SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH ANTIBIOGRAM, ANTIBIOTIC AND DISINFECTANT RESISTANCE GENES, BIOFILM-PRODUCING AND -ASSOCIATED GENES, AND GENOTYPE OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS CLINIcaL ISOLATES FROM NORTHERN THAILAND Rathanin Seng1, Thawatchai Kitti2, Rapee Thummeepak3, Autchasai Siriprayong3, Thitipan Phukao3, Phattaraporn Kongthai3, Thanyasiri Jindayok4, Kwanjai Ketwong5, Chalermchai Boonlao6 and Sutthirat Sitthisak3,7 1Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok; 2Faculty of Oriental Medicine, Chiang Rai College, Chiang Rai; 3Department of Microbiology and Parasitology, Faculty of Medical Science, 4Division of Clinical Pathology, Faculty of Medicine, Naresuan University, Phitsanulok; 5Sawanpracharak Hospital, Amphoe Meuang, Nakhon Sawan; 6Chiang Rai Prachanukroh Hospital, Amphoe Meuang, Chiang Rai; 7Centre of Excellence in Medical Biotechnology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand Abstract. Methicillin-resistant Staphylococcus aureus (MRSA) causes a variety of infectious diseases in both hospital and community. The study determined preva- lence of antibiotic resistance and associated genes, biofilm-producing phenotype and associated genes, SCCmec types, and clonal subtype ST239 of MRSA clinical isolates obtained from three hospitals in northern Thailand during January 2013 to October 2015. Some 95% of MRSA isolates were multidrug resistant, with 82%, 60% and 47% harboring ermA, ermB and qacAB, respectively. Although all MRSA isolates were positive for slime (biofilm) production on Congo red agar, quan- titative measurement of biofilm generation using microtiter plate assay (MTP) indicated 60% were low biofilm producers, with prevalence of biofilm-associated genes, bab, cna, fnbA, and icaAD, ranging from 50% to 100%. MRSA SCCmec type III was predominant, but the presence of SCCmec type IV and type V (albeit at low frequency) indicated acquisition of community-acquired infection. Clonal subtype ST239 was detected in 29% of MRSA isolates in hospitals located in the lower and upper northern regions. The information provided by this study should be useful for future active surveillance of MRSA and in the development of the strategies to lower prevalence and to control the spread of this virulent staphylococcal infec- tion in hospitals and the community at large. Keywords: Staphylococcus aureus, antibiogram, biofilm, SCCmec type, MRSA, Thailand Correspondence: Dr Sutthirat Sitthisak, Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand. Tel: +66 (0) 55 964626, +66 (0) 84 5734203; Fax: +66 (0) 55 964770 E-mail: [email protected] 1060 Vol 49 No. 6 November 2018 PHENOTYPES AND GENOTYPES OF MRSA INTRODUCTION tion accumulation phase through form- ing homophilic interactions or through Methicillin-resistant Staphylococcus binding of proteins to surface receptors aureus (MRSA) is a gram-positive bacte- on adjacent cells (Herman-Bausier et al, rium causing infection in both commu- 2015); and collagen adhesin is a virulence nity and hospital. Most strains of MRSA factor associated with S. aureus adhesion have developed resistance to most of (Saei, 2012). Limited research has been the other beta-lactam antibiotics (Stefani conducted on S. aureus biofilm formation et al, 2012). Resistance to methicillin and in Thailand. From 73% to 95% of Thai S. other beta-lactam antibiotics is caused aureus isolates form biofilm (Indrawat- by an acquisition of mecA located within tana et al, 2013; Tangchaisuriya et al, 2014; staphylococcal cassette chromosome mec Daoda et al, 2015), but 44.4% of clinical element (SCCmec), with SCCmec types MRSA isolates only demonstrated weak I, II and III carried by hospital-acquired biofilm production and there is no as- (HA)-MRSA, rendering resistance to sociation between biofilm formation and non-beta-lactam antibiotic, while SCCmec MRSA genotypes (Boontha et al, 2015). types IV, V and VI (encoding beta-lactam Study of biofilm generation in clinical resistance factors) are carried by commu- MRSA isolates from different hospitals nity-acquired (CA)-MRSA (encoding beta- in Thailand is necessary to broaden our lactam resistance (Tulinski et al, 2012; Eed understanding of biofilm formation. et al, 2015). Multilocus sequence typing In this study, phenotypic properties (MLST) has allowed characterization of of biofilm and biofilm-associated genes approximately 90% of HA-MRSA in nine of clinical MRSA isolates from three hos- Southeast Asian countries into a single pitals in Thailand were characterized, in- clonal subgroup ST239 (Chongtrakool cluding prevalence of SCCmec type, clonal et al, 2006). However, there is little infor- subgroup ST239, antibiogram profile, mation of MRSA SCCmec types as well as resistance to disinfectants and carriage their clonal distribution in Thailand. of antibiotic and disinfectant resistance The majority of MRSA possess an abi- genes. Understanding virulence proper- lity to generate biofilm, a crucial property ties, such as biofilm formation, in such for intracellular persistence and virulence pathogenic microbes can provide insight- involved in pathogenesis (Oyama et al, ful information towards development 2016). Four major genes play key roles of new strategies to ameliorate severity in biofilm development and adhesion, and prevalence of infectious diseases. namely, bap (encoding biofilm-associated Molecular characteristics, such as clonal protein; BAP), cna (encoding collagen distribution, should be useful for future adhesin), fnbA (encoding fibronectin- active surveillance aimed at controlling binding protein FnbA), and icaAD (lo- the spread of existing antimicrobial resis- cated within the intracellular adhesion tant bacteria such as MRSA. (ica) operon and encoding polysaccharide intracellular adhesin PIA). Bap is involved MATERIALS AND METHODS in the initial attachment and intracellu- lar adhesion (Cucarella et al, 2004); PIA Isolation and identification of MRSA iso- mediates cell-to-cell adhesion (Becker lates et al, 2014); FnbA mediates biofilm forma- A total of 38 MRSA isolates were col- Vol 49 No. 6 November 2018 1061 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH lected from three hospitals in Thailand, mM Mg2+, 2.5 μl of 2.5 mM dNTPs, 0.2 μl of namely, Chiangrai Prachanukroh Hospital 5 U Taq polymerase (RBC Bioscience), var- (HA), a large tertiary hospital located in ious concentrations of each primer (Zhang the northern region, Naresuan University et al, 2005) and 3 μl of DNA template. Hospital (HB) and Sawanpracharak Hos- Thermocycling (GeneMate thermal cycler, pital (HC) located in the lower northern Hangzhou, China) was conducted as fol- region (see Fig 2). Samples from HA (n = lows: 94ºC for 4 minutes; followed by 30 23) and HB (n = 9) were collected from cycles of 94ºC for 20 seconds, 55ºC for 30 November 2014 to October 2015 and from seconds and 72ºC for 30 minutes; and a HC (n = 6) in January 2013. Isolates were final step of 72ºC for 5 minutes. collected from multiple sites, such as Clonal subgroup ST239 was identified blood, pus, sputum, urine and other body by a PCR method using primer sets and fluids, and identified as S. aureus using a conditions as described previously (Feil coagulase test, detection of 16S rDNA and et al, 2008). In short, a total volume of 25 μl nuc by PCR amplification (Sasaki et al, 2010) and as MRSA using a cefoxitin disk containing 1X PCR buffer (RBC Biosci- (30 μg) (Oxoid, Hampshire, UK) diffusion ence), 1.5 mM MgCl2, 0.2 mM dNTPs, test on Mueller-Hinton agar (Hi-Media, 0.7 μM each of the four primers, 1.25 U Mumbai, India) and PCR-based detection Taq polymerase (RBC Bioscience), and of mecA (Kitti et al, 2011). 2.5 μl of DNA was amplified with the following thermocycling (GeneMate ther- Antibiogram determination mal cycler, Hangzhou, China) conditions: Antibiotic susceptibilities to chloram- 95°C for 15 minutes; followed by 30 cycles phenicol (C; 30 μg), ciprofloxacin (CIP; 5 of 95°C for 30 seconds, 55°C for 30 seconds μg), clindamycin (DA; 2 μg), erythromy- and 72°C for 30 seconds; and a final step of cin (E; 15 μg), fusidic acid (FD; 10 μg), 72°C for 7 minutes. Amplicons were ana- gentamicin (CN; 10 μg), linezolid (LZD; 30 lyzed by 1% agarose gel-electrophoresis μg), mupirocin (MUP; 5 μg), novobiocin and staining with ethidium bromide. (NV; 5 μg), oxacillin (OX, 1 μg), penicil- lin (P; 10 units), rifampicin (RD; 5 μg), Detection of biofilm-associated, antibiotic sulfamethoxazole/trimethoprim (SXT; and disinfectant resistance genes in MRSA 1.25/23.75 μg), and vancomycin (VA; 30 isolates μg) (Oxoid, Hampshire, UK) were de- Primers employed in detection of termined using a disk diffusion method MRSA biofilm-associated genes bab, fnbA (CLSI, 2014). An isolate is categorized as and icaAD, cna and antibiotic and disin- multidrug resistant (MDR) if resistant to fectant resistance genes ermA, ermB, ermC, at least three classes of antibiotics. and qacAB are listed in Table 1. Bacteria Detection of SCCmec types and clonal sub- were grown on tryptone soya agar (Hi- group ST239 Media, Mumbai, India) and DNA was SCCmec types were determined by extracted using a boiling method. For bab, multi- and uniplex PCR according to a fnbA, icaAD and cna, PCR mixture (20 μl) method modified from a previous study contained 2 μl of 10X PCR buffer, 0.2 μl (Zhang et al, 2005). In brief, amplification of 25 mM MgCl2, 2 μl of 2.5 mM dNTPs, was performed in a total volume of 25 μl 0.2 μl