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Methicillin resistant Staphylococcus aureus (MRSA) and the means of controlling it, continue to be of major interest to the healthcare community. Antiseptics are increasingly being used as part of intervention programs to prevent MRSA transmission.
The susceptibility of 60 methicillin-resistant Staphylococcus aureus (MRSA) isolated from Malaysia towards three antiseptics agents; Benzalkonium chloride (BAC) and benzethonium chloride (BZT) and Chlorhexidine digluconate (CHG) were determined by broth microdilution method. The Minimum Inhibitory Concentration (MIC) for all antiseptics was comparable and ranging from 0.5-2µg/ml all isolates were susceptible for all tested antiseptic agents.
All strains were examined concerning the presence of antiseptic resistant genes qacA/B and smr. By PCR, the qacA/B and smr were found in 82.8% and 2.6% respectively. It is the first time that qacA/B and smr genes are reported in Malaysia with a high rate for qacA/B gene. The presence of these antimicrobial resistance determinants is a serious concern. It is important to determine the susceptibility of clinical MRSA to various biocides to assess the control and preventive measures currently implemented in hospitals and the surveillance of antiseptic-resistant MRSA could provide important information on the control of nosocomial infection.
Methicillin-resintant Staphylococcus aureus (MRSA) is a major nosocomial pathogen causing a wide range of infections ranging from localized skin conditions to life-threatening conditions such as pneumonia and endocarditis (DeMarco et al. 2007).
The emergence of this pathogen is a great concern in hospital environments for infection control team and use of biocides including antiseptics is essential in infection control practices in hospital and other health care settings in order to prevent its infections and spreading.
Antiseptics are anti-infective substances that, after topical administration, destroy or inhibit the growth of microorganisms in or on living tissue (skin, mucous membrane and wound). Antiseptics are applied externally and, to prevent the development of biocide resistance, they are used at concentrations considerably higher than minimal bactericidal concentrations (Muller and Kramer 2008)
A wide variety of chemical agents including Quaternary ammonium compounds (QACs) such as Benzalkonium chloride (BAC) and benzethonium chloride (BZT), cationic biocides such as chlorhexidine digluconate (CHG) are used widely in antiseptic preparations in healthcare settings.
BAC and BZT are synthetic quaternary ammonium compounds exhibits a broad spectrum of microbiocidal activity against bacteria bacteria, fungi and viruses. CHG is a biguanidine derivative and one of the most widely used biocides for antiseptic purposes. CHG is a bactericidal agent with broad-spectrum activity and low skin irritation. CHG disrupts the outer cell membrane and attacks the bacterial cytoplasm (Karami, Alsterholm and Faergemann 2009).
Unfortunately, Overuse of antiseptic agents by various health settings has led to the emergence of MRSA with decreased antiseptic susceptibility or antiseptic-resistant MRSA (Wang et al. 2008a). At least 12 antiseptic resistance genes (qacA to qacJ, smr, and norA) have been identified in Staphylococcus species. Four antiseptic resistance genes, qacA, qacB, smr, and norA, are found mainly in clinical isolates of S. aureus (Noguchi et al. 2006). It has been reported that two genes, qacA and qacB, confer high-level resistance to antiseptics, and smr, confer low-level resistance due to the energy-dependent drug efflux mechanisms. (Sekiguchi et al. 2004)
The qacB gene is closely related to the qacA gene and Analysis of nucleotide sequences indicates that qacA and qacB differs at the nucleotide level by seven or nine nucleotides. Therefore, the simple PCRs cannot discriminate between them , and qacA and qacB are considered to be the same and designated as qacA/B gene. smr (staphylococcal multidrug resistance, also known as qacC/D) which is identical to the qacD and ebr genes, encodes a small protein that belongs to a small multidrug resistance family and confers resistance to quaternary ammonium compounds and ethidium bromide.
The multidrug resistance gene qacA/B is encoded on multiresistance plasmids from clinical isolates of S. aureus and confers resistance to a wide range of antimicrobial organic cations, including Qacs, diamidines, biguanidines, and guanylhydrazones (Bernadette A. Mitchell 1998)
Compared to qacA/B and smr, the norA is considered has a very low contribution to the resistance of cationic antiseptic agents. The qacA/B gene has been reported to be associated with high-level resistance to antiseptic agents and to be widely prevalent among MRSA isolates found in Europe and Asia (2, 24, 32, 35). Therefore, the qacA/B and smr genes have become a major antiseptic resistance gene in MRSA. (Noguchi et al. 2006)
At present, biocides are an integral and essential component of clinical medicine practice and the emergence of antisepticresistant MRSA strains is likely to pose a major challenge to hospital infection control teams. Therefore, it is important for infection control team to know the susceptibilities of MRSA to various biocides.
Based on the scare data available about MRSA resistance for antiseptics in Malaysia, the aim of this study was to assess the efficacy of three different antiseptics agents and to investigate the prevalence of antiseptic resistance genes qacA/B and smr determinants in clinical isolates of MRSA in Malaysia.
Material and Methodology:
Sixty of non-repetitive clinical isolates identified as MRSA were collected from the clinical laboratory of the tertiary hospital in Kuala Lampur from February to May 2009. They were isolated from pus (19), tracheal aspirate (9), blood (8), nasal swab (8), abscess (6), sputum(5),tissue (4) and wounds (1). Only 1 isolate was included per patient.
All antiseptics benzalkonium chloride (BAC), benzethonium chloride (BZT) and Chlorhexidine digluconate (CHG) were purchased from Sigma Aldrich,
Stock of antiseptics containing 100µg/ml of BAC and BZT in deionized water was prepared and stored at 4°C. CHX was used as manufacturer concentration 20µg/ml. Additional dilutions were prepared in Muller Hinton Broth before each experiment as required.
Minimum inhibitory concentration determination:
MICs of the isolates towards several antiseptics were determined by broth microdilution method according to CLSI standards (CLSI, 2009). The growth resulting from the plates with MRSA was scraped with a platinum loop, and diluted in Mueller Hinton Broth (MHB; Merck, Germany) to an absorbance of 0.5 at 560 nm (Å560 = 0.5).
Working solutions of antiseptics were made by serial assay at 1µg/ml intervals from 0 to 6 µg/ml for BAC and BZT. For CHX the interval was 0.5 µg/ml from 0 to 4 µg/ml. MRSA cultures at 0.5 McFarland concentration were added into each tube containing the antiseptics at different concentrations (0 to 10 µg/ml for BAC and BZT and from 0 to 4 µg/ml for CHX) and incubated overnight at 37°C with constant shaking on the shaking incubator. Then, the incubated mixtures were plated out onto Mueller-Hinton agar (Merck, Germany) plates and incubated at 37°C for 24 h. The bacterial colonies that grew on each of the agar plates were counted. Plates containing no antiseptics were used as positive control. The MIC was defined as the lowest concentration of antiseptics that prevented visible bacterial growth after incubation at 37°C for 24 h. (do Amorim, Aun and Mayer 2004). Susceptibility tests for the isolates against to antiseptics were repeated two times on different days.
Total genomic DNA of all isolates in this study was extracted using GF-1 Bacterial DNA Extraction Kit (Vivantis Technologies, Malaysia), as described by the manufacturer. The MRSA strains were confirmed by detection of the mecA gene by PCR.
Searches for mecA, qacA/B, and smr were performed with sets of primers as shown in Table 2. All primers used in this study were synthesized by First Base Laboratories, Malaysia
Product size (bp)