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

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, , 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 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 , 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 of 10 μM each primer, 0.15 μl of 5 containing 3 μl of 10X buffer (RBC Biosci- U Taq polymerase (RBC Bioscience), and ence corp, Taipei, Taiwan) containing 15 2 μl of DNA. Thermocycling of each gene

1062 Vol 49 No. 6 November 2018 Phenotypes and Genotypes of MRSA et al (2003) et al (2003) et al (2001) et al (2005) et al (2007) et al (2010) et al (2017) et al (2017) et al (2017) Reference et al (2008) Seng Seng Seng Smith Arciola Arciola This study Cucarella Cucarella Feil Feil et al (2008) Zhang et al (2011) Strommenger Strommenger Chaieb Strommenger Strommenger Tm 55 58 63 55 55 61 58 53 57.3 62 55.9 56.7 (ºC) 211 422 901 192 191 971 220 484 57 361 299 142 190 1,216 size (bp) Amplicon Table 1 Table

Primer used in the study. the in used Primer

F: GACAGTCGCTACGAAAAG R: AATAAGCTCCTAACTA F: CCCTCTTCGTTATTCAGCC R: CAGGAGGCAAGTCACCTTG F: GGCGCAAGCAGCAGAATTA R: CATAGTTCTTTGTGGTGTTGC F: AATGTGCTTGGATGCAGATACTAT C AATGTGCTTGGATGCAGATACTAT F: R: GAATCGTCATCTGCATTTGCA F: AAAGCGTTGCCTAGTGGAGA R: AGTGCCTTCCCAAACCTTTT F: GATACA AACCCAGGTGGTGG F: GATACA R: TGTGCTTGACCATGCTCTTC F: CCCTATATCGAAGGTGTAGAATTGCAC R: GCTGTTGAAGTTAATACTGTACCTGC F: CACTTTAAATACTGACGAAAAT R: TTGAAAATTGATCATTCAGCAA F: TCGCACTCTCGTTGAACA R: AAATCCGCTTCGACAAACATT F: GCAGAAAGTGCAGAGTTCG R: CCAGTCCAATCATGCCTG F: AATCGTCAATTCCTGCATGT AATACGGGTTTG R: TAATCGTGG Primer

F: AATCGTCAATTCCTGCATGT AATACGGGTTTG R: TAATCGTGG F: AAGCGGTAAACCCCTCTGA F: AAGCGGTAAACCCCTCTGA R: TTCGCAAATCCCTTCTCAAC

Target gene Target ica AD (MR-CoNS) A (MR-CoNS) fnb A bap (MR-CoNS) ica AD (MRSA) cna (MRSA) A (MRSA) fnb A bap (MRSA) qac AB erm C erm A erm B ; MRSA, methicillin-resistant Staphylococcus aureus. coagulase negative Staphylococcus ; MRSA, methicillin-resistant MR-CoNS, methicillin-resistant

ST239 / ST 8 ST239 / ST 30

Vol 49 No. 6 November 2018 1063 Southeast Asian J Trop Med Public Health was conducted in DNA thermal cycler with 0.25% glucose in 96-well polystyrene (GeneMate thermal cycler) individually as tissue culture microtiter plates (Nunc, previously described (Table 1) and ampli- Roskilde, Denmark) overnight at 37°C, cons were analyzed as described above. each experiment being conducted in trip- The other antibiotic and disinfectant licate. Culture medium then was removed resistance genes, ermA, ermB, ermC, and and adherent cells were treated with 95% qacAB were detected by PCR modified as ethanol and stained with 1% crystal violet. previously described (Ardic et al, 2005; A570 nm was measured and data interpreted Zhang et al, 2011; Youn et al, 2014). In as follows: highly positive (A570 nm ≥1), brief, PCR mixture (20 μl) consisted of 2.4 low grade positive (0.1≤ A570 nm <1) and μl of 10X PCR buffer containing 15 mM negative (A570 nm <0.1).

MgCl2, (RBC Biosience) 2 μl of 2.5 mM dNTPs, 0.2 μl of 10 μM each primer, 0.2 RESULTS μl of 5 U HotStart Taq DNA polymerase (RBC Biosience), and 2 μl of DNA. Ther- Antibiogram profile mocycling was performed in a GeneMate Based on a disc diffusion assay, 76- thermal cycler as follows: 95ºC for 5 min- 96% of the MRSA isolates were resistant to utes; followed by 30 cycles of 94ºC for 25 ciprofloxacin, clindamycin, erythromycin, seconds, 57ºC for 30 seconds and 72ºC for gentamicin, and penicillin; <10% resistant 30 seconds; with a final step of 72ºC for 5 to chloramphenicol, mupirocin and rifam- minutes. Amplicons from representative picin; and none resistant to fusidic acid, isolates were purified from gels using linezolid, novobiocin, and vancomycin QIAquick PCR Purification Kit (Qiagen, (Fig 1). MDR-MRSA constituted 95% of Valencia, CA) and directly sequenced by the isolates. Macrogen (Seoul, Korea). Prevalence of antibiotic and disinfectant resistance genes Detection of biofilm formation Employing PCR to identify the target Biofilm formation of MRSA isolates genes, prevalence of ermA, ermB, ermC, was detected on Congo red agar (CRA) and qacAB among the MRSA isolates was plates as previously described (Seng et al, 82, 60, 8 and 47%, respectively (Table 2). 2017). In brief, MRSA isolate was streaked on CRA plate (Hi-Media) and incubated SCCmec types and prevalence of clonal at 35°C under aerobic conditions for 24-48 subgroup ST239 hours. Slime (biofilm)-producing MRSA Using multi- and uniplex PCR, MRSA formed black (indicated by a darkening isolates were characterized as SCCmec of colony with an absence of dry crystal- type I (n = 10; 26%), type II (n = 10; 26%), line colonial morphology) or very black type III (n = 15; 39%), type IV (n = 2; 5%), (indicated by a darkening of colony and type V (n = 1; 3%) (Fig 2). Clonal with presence of dry crystalline colonial subgroup ST239 was detected in 11 (29%) morphology) colonies while non-slime isolates, 10 from HA and 1 from HB. (biofilm) producer strain formed red Biofilm formation and biofilm-associated colonies. Qauntification of biofilm forma- genes tion was performed by a microtiter plate Observation of colony phenotypes on assay (MTP) (Bekir et al, 2011). In short, CRA plates, 7 (18%) isolates formed very MRSA isolates were cultured in trypti- black colonies, 31 (82%) black colonies case soy broth (Hi-Media) supplemented and none red colonies, indicating that all

1064 Vol 49 No. 6 November 2018 Phenotypes and Genotypes of MRSA

Table 2 Prevalence of antibiotic and disinfectant resistance genes of clinical methicillin- resistant Staphylococcus aureus (MRSA) isolates from three hospitals (HA, HB and HC), Thailand, January 2013 - October 2015.

Antibiotic/disinfectant MRSA from HA MRSA from HB MRSA from HC Total resistance gene Number (%) Number (%) Number (%) (%) (n = 38) (n = 23) (n = 9) (n = 6)

ermA 18 (78) 8 (89) 5 (83) 31 (82) ermB 17 (74) 1 (11) 5 (83) 23 (60) ermC 1 (4) 1 (11) 1 (17) 3 (8) qacAB 6 (26) 7 (78) 5 (83) 18 (47) SCCmec I 6 (26) 2 (22) 2 (33) 10 (26) SCCmec II 1 (4) 5 (56) 4 (67) 10 (26) SCCmec III 13 (56) 2 (22) 0 (0) 15 (39) SCCmec IV 2 (9) 0 (0) 0 (0) 2 (5) SCCmec V 1 (4) 0 (0) 0 (0) 1 (3) ST239 10 (43) 1 (11) 0 (0) 11 (29) isolates were slime (biofilm) producers. DISCUSSION However, quantification of biofilm forma- tion using a MTP assay showed 23 isolates MRSA is a pervasive pathogen recog- (60%) were low producers and the remain- nized as a major health threat worldwide ing non-biofilm producers (Table 3). Only that possesses a variety of virulence fac- 1 isolate (11%) of MRSA isolated from HB tors involved in pathogenesis such as was a non-biofilm producer, while almost biofilm formation (Oyama et al, 2016). In 50% of isolates from HA and HC were order to strengthen an understanding of negative for biofilm production. MRSA antibiotic resistance, dissemination Two sets of primers including the and virulence, presence, phenotypic and primers designed from MRSA and meth- genotypic antibiotic resistance, and bio- icillin-resistant coagulase negative Staphy- film formation were examined in 38 MRSA lococcus (MR-CoNS) were used to am- isolates from three different hospitals in plify bab, fnbA and icaAD. Only a pair northern Thailand. of primers was used to detect cna. PCR High rates of MRSA resistance to amplification of target genes using MRSA- ciprofloxacin, clindamycin, erythromy- based primers showed prevalence of bab, cin, and gentamicin in this study were fnbA, icaAD, and cna was 100%, 63%, 53% and 50%, respectively; and employing similar to other reports in middle region MR-CoNS-based primers, prevalence of of Thailand (Phokhaphan et al, 2017) and bab, fnbA and icaAD was 100%, 81% and Asia (Song et al, 2011), but the rate was 100%, respectively (Table 4). It is worth approximately 40-50% higher than clinical noting that the frequencies of fnbA and MRSA isolated in India (Pai et al, 2010). A icaAD detected using MR-CoNS-primers recent study found only 48% and 29% of were higher than those of MRSA-based MRSA isolated from Oman were resistant primers. to erythromycin and clindamycin, respec-

Vol 49 No. 6 November 2018 1065 Southeast Asian J Trop Med Public Health Penicillin Gentamicin Sulfamethoxazole/ Trimethoprim Mupirocin Cefoxitin Fusidic acid Novobiocin Linezolid Vancomycin Chloramphenicol Rifampicin Erythromycin Clindamycin Ciprofloxacin MDR

HB

HC

HA

Total

Resistance rate (%)

Fig 1 - Antibiogram profiles of clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates from three hospitals (HA, HB and HC), Thailand, January 2013 - October 2015. Antibiotic susceptibility was determined using a disc diffusion assay. Number of isolates from HA, HB and HC was 23, 9 and 6, respectively. MDR, multidrug resistant.

HA isolates (n=23)

ST 239 (n=1)

HB isolates (n=9)

HC isolates (n=6)

Fig 2 - Location of the three hospitals (HA, HB and HC) in northern Thailand, and frequency of SCCmec types and number of clonal subtype ST239 of clinical methicillin-resistant Staphylococcus aureus isolates at each hospital, January 2013 - October 2015. SCCmec types clonal subtype ST239 were identified using PCR-based methods.

1066 Vol 49 No. 6 November 2018 Phenotypes and Genotypes of MRSA

Table 3 Biofilm formation using microtiter plate (MPT) and Congo red agar (CRA) assays of clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates from three hospitals (HA, HB and HC), Thailand, January 2013 - October 2015.

Assay MRSA from HA MRSA from HB MRSA from HC Total Number (%) Number (%) Number (%) (%) (n = 38) (n = 23) (n = 9) (n = 6)

CRA Very black 5 (22) 2 (22) 0 (0) 7 (18) Black 18 (78) 7 (78) 6 (100) 31 (82) Red 0 (0) 0 (0) 0 (0) 0 (0) MTP Strong biofilm producer 0 (0.0) 0 (0) 0 (0) 0 (0) Low biofilm producer 12 (52) 8 (81) 3 (50) 23 (60.5) Non-biofilm producer 11 (48) 1 (11) 3 (50) 15 (39.5)

Table 4 Biofilm-associated genes of clinical methicillin-resistantStaphylococcus aureus (MRSA) isolates from three hospitals (HA, HB and HC), Thailand, January 2013 - October 2015.

Biofilm-associated MRSA from HA MRSA from HB MRSA from HC Total gene Number (%) Number (%) Number (%) (%) (n = 38) (n = 23) (n = 9) (n = 6)

MRSA-based primers bap 23 (100) 9 (100) 6 (100) 38 (100) fnbA 23 (100) 0 (0) 1 (17) 24 (63) icaAD 14 (61) 3 (33) 3 (50) 20 (53) cna 6 (26) 7 (78) 6 (100) 19 (50) MR-CoNS-based primers bap 23 (100) 9 (100) 6 (100) 38 (100) fnbA 20 (87) 7 (78) 4 (67) 31 (82) icaAD 23 (100) 9 (100) 6 (100) 38 (100)

MR-CoNS, methicillin resistant coagulase negative Staphylococcus. tively (Pathare et al, 2016). Therefore, re- mycin ribosomal methylase A and B) were sistance to erythromycin and clindamycin detected, in agreement with studies of S. in MRSA are likely related to collection aureus from patients in Malaysia (Lim et al, period, source and geographical region. 2012). Reduced susceptibility to disinfec- Consistent with the high frequency tant chlorhexidine is associated with the of erythromycin resistance, high carriage carriage of qacAB (encoding QacA protein) rates of ermA and ermB (encoding erythro- (Horner et al, 2012). Although suscepti-

Vol 49 No. 6 November 2018 1067 Southeast Asian J Trop Med Public Health bility of MRSA isolates to chlorhexidine et al, 2014). However, quantitative biofilm was not determined, the proportion of assay indicated no MRSA isolates were isolates harboring qacAB was similar strong biofilm producers and less than to that in West African (Conceição et al, two thirds were low biofilm producer, 2016). MRSA carrying qacAB is more often consisted with that (44%) reported by diagnosed in patients with nosocomial Boontha et al (2015), but much lower and catheter-related blood than bone and than other findings (73-95%) in Thailand soft-tissue infections (Cho et al, 2017). (Indrawattana et al, 2013; Tangchaisuriya MRSA with qacAB also is associated with et al, 2014; Daoda et al, 2015), suggesting resistance to ciprofloxacin, clindamycin a possible decline in biofilm-producing and mupirocin resistance (Lee et al, 2013). MRSA in the country. Although CRA Thus, the presence of qacAB may play a method is easy and takes little time to role in the virulence of MRSA. perform, the poor agreement between MRSA isolates collected from Thai CRA and MTP methods was found in hospitals previously were demonstrated the present study and this phenomenon to carry only SCCmec type II and III (Lu- also was reported by previous studies litanond et al, 2010), while SCCmec type (Darwish and Asfour, 2013; Mathur et al, IV and V were also detected in this study, 2013). Therefore, CRA alone could not be suggesting the spread of CA-MRSA infec- used to detect the biofilm formation. It is tion into hospitals. The invasive MRSA worth pointing out the majority of biofilm clonal subtype ST239 causes a wide range producers were SCCmec types I and II, of infections across the world, particularly indicators of HA-MRSA. in Asia (Feil et al, 2008). Nearly 50% of ST S. aureus adherence to a biotic or an 239 isolates were biofilm producers. Bio- abiotic surface is mediated by a number film producing- ST 239 has been reported of microbial surface components-recog- to resistant to ciprofloxacin, erythromy- nizing adhesive matrix compounds and cin, gentamicin, tetracycline, and trime- the expression of such matrix-attachment thoprim-sulfamethoxazole (Shahsavan et molecules has received increasing atten- al, 2011; Aparecida et al, 2012). Some 30% tion following the discovery of biofilm of MRSA isolates in this study were ST239, associated genes (Otto, 2008). We detected almost all obtained from hospitals located four major biofilm associated genes (bab, in both the upper and lower northern fnbA, icaAD and cna) using two sets of regions of the country. It is imperative to primers (MRSA and MR-CoNS primers). determine how widespread this MRSA Using MRSA-based primers, high preva- strain in hospitals is across the country. lence of biofilm associated genes bab, cna, Biofilm formation is one of the viru- fnbA, and icaAD has been observed, rang- lence factors of staphylococci that de- ing from 50% to 100%. Daoda et al (2015) crease the treatment effectiveness by reported prevalence of fnbA and icaAD reducing sensitivity to antibiotics and of MSSA was equal to those of MRSA. immune response (Stewart and Coster- Tangchaisuriya et al (2014) previously ton, 2001). All MRSA isolates exhibited observed 93%, 92% and 61% of S. aureus phenotypic slime (biofilm) production by carry fnbA, icaAD and cna, higher than the the presence of black or very black colo- current study. In addition, the presence nies on CRA plates as has been reported of icaAD, amplified using MRSA-based in Iran where 97.5% of MRSA isolates are primers, was approximately half of that slime (biofilm) producers (Moghadam detected using MR-CoNS primers while

1068 Vol 49 No. 6 November 2018 Phenotypes and Genotypes of MRSA the frequency of other genes detected was Ardic N, Ozyurt M, Sareyyupoglu B, Hazneda- unchanged. This difference may be due roglu T. Investigation of erythromycin and to cross-hybridization of the MR-CoNS tetracycline resistance genes in methicillin- primers. resistant staphylococci. Int J Antimicrob Agents 2005; 26: 213-8. In conculsion, this study reveals 95% Becker K, Heilmann C, Peters G. Coagulase- of MRSA isolates from three hospitals in negative staphylococci. Clin Microbiol Rev northern Thailand were MDR and carried 2014; 27: 870-926. a variety of SCCmec types, including those Bekir K, Ben Abdallah F, Ellafi A, et al. Ad- associated with community-acquired in- herence assays and slime production of fection, and virulence genes. MRSA clonal Staphylococcus aureus strains after their subtype ST239 presented in two hospitals incubation in seawater microcosms. Ann located in the lower and higher northern Microbiol 2011; 61: 819-23. regions suggesting dissemination of this Beleir K, Abdallah FB, Ellafi A, Bakhrouf A. clone in northern Thailand. High preva- Adherence assays and slime production lence of phenotypic and genotypic bio- of Staphylococcus aureus strains after their film formation also were detected. These incubation in seawater microcosms. Ann data are essential in the development Microbiol 2011; 61: 819-23. new strategies to lower the severity and Boontha W, Wilailuckana C, Charoensri N, prevalence of infectious diseases caused et al. Biofilm formation and surface protein by pathogenic MRSA. encoding genes in methicillin-resistant Staphylococcus aureus isolated from patients ACKNOWLEDGEMENTS in Srinagarind Hospital, Khon Kaen prov- ince. J Med Tech Phy Ther 2015; 26: 212-21. The authors acknowledge the micro- Chaieb K, Zmantar T, Chehab O, et al. Antibiotic biology staff, Chiangrai Prachanukroh resistance genes detected by multiplex Hospital, Naresuan University Hospital PCR assays in Staphylococcus epidermidis and Sawanpracharak Hospital for speci- strains isolated from dialysis fluid and men collections, and Ms. Khundaw Modla needles in a dialysis service. Jpn J Infect for assistance in performing antimicrobial Dis 2007; 60: 183. susceptibility tests. Cho OH, Park KH, Moon SM, Bae IG. Preva- lence and characteristics of qacA/B-pos- REFERENCES itive methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection Aparecida Guimarães M, Rocchetto Coelho isolates in a tertiary hospital. Open Forum L, Rodrigues Souza R, Ferreira-Carvalho Infect Dis 2017; 4, 640. BT, Marie Sá Figueiredo A. Impact of bio- Chongtrakool P, Ito T, Ma XX, et al. Staphylo- cides on biofilm formation by methicillin- coccal cassette chromosome mec (SCCmec) resistant Staphylococcus aureus (ST239- typing of methicillin-resistant Staphylococ- SCCmecIII) isolates. Microbiol Immunol cus aureus strains isolated in 11 Asian coun- 2012; 56: 203-7. tries: a proposal for a new nomenclature Arciola CR, Campoccia D, Gamberini S, Bal- for SCCmec elements. Antimicrob Agents dassarri L, Montanaro L. Prevalence of Chemother 2006; 50: 1001-12. cna, fnbA and fnbB adhesin genes among Clinical Laboratory Standard Institute (CLSI). Staphylococcus aureus isolates from ortho- Performance Standards for Antimicrobial pedic infections associated to different Susceptibility Testing; Twenty-Fourth In- types of implant. FEMS Microbiol Lett 2005; formational Supplement: CLSI document 246: 81-6. M100-S24, Wayne: CLSI, 2014.

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