Dendrogram based on PFGE SmaI restriction pattern analysis using the unweighted pair group method for 65 MRSA isolates. Antiseptic-resistance genes were detected (+) or not detected (–) by PCR. MICs are given in µg ml–1.

 

Our results indicate that qacA/B is functionally the most important gene mediating antiseptic resistance in the MRSA strains of Asia and it is distributed widely across Asia.


   DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
We examined in the present study the susceptibility to antiseptic agents and distribution of antiseptic-resistance genes of MRSA collected in Asian countries between 1998 and 1999. Since the MIC ranges of the antiseptic agents were narrow except for acriflavine, it was difficult to define the criteria for resistance to each antiseptic agent.

The qacA/B genes were found in four acriflavine-intermediate strains and two acriflavine-susceptible strains (Table 2). A minimum of two independent experiments confirmed the susceptibility to acriflavine and the presence of qacA/B. We are currently investigating how strains with qacA/B could lack resistance to acriflavine.

The percentage of acriflavine-resistant strains was similar to that of the strains with qacA/B in Thailand, Sri Lanka, Vietnam and Singapore. The results strongly suggest that the acriflavine resistance of isolates from those countries was mediated by the acquisition of qacA/B. In contrast, 17.0 % (48/283) and 37.7 % (26/69) of the acriflavine-resistant strains isolated from Japan and Korea, respectively, carried no qacA/B. Furthermore, acriflavine-resistant MRSA without qacA/B and smr totalled 26.5 % (137/516) of isolates and also exhibited low-level resistance to QACs and chlorhexidine. The result indicates that acriflavine-resistant MRSA without qacA/B and smr are not intrinsically tolerant to acriflavine. Plasmid-borne antiseptic-resistance genes qacG, qacH and qacJ, which encode efflux proteins belonging to the small multidrug-resistance family, as well as smr, were found in staphylococcal species isolated from the food industry and horses (Bjorland et al., 2003; Heir et al., 1998, 1999). In addition, two chromosomal genes, sepA and mdeA, which encode a multidrug-efflux transporter, were identified in S. aureus (Huang et al., 2004; Narui et al., 2002). Therefore, the genes other than qacA/B and smr encoding the multidrug-efflux transporter may be responsible for the antiseptic resistance of MRSA lacking qacA/B and smr.

Most strains with only smr (22/28) were susceptible or showed low-level resistance to acriflavine (MIC <=128 µg ml–1) (Fig. 1). Strains containing the smr gene exhibit resistance to QACs and ethidium bromide, but low-level or no resistance to other dyes (Littlejohn et al., 1992; Noguchi et al., 1999). These results support our data. However, 21.4 % (6/28) of the strains with only smr exhibited high-level resistance to acriflavine (Fig. 1, acriflavine MIC >=256 µg ml–1). The high-level resistance to acriflavine in the strains with only smr might be due to the mutation of a chromosomal multidrug-efflux gene(s) such as norA and the acquisition of other plasmid-borne antiseptic-resistance genes (Noguchi et al., 1999, 2002, 2004).

To study the diversity of the MRSA used, the types of 65 isolates of 894 MRSA were analysed by PFGE. At least 64.6 % (42/65) of the MRSA tested were of a different PFGE type. Although MRSA isolated from Japan, South Korea and the Philippines had different PFGE types, strains having identical PFGE types were found from other Asian counties. PFGE types of isolates from Japan were different from those of MRSA isolated from Southeast Asian countries, though some MRSA from South Korea were included in the PFGE group of Japan (Fig. 2). Furthermore, PFGE analysis of MRSA with qacA/B divided the 30 isolates into 21 PFGE types. In two of the four PFGE types of MRSA with qacA/B found in Singapore, one was identical to that of MRSA without qacA/B from India and another was identical to that of MRSA from Saudi Arabia and Indonesia. PFGE typing of MRSA with qacA/B indicated that a specific MRSA clone with qacA/B was not prevalent in Asia but that qacA/B were spread among MRSA.

When the antiseptic susceptibility and the distributions of antiseptic-resistance genes of MRSA isolated from Japan in 1992 were studied (Noguchi et al., 1999), 71.4 % (70/98) of MRSA exhibited resistance to acriflavine, and qacA/B and smr were detected in 10.2 % (10/98) and 20.4 %(20/98) of those MRSA, respectively. However, 7 years later, the genes qacA/B and smr were detected in 47.9 % (198/413) and 3 % (14/413), respectively, of MRSA isolates from Japan. Mayer et al. (2001) detected qacA/B and smr at rates of 62.6 % (186/297) and 6.4 % (19/297), respectively, in MRSA isolated from Europe between 1997 and 1999. The qacA/B genes are typically located on a transposon of a transmissible multidrug-resistance plasmid such as pSK1 (Lyon & Skurry, 1987). These results strongly suggest that qacA/B is spreading rapidly in MRSA throughout Asia. Therefore, surveillance of antiseptic-resistant MRSA could provide important information on the control of nosocomial infection.


   ACKNOWLEDGEMENTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
We thank Y. Namiki, T. Okihara, A. Nakamura, A. Higashi, A. Tajima, Y. Sasaki and Y. Handa for their technical assistance. This work was supported by grants for private universities provided by the Ministry of Education, Culture, Sports, Science and Technology, and by the Promotion and Mutual Aid Corporation for Private School of Japan.


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 METHODS
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 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

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