1 Abtract

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2 Objectives: Staphylococcus aureus (S. aureus), especially Methicillin-resistant S. aureus
3 (MRSA), remains a major public health concern. This study reports the antimicrobial resistance
4 profiles and molecular characteristics of S. aureus isolated during 2017-2018 from Queen Sirikit
5 Naval Hospital (QSH) in Chonburi province, Thailand.

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7 Methods: S. aureus were isolated from clinical specimens of inpatient and outpatient at QSH. All
8 strains were tested for antimicrobial susceptibility. Staphylococcal cassette chromosome mec
9 (SCCmec) typing, Panton-Valentine leukocidin (pvl) toxin, pulsed-field gel electrophoresis

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10 (PFGE), multilocus sequence typing (MLST) and staphylococcal protein A (spa) typing were
11 performed to determine molecular characteristics and relatedness of these isolates.
12

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13 Results: Thirty one S. aureus isolates were identified and 27 isolates were confirmed to be
14 MRSA. The isolated MRSA exhibited resistance up to 7 antibiotics classes. SCCmec type II was
15
16
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predominant (51.9%) and ST764 (55.6%) was the main MLST type. Five spa types were
identified with t045 (55.6%) as the major type. All 31 S. aureus isolates were grouped into 7
types by PFGE with SCCmecII-ST764-t045 clone being the most prevalent.
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19 Conclusions: The molecular characteristics of S. aureus isolates in this study differ from
20 previous reports in Thailand, which could indicate a shift in local strains. The MRSA isolates
21 demonstrated a diverse antimicrobial resistance pattern. The observed genotypic shift and drug
22 resistance of MRSA should be regularly monitored to track the evolving MRSA. This will
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23 provide epidemiological data to aid with effective prevention and control measures to decrease
24 further spread of drug-resistant MRSA in all settings.
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25

26 Keywords: MRSA, SCCmec, MLST, PFGE, spa, PVL

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 27 Introduction

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28 Staphylococcus aureus (S. aureus) is a Gram-positive bacterium, and a major cause of

29 various infections in both communities and healthcare facilities. S. aureus is responsible for

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30 infections ranging from folliculitis to life-threatening invasive diseases such as endocarditis and

31 osteomyelitis [1,2]. S. aureus, especially methicillin-resistant S. aureus (MRSA), is one of the

32 most common hospital and community acquired pathogens associated with morbidity and

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33 mortality. According to the Centers for Disease Control and Prevention (CDC), infection by

34 MRSA resulted in almost 11,000 deaths in 2011 in the US [3]. MRSA was also attributed to

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35 100,000 deaths worldwide in 2019 [4]. Methicillin was first introduced for treatment of

36

37
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staphylococcal infections in 1959 and the first MRSA was reported in London within two years

of its introduction [5]. In recent years, therapeutic treatment options for MRSA infections have
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38 been greatly reduced due to increased resistance to multiple classes of antimicrobial agents [1].

39 Generally, MRSA is divided into two main groups: hospital associated (HA) and community

40 associated (CA) [6]. Clinically, the infections caused by HA-MRSA strains are associated with
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41 high mortality and morbidity. These strains are usually multidrug resistant (MDR), a resistant to
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42 at least one agent in three or more antimicrobial categories, which limits the number of

43 antibiotics available to effectively treat the infections [7]. A growing population of CA-MRSA

44 strains were reported to express virulence factors, such as Panton-Valentine leucocidin (PVL),
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45 encoded by lukS-PV and lukF-PV, which is well known for its induction of apoptosis in human

46 polymorphic leukocytes causing cell lysis [8].

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47 The molecular characterization of S. aureus is mainly based on SCCmec types, a mobile

48 genetic element that is the defining feature of MRSA, that have been classified into major types I
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49 to XIII, along with various subtypes [1]. Multiplex PCR assays were developed to identify all

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 50 major types [9,10]. Other methods, such as pulsed-field gel electrophoresis (PFGE), multilocus

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51 sequence typing (MLST), spa typing, and advanced whole genome sequencing (WGS) are also

52 employed to characterize MRSA. Molecular characterization to study strain relatedness, clonal

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53 distribution, and genetic diversity is required to keep pace with the changing epidemiology of

54 MRSA.

55 This study describes the multidrug resistance of S. aureus-associated infections from

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56 clinical samples obtained from Queen Sirikit Hospital, Chonburi, Thailand and the molecular

57 characterization of these representative S. aureus isolates.

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4584919
 58 Methods

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59 Bacterial Strains

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60 S. aureus was isolated and identified as part of a surveillance study of multidrug resistant

61 ‘ESKAPEE’ pathogens, Enterococcus faecium, Stphylococcus aureus, Klebsiella pneumonia,

62 Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli, at

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63 the Microbiology laboratory at Queen Sirikit Naval Hospital, Chonburi, Thailand from April

64 2017 to May 2018 [11]. Isolated MDR S. aureus isolates were transferred to the Armed Forces

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65 Research Institute of Medical Sciences (AFRIMS) in Bangkok, Thailand for further molecular

66 analysis. Isolates were grown on sheep blood agar plates and trypticase soy agar plates followed

67 er
by an incubation in an aerobic condition at 35± 2 °C for 18-24 hours in preparation for
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68 downstream molecular analysis.

69 S. aureus phenotypic characterization and antimicrobial susceptibility testing

70 Biochemical identification and antimicrobial susceptibility testing were performed with

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71 the BD Phoenix M50 using the NMIC/ID 55 panel, according to the manufacturer’s instructions
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72 (BD Diagnostics, Sparks, MD). Clinical and Laboratory Standards Institute (CLSI) guidelines in

73 2017 were followed for results interpretation [12]. The antimicrobial susceptibility tests for S.

74 aureus isolates using the NMIC/ID included amikacin, ampicillin, ciprofloxacin, clindamycin,
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75 erythromycin, gentamicin, linezolid, high level mupirocin, oxacillin, penicillin, rifampin,

76 teicoplanin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin. For a screening of

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77 MRSA, the oxacillin and cefoxitin minimum inhibitory concentration (MIC) was used to screen

78 all S. aureus isolates. Isolates with oxacillin MIC ≥ 4 µg/mL and cefoxitin ≥ 8 µg/mL were
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79 defined as methicillin-resistant S. aureus (MRSA).

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 80 Genomic DNA purification of S. aureus isolates

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81 Genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden,

82 Germany) according to the manufacturer instruction, with a pretreatment lysis of Gram-positive

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83 bacterial cell walls. DNA concentration and quality assessment was determined by using a

84 NanoDrop spectrophotometer (Thermo scientific, MA, USA). DNA was stored at -20 °C until

85 analysis.

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86 Real-time PCR detection of mecA gene

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87 All S. aureus isolates were subjected to mecA (methicillin resistance gene) detection by

88 real-time PCR. The real-time PCR assays were performed as previously described [13] on a

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CFX96 Touch Deep Well™ Real-Time PCR Detection System (Bio-Rad, CA, USA) using the
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90 SensiFAST Probe No-ROX Mix (Bioline, London, UK). Real-time PCR results were analyzed

91 by Bio-Rad CFX Manager software version 3.1. The threshold cycle (Ct) was set at 1,000

92 relative fluorescence units (RFU). The S. aureus ATCC BAA-1768, S. aureus ATCC 25923, and
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93 sterile water were used as positive control, negative control, and blank, respectively.

94 PCR detection of Panton-Valentine leukocidin (pvl) toxin

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95 All S. aureus isolates were tested for the presence of pvl genes (433 bp) by PCR as
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96 previously described [2] using a Mastercycler® Nexus GX2 (Eppendorf, NY, USA). Briefly, a

97 25 μL reaction contained 2 µL of DNA template, 1x AmpliTaq PCR buffer, 1.5 mM MgCl2, 100

98 µM of each dNTP, 0.4 µM of each primer, pvl-F and pvl-R, and 1.25 U of AmpliTaq DNA
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99 polymerase (Thermo Fisher Scientific, MA, USA). The thermal cycling condition was as

100 follows: denaturation at 94 ºC for 5 min, followed by 28 cycles of 94 ºC for 1 min, 55 ºC for 1
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101 min, and 72 ºC for 1 min, with a final extension at 72 ºC for 10 min.

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102 Identification of SCCmec types

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103 SCCmec typing was performed following Boye et al., 2007 for SCCmec type I to V [10].

104 Briefly, an amplification of SCCmec genes was performed in a total volume of 25 µL containing

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105 2 µL of DNA template, 1x AmpliTaq PCR buffer, 1.5 mM MgCl2, 100 µM of each dNTP and

106 1.25 U of AmpliTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and

107 primer concentrations were as follows: primers β and α3, 0.2 µM each; ccrC-F and ccrC-R, 0.25

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108 µM each; 1272F1 and 1272R1, 0.08 µM each; and 5RmecA and 5R431, 0.1 µM each. The

109 reaction was carried out on a Mastercycler® Nexus GX2 (Eppendorf, NY, USA) and a thermal

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110 cycling condition comprised of denaturation at 94 ºC for 5 min, followed by 28 cycles of 94 ºC

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for 1 min, 55 ºC for 1 min, and 72 ºC for 1 min, with a final extension of 72 ºC for 10 min.

SCCmec types were identified by comparing the banding patterns of MRSA to reference strains:
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113 ATCC BAA-44 (SCCmec type I), ATCC BAA-41 (SCCmec type II), NR-45889_BEI Resources

114 (SCCmec type III), ATCC BAA-1768 (SCCmec type IV), and ATCC BAA-2094 (SCCmec type

115 V).
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116 Multiplex PCR assays for SCCmec type VI to XII were performed using two sets of
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117 primers that target SCCmec type VI to VIII and SCCmec type IX to XII [9]. Briefly, a 25 μL

118 reaction contained 2 µL of DNA template, 1x AmpliTaq PCR buffer, 1.5 mM MgCl2, 200 µM
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119 each dNTP, and 1.25 U of AmpliTaq DNA polymerase (Thermo Fisher Scientific, MA, USA).

120 For SCCmec type VI to VIII, 0.1 µM of each primer was used except 0.2 µM of Type VII. For

121 SCCmec type IX to XII, 0.1 µM of each primer was used except 0.2 µM of Type IX and Type X.
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122 The thermal cycling condition was as follows: an initial denaturation at 94 °C for 5 min followed

123 by 30 cycles of 94 °C for 1 min, gradient annealing at 50-60 °C for 1 min, and 72 °C for 1 min
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124 with a final extension at 72 °C for 10 min. All of the amplified products were sent for sequencing

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125 for confirmation (First BASE, Malaysia).

126 Analysis of whole genome sequencing (WGS) data was performed on a strain that was

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127 not typeable by multiplex PCR assays described above. Sequences of the untypeable isolates

128 were edited and assembled by BioNumerics software version 7.6 (Applied Maths, Ghent,

129 Belgium). The edited sequences were queried in SCCmecFinder, a web-based tool for Typing of

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130 Staphylococcal Cassette Chromosome mec in Staphylococcus aureus

131 (https://cge.food.dtu.dk/services/SCCmecFinder/).

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132 Polymorphism of the X region encoding S. aureus protein A (spa typing)

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The PCR assay was performed as previously described [14] in a final volume of 50 μL
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134 containing 4 µL of DNA template, 1x AmpliTaq PCR buffer, 1.5 mM MgCl2, 100 µM of each

135 dNTP, 0.1 µM of spa-1113 F and spa-1514 R primer, each, and 1.25 U of AmpliTaq DNA

136 polymerase (ThermoFisher Scientific, Waltham, MA, USA) on a Mastercycler® Nexus GX2
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137 (Eppendorf, NY, USA) according to Ridom SpaServer recommendations

138 (https://spaserver.ridom.de/). Amplified products were purified and commercially sequenced

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139 (First BASE, Malaysia). The isolates were assigned a repeat profile and a specific spa type using

140 BioNumerics software version 7.6 (Applied Maths, Ghent, Belgium) which was based on the
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141 Ridom SpaServer database (http://www.spaserver.ridom.de).

142 Multi-Locus Sequence Typing (MLST)

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143 MLST analysis was performed as described previously [15]. Briefly, gene fragments

144 comprising seven housekeeping genes (approximately 500 bp in length, each): carbamate kinase
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145 (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glp), guanylate kinase (gmk),

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146 phosphate acetyltransferase (pta), triosephosphate isomerase (tpi), and acetyl coenzyme A

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147 acetyltransferase (yqiL) were amplified by PCR using AmpliTaq Gold Master mixtures (Thermo

148 Fisher Scientific, Waltham, MA, USA). Allele numbers and sequence types (STs) were assigned

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149 by querying the sequences onto S. aureus PubMLST website (http://pubmlst.org/saureus). New

150 alleles and STs were submitted to the database.

151 Visualization of PCR products and analysis of sequenced PCR products

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152 PCR products (6-8 µL) were visualized by electrophoresis on 1.5-1.8% w/v agarose gels

153 with ethidium bromide (0.5 µg/ml) staining and the images were captured via a gel

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154 documentation system (Syngene, United Kingdom). PCR products were purified for sequencing

155

156
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using the Wizard SV Gel and PCR clean up system (Promega, WI, USA). After purification, the

PCR products were sent to be commercially sequenced by First BASE, Malaysia. Sequences for
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157 MLST and spa typing were edited and analyzed by Bionumerics V.7.6 software (Applied Maths,

158 Ghent, Belgium).

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159 Pulsed-field gel electrophoresis (PFGE)

160 PFGE was performed on all of the S. aureus according to previously described method
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161 [16] with a few modifications. Briefly, a single colony of the S. aureus isolate was inoculated

162 into 5 ml of Brain heart Infusion Broth and incubated with shaking at 37 °C for 18-24 hrs. The
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163 genomic plugs were prepared from whole cell plug lysis. Cleavage of the agarose DNA plug was

164 digested with 40 U smaI (Roche Diagnostics, Indianapolis, IN, USA) according to the
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165 manufacturer’s instructions. Digested plugs were loaded into the wells of a 1.8% agarose gel and

166 run in 0.5x TBE using a CHEF Mapper system (Bio-Rad, Hercules, CA, USA) with the
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167 following parameters: 6V, temperature 14 °C, initial switch time 5s, final switch time 40s,

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168 included angle 120, and a run time of 21 hrs. The Salmonella Braenderup H9812 strain (ATCC

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169 BAA664) restrict-digested with XbaI was included as a control marker. After electrophoresis,

170 gels were stained with ethidium bromide solution (0.5 µg/ml), and DNA band patterns were

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171 visualized by a Gel Documentation System (Syngene, Cambridge, United Kingdom). Gel images

172 were saved as TIFF files and analyzed using the BioNumerics software version 7.6 (Applied

173 Maths, Ghent, Belgium). The phylogenetic tree was generated to describe the relationship among

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174 S. aureus isolates by cluster analysis using the Dice coefficient and the unweighted pair group

175 method (UPGMA). The dendrogram of isolates with the similarity over 80% were clustered as

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176 having the same pattern.

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177 Results and discussion

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178 A total of 31 MDR S. aureus isolates were received and confirmed, accounting for 4.6%

179 of the 670 ESKAPEE isolates received during the reporting period. A subset of these isolates

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180 (25) was included in the overall surveillance report [11]. Phenotypic and genotypic

181 characterization identified 27/31 (87%) to be MRSA based on MIC results and the presence of

182 mecA by real time PCR and the remaining 4/31 (13%) MDR S. aureus isolates were methicillin-

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183 susceptible S. aureus (MSSA). The distribution of MRSA according to specimen types were as

184 follows: sputum 13/27 (48.1%), pus 6/27 (22.2%), blood 2/27 (7.4%), urine 2/27 (7.4%), wound

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185 exudate 2/27 (7.4%), central line 1/27 (3.7%), and radivac drain 1/27 (3.7%).

186 er
MRSA isolates in this study exhibited drug resistance against non-β-lactam antibiotics at
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187 varying degrees (Table 1). All isolates were resistant to ampicillin and penicillin. In addition,

188 MRSA isolates were resistant to clindamycin (88.9%), erythromycin (88.9%), ciprofloxacin

189 (85.2%), gentamicin (59.3%), tetracycline (55.6%), trimethoprim-sulfamethoxazole (11.1%),

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190 rifampin (7.4%), and mupirocin (3.7%) (Table 1). All but one of the 27 MRSA isolates were

191 multidrug resistant (MDR), with resistance up to 7 antibiotic classes, and one MSSA isolate was
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192 MDR with resistance to clindamycin and erythromycin. All isolates were susceptible to

193 amikacin, linezolid, teicoplanin and vancomycin, supporting the use of these drugs as treatment
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194 options for S. aureus infection (Fig. 2). However, the National Antimicrobial Resistant

195 Surveillance Center (NARST) has shown a declining trend to oxacillin in MRSA (28% to 8.3%)
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196 from 2006 to 2021[17].

197 SCCmec types I, II, IV, and V were identified among 26 MRSA isolates with 1 isolate
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198 that was not typeable. SCCmec type II was the most common 14/27 (51.9%), followed by

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199 SCCmec type I 5/27 (18.5%), SCCmec type V 4/27 (14.8%), and SCCmec type IV 3/27 (11.1%).

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200 The gel band patterns of SCCmec types I-V in Fig. 1. The untypeable strain was further

201 characterized for SCCmec type VI to XII but it remained untypeable. Further characterization by

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202 SCCmecFinder using WGS data (BioProject no. PRJNA814829) [11] did not yield any typing

203 data. The isolates contained mecA but a key SCCmec element was not detected, namely, ccrA2

204 and ccrB2. It also contained the key type II genes (GenBank no. CP000736): mecI and mecR1

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205 with 100% identity but only had a 51.09% sequence coverage to SCCmec type II. This isolate

206 may represent a new or a variant of an existing SCCmec types where further characterization is

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207 needed. SCCmec type II carries various drug resistance genes, an integrated copy of

208

209
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staphylococcal plasmid pUB110 in the J3 region and Tn554 transposon in the J2 region

conferring resistance to aminoglycoside, macrolide, lincosamide, and streptogramin (MLSB)

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210 [18,19]. Thus, it was not surprising that all SCCmec type II isolates were MDR (Fig. 2).

211 However, the prevalence of SCCmec type II in this study differs from other previous reports

212 where SCCmec type III was the most commonly identified SCCmec type among MRSA isolates
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213 from various regions of Thailand [20-22]. The difference in this observation may be due to

214 geographic and temporal differences which could suggest an epidemiological shift of the
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215 circulating SCCmec types in Thailand [22]. SCCmec type I, a HA-MRSA, was reported to be

216 prevalent in several countries (56.9%) but it was only found to be 18.5% in our study [23]. Seven
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217 strains were SCCmec types IV and V which are reported to be common in community-associated

218 MRSA (CA-MRSA). Unlike SCCmec types I, II, and III, SCCmec type IV and V do not have
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219 any identifiable antibiotic resistance genes other than mecA which has been used to explain its

220 ability to achieve greater fitness and outcompete HA-MRSA strains that carry a larger number of
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221 genes on their mobile genetic element [24]. The prevalence of SCCmec type differs

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222 geographically with reports including type I in southern Germany and Belgium, type II in

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223 England, type III in India, Iran and China, and type IV in Lebanon. This provides a basis for the

224 monitoring of SCCmec type among isolated MRSA [5,25,26].

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225 Panton-Valentine leucocidin (PVL), a cytotoxic toxin encoded by lukS-PV and lukF-PV,

226 is associated with severe pneumonia and skin/soft-tissue infections caused by S. aureus [27].

227 PVL mainly destroys polymorphonuclear leukocytes, monocytes, and macrophages, leading to

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228 cell destruction and the release of pro-inflammatory cytokines and nuclear factor-kappa B. PVL

229 production results in necrotizing damage such as lung injury and deep-seated skin and soft-tissue

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230 infections [27]. S. aureus with PVL are reported to be more virulent, highly transmissible, more

231

232
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toxic, with a high association with mortality and morbidity compared to PVL negative S. aureus

[28]. In this study, the pvl genes were present in 4/27 (14.8%) and 2/4 (50%) of MRSA and
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233 MSSA, respectively. pvl genes have been reported to be associated with CA-MRSA strains

234 harboring SCCmec type IV, V, VI, VII, and VIII [6]. In this study, all 4 PVL positive MRSA

235 isolates harbored SCCmec type V, while 3 isolates harboring the SCCmec type IV were PVL-
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236 negative (Table 1 and Fig. 2). The PVL-positive rate of MRSA strains in this study is low,
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237 similar to a report from Japan [6]. However, a higher prevalence of PVL (>48%) has been

238 reported in Colombia, India and Saudi Arabia [6]. The variability of PVL prevalence may be

239 dependent on geography. The patients’ clinical data from this study, however, were not available
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240 to make any correlation between the presence of PVL and severity of patients’ symptoms.

241 Resistance to clindamycin, erythromycin and trimethoprim/sulfamethoxazole was an added

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242 character in PVL positive MSSA isolates when compared to PVL negative MSSA isolates,

243 which can increase their virulence. There was no observable correlation between the presence of
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244 PVL and drug resistance pattern among MRSA except 2 out of 4 MRSA SCCmec type V isolates

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245 with a trimethoprim/sulfamethoxazole resistance, which is a rare observation among MRSA in

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246 this study. The combined virulence of PVL and MDR MRSA/MSSA could prove to be a

247 challenge for an effective therapeutic treatment.

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248 Protein A (spa) is one of the virulence factors found on the surface of S. aureus which

249 prevents the bacteria from phagocytosis by the host immune system. A hypervariable region, Xr,

250 in spa gene is conserved among S. aureus strains and has been used for the development of a

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251 single-locus sequence typing technique for MRSA [14]. Typing of spa gene, which contains

252 variable numbers of 21- 27-bp repeats, with 24-bp repeat being the most common using PCR,

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253 revealed 5 different spa types among the 27 MRSA isolates with spa type t045 (15/27, 55.6%) as

254

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the most common type, similar to a previous report from a hospital in western Thailand by

Santimaleeworagun et al. 2021, followed by t001 5/27 (18.5%), t034 4/27 (14.8%), t032 2/27
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256 (7.4%), and t1379 1/27 (3.7%). The 4 MSSA isolates contained different spa types (t127, t084

257 and t034) and a new spa type, t18301, was identified and submitted to

258 https://www.spaserver.ridom.de/ with GenBank accession no. MW118058 (Fig. 2) and this is the
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259 first report of the t18301 spa type.

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260 To further characterize MRSA and to investigate the clonal relatedness of MRSA in this

261 study, MLST was performed through an analysis of allelic sequence of short DNA fragments of
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262 7 S. aureus housekeeping genes [15]. All 27 MRSA isolates were assigned into 6 different STs

263 (Fig. 2). ST764 was the most common (15, 55.6%), followed by ST1232 and ST228 at 4 strains

264 each (14.8%), ST22 at 2 strains (7.4%), and ST834 at 1 strain (3.7%). A new ST type, ST4753,
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265 was identified in 1 strain (3.7%, BioProject no. PRJNA814829) [11]. ST1, ST188, ST4751, and

266 ST1232 were identified among MSSA. ST764 was recently recognized as a hybrid of ST5
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267 lineage of HA-MRSA with few virulence traits of CA-MRSA in Japan causing invasive

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268 infections and were carried among medical students [29]. ST764 SCCmec type II, with similar

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269 genetic characteristics to ST764-MRSA-II in Japan, was recently reported to be associated with

270 severe hospital-acquired invasive diseases in Thailand as well [30], suggesting that it may have

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271 spread from Japan through healthy carriers [29,30]. Such spread was evident in reports that

272 ST764-MRSA-II was also recently reported in Shanghai and Eastern China [31,32].

273 PFGE, the gold standard of MRSA typing, is a very discriminative approach for S. aureus

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274 typing [1]. A dendrogram of percent similarity, calculated with Dice coefficients from the PFGE

275 data using a cutoff of 80%, revealed 7 major clusters of isolates, designated as patterns 1 to 7

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276 (Fig. 2). The combined analysis of PFGE pattern, MLST, spa type, SCCmec type, mecA gene,

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pvl gene, and drug resistance pattern are shown in Fig. 2. Five S. aureus isolates (4- ST1232-

SCCmec type V-t034 and 1- ST1232-SCCmec type negative-t034) could not be typed. The
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279 untypeable strains were t034 spa type which is consistent with other reports of untypeable S.

280 aureus isolates [33,34]. The untypeability could be due to DNA methylation at the SmaI

281 restriction site, CCCGGG, preventing it from being digested [35,36]. This hypervariable region
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282 of the spa gene and the epigenetic nature of this region may be a contributing factor to S. aureus
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283 virulence, in specific, its ability to evade the host immune system [37]. Pattern 1 was the main

284 PFGE cluster in this study (12/31, 38.7%, ST764-SCCmec II-t045), followed by pattern 3 (5/31,

285 16%, ST228-SCCmec I-t001 and ST4753-SCCmec I-t001) (Fig. 2). In contrast to previous
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286 reports that HA-MRSA strains ST239 SCCmec III and ST5-SCCmecII were the most common

287 types in Thailand, this study reports ST764-SCCmec II-t045 to be common [21-23,38,39]. This
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288 is similar to Kondo et al. 2022, in which this strain is highly similar to the recently emerged

289 strain in Japan indicating the intercontinental spread of this strain [30].
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290 In summary, this study was performed to characterize multidrug-resistant S. aureus

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291 isolates. All MRSA (27/31) isolates in this study exhibited resistance against non-β-lactam

292 antibiotics at varying degrees, up to 7 groups of antibiotic classes. Almost all of the MRSA

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293 isolates carry the same SCCmec and have similar antimicrobial susceptibility patterns. ST764-

294 SCCmec II-t045 (14/27, 51.9%) was the main clone identified. This study provides antimicrobial

295 resistance patterns and molecular characteristics of local MRSA in this region of Thailand. This

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296 contributes to the existing local epidemiological knowledge of MRSA of a potential shift of the

297 common strains, an identification of a new (untypeable) strain, as well as the potential of an

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298 international spread of an emerging strain. This highlights an importance of a continued

299

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surveillance effort of MRSA as its antimicrobial resistance capability continues to evolve,

maintaining its status as one of the major causes of hospital- and community-acquired infections.
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301 Funding

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302 This work was supported by The Armed Forces Health Surveillance Division – Global

303 Emerging Infections Surveillance (AFHSD-GEIS), ProMIS ID P01076_17_AF.

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304 Competing interests

305 None declared.

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306 Ethical approval

307 The study was approved by the Research Ethic Committee, Naval Medical Department,

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308 Royal Thai Navy under protocol number RP048/59 and Walter Reed Army Institute of Research

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(WRAIR), Silver Spring, MD, USA under protocol number WR# 2372.
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310 Disclaimer/Disclosure

311 Material has been reviewed by the Walter Reed Army Institute of Research. There is no

312 objection to its presentation and/or publication. The opinions or assertions contained herein are
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313 the private views of the author, and are not to be construed as official, or as reflecting true views

314 of the Department of the Army or the Department of Defense.

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315 Acknowledgements
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316 The authors would like to acknowledge and thank the staffs at the Queen Sirikit Naval

317 Hospital for providing bacterial isolates and the staff at the Walter Reed Army Institute of

318 Research - Multidrug-Resistant Organism Repository and Surveillance Network

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319 (WRAIR/MRSN) for whole-genome sequencing and bioinformatics analyses.

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320 References

ed
321 [1] Lakhundi S, Zhang K. Methicillin-Resistant Staphylococcus aureus: Molecular

322 Characterization, Evolution, and Epidemiology. Clin Microbiol Rev 2018;31(4). DOI:

iew
323 10.1128/cmr.00020-18.

324 [2] Goudarzi M, Goudarzi H, AM SF, Udo EE, Fazeli M, Asadzadeh M et al. Molecular

325 Characterization of Methicillin Resistant Staphylococcus aureus Strains Isolated from

ev
326 Intensive Care Units in Iran: ST22-SCCmec IV/t790 Emerges as the Major Clone. PLoS

327 One 2016;11(5):e0155529. DOI: 10.1371/journal.pone.0155529.

r
328 [3] Albarrag A, Shami A, Almutairi A, Alsudairi S, Aldakeel S, Al-Amodi A. Prevalence

329

330
er
and Molecular Genetics of Methicillin-Resistant Staphylococcus aureus Colonization in

Nursing Homes in Saudi Arabia. Can J Infect Dis Med Microbiol 2020;2020:2434350.
pe
331 DOI: 10.1155/2020/2434350.

332 [4] Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet

333 2022;399(10325):629-655. DOI: 10.1016/s0140-6736(21)02724-0.

ot

334 [5] Moosavian M, Shahin M, Navidifar T, Torabipour M. Typing of staphylococcal cassette

335 chromosome mec encoding methicillin resistance in Staphylococcus aureus isolates in

tn

336 Ahvaz, Iran. New Microbes New Infect 2018;21:90-94. DOI:

337 10.1016/j.nmni.2017.11.006.
rin

338 [6] Funaki T, Yasuhara T, Kugawa S, Yamazaki Y, Sugano E, Nagakura Y et al. SCCmec

339 typing of PVL-positive community-acquired Staphylococcus aureus (CA-MRSA) at a

ep

340 Japanese hospital. Heliyon 2019;5(3):e01415. DOI: 10.1016/j.heliyon.2019.e01415.

341 [7] Appelbaum PC. Microbiology of Antibiotic Resistance in Staphylococcus aureus.

Pr

342 Clinical Infectious Diseases 2007;45(Supplement_3):S165-S170. DOI: 10.1086/519474.

17

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4584919
343 [8] Xie X, Bao Y, Ouyang N, Dai X, Pan K, Chen B et al. Molecular epidemiology and

ed
344 characteristic of virulence gene of community-acquired and hospital-acquired methicillin-

345 resistant Staphylococcus aureus isolates in Sun Yat-sen Memorial hospital, Guangzhou,

iew
346 Southern China. BMC Infect Dis 2016;16:339. DOI: 10.1186/s12879-016-1684-y.

347 [9] Bhowmik D, Das BJ, Pandey P, Chetri S, Chanda DD, Bhattacharjee A. An array of

348 multiplex PCR assays for detection of staphylococcal chromosomal cassette mec

ev
349 (SCCmec) types among staphylococcal isolates. J Microbiol Methods 2019;166:105733.

350 DOI: 10.1016/j.mimet.2019.105733.

r
351 [10] Boye K, Bartels MD, Andersen IS, Møller JA, Westh H. A new multiplex PCR for easy

352

353
er
screening of methicillin-resistant Staphylococcus aureus SCCmec types I-V. Clin

Microbiol Infect 2007;13(7):725-7. DOI: 10.1111/j.1469-0691.2007.01720.x.

pe
354 [11] Ruekit S, Srijan A, Serichantalergs O, Margulieux KR, Mc Gann P, Mills EG et al.

355 Molecular characterization of multidrug-resistant ESKAPEE pathogens from clinical

356 samples in Chonburi, Thailand (2017-2018). BMC Infect Dis 2022;22(1):695. DOI:
ot

357 10.1186/s12879-022-07678-8.

358 [12] Clinical and Laboratory Standards Institute: Performance standards for antimicrobial
tn

359 susceptibility testing, 27th edition. In Edited by Institute CaLS. Wayne, PA; 2017.

360 [13] Mc Gann P, Milillo M, Kwak YI, Quintero R, Waterman PE, Lesho E. Rapid and
rin

361 simultaneous detection of the chlorhexidine and mupirocin resistance genes qacA/B and

362 mupA in clinical isolates of methicillin-resistant Staphylococcus aureus. Diagn Microbiol

ep

363 Infect Dis 2013;77(3):270-2. DOI: 10.1016/j.diagmicrobio.2013.06.006.

364 [14] Fasihi Y, Kiaei S, Kalantar-Neyestanaki D. Characterization of SCCmec and spa types of
Pr

365 methicillin-resistant Staphylococcus aureus isolates from health-care and community-

18

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4584919
366 acquired infections in Kerman, Iran. J Epidemiol Glob Health 2017;7(4):263-267. DOI:

ed
367 10.1016/j.jegh.2017.08.004.

368 [15] Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for

iew
369 characterization of methicillin-resistant and methicillin-susceptible clones of

370 Staphylococcus aureus. J Clin Microbiol 2000;38(3):1008-15. DOI:

371 10.1128/jcm.38.3.1008-1015.2000.

ev
372 [16] McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, Tenover FC.

373 Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus

r
374 isolates from the United States: establishing a national database. J Clin Microbiol

375

376 [17]
er
2003;41(11):5113-20. DOI: 10.1128/jcm.41.11.5113-5120.2003.

National Antimicrobial Resistance Surveillance Thailand: Antimicrobial Resistance

pe
377 2000-2021(12M). In Internet Available from:

378 http://narst.dmsc.moph.go.th/data/AMR%202000-2021-12M.pdf; 2021.

379 [18] Ito T, Okuma K, Ma XX, Yuzawa H, Hiramatsu K. Insights on antibiotic resistance of
ot

380 Staphylococcus aureus from its whole genome: genomic island SCC. Drug Resist Updat

381 2003;6(1):41-52. DOI: 10.1016/s1368-7646(03)00003-7.

tn

382 [19] Malachowa N, DeLeo FR. Mobile genetic elements of Staphylococcus aureus. Cell Mol

383 Life Sci 2010;67(18):3057-71. DOI: 10.1007/s00018-010-0389-4.

rin

384 [20] Jariyasethpong T, Tribuddharat C, Dejsirilert S, Kerdsin A, Tishyadhigama P, Rahule S

385 et al. MRSA carriage in a tertiary governmental hospital in Thailand: emphasis on

ep

386 prevalence and molecular epidemiology. Eur J Clin Microbiol Infect Dis 2010;29(8):977-

387 85. DOI: 10.1007/s10096-010-0954-7.

Pr

19

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4584919
388 [21] Lulitanond A, Chanawong A, Sribenjalux P, Wilailuckana C, Kaewkes W, Vorachit M et

ed
389 al. Preliminary report of SCCmec-types and antimicrobial susceptibilities of methicillin-

390 resistant Staphylococcus aureus isolates from a university hospital in Thailand. Southeast

iew
391 Asian J Trop Med Public Health 2010;41(4):920-7.

392 [22] Lawung R, Chuong LV, Cherdtrakulkiat R, Srisarin A, Prachayasittikul V. Revelation of

393 staphylococcal cassette chromosome mec types in methicillin-resistant Staphylococcus

ev
394 aureus isolates from Thailand and Vietnam. J Microbiol Methods 2014;107:8-12. DOI:

395 10.1016/j.mimet.2014.08.024.

r
396 [23] Kitti T, Seng R, Saiprom N, Thummeepak R, Chantratita N, Boonlao C et al. Molecular

397

398
er
Characteristics of Methicillin-Resistant Staphylococci Clinical Isolates from a Tertiary

Hospital in Northern Thailand. Can J Infect Dis Med Microbiol 2018;2018:8457012.

pe
399 DOI: 10.1155/2018/8457012.

400 [24] Valsesia G, Rossi M, Bertschy S, Pfyffer GE. Emergence of SCCmec type IV and

401 SCCmec type V methicillin-resistant Staphylococcus aureus containing the Panton-

ot

402 Valentine leukocidin genes in a large academic teaching hospital in central Switzerland:

403 external invaders or persisting circulators? J Clin Microbiol 2010;48(3):720-7. DOI:

tn

404 10.1128/jcm.01890-09.

405 [25] Tang YT, Cao R, Xiao N, Li ZS, Wang R, Zou JM et al. Molecular epidemiology and
rin

406 antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus isolates in

407 Xiangyang, China. J Glob Antimicrob Resist 2018;12:31-36. DOI:

ep

408 10.1016/j.jgar.2017.08.016.
Pr

20

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4584919
409 [26] Harastani HH, Araj GF, Tokajian ST. Molecular characteristics of Staphylococcus aureus

ed
410 isolated from a major hospital in Lebanon. Int J Infect Dis 2014;19:33-8. DOI:

411 10.1016/j.ijid.2013.10.007.

iew
412 [27] Santimaleeworagun W, Preechachuawong P, Samret W, Jitwasinkul T. The First Report

413 of a Methicillin-Resistant Staphylococcus aureus Isolate Harboring Type IV SCCmec in

414 Thailand. Pathogens 2021;10(4). DOI: 10.3390/pathogens10040430.

ev
415 [28] Jaiswal R, Garg A, Tripathi P, Venkatesh V. Epidemiology of Panton Valentine

416 Leukocidin in clinical Staphylococcus aureus isolates - A prospective study at a tertiary

r
417 care centre in North India. Clinical Epidemiology and Global Health 2022;15:101006.

418

419 [29]
er
DOI: https://doi.org/10.1016/j.cegh.2022.101006.

Takano T, Hung WC, Shibuya M, Higuchi W, Iwao Y, Nishiyama A et al. A new local
pe
420 variant (ST764) of the globally disseminated ST5 lineage of hospital-associated

421 methicillin-resistant Staphylococcus aureus (MRSA) carrying the virulence determinants

422 of community-associated MRSA. Antimicrob Agents Chemother 2013;57(4):1589-95.

ot

423 DOI: 10.1128/aac.01147-12.

424 [30] Kondo S, Phokhaphan P, Tongsima S, Ngamphiw C, Phornsiricharoenphant W,

tn

425 Ruangchai W et al. Molecular characterization of methicillin-resistant Staphylococcus

426 aureus genotype ST764-SCCmec type II in Thailand. Sci Rep 2022;12(1):2085. DOI:
rin

427 10.1038/s41598-022-05898-1.

428 [31] Chen W, He C, Yang H, Shu W, Cui Z, Tang R et al. Prevalence and molecular
ep

429 characterization of methicillin-resistant Staphylococcus aureus with mupirocin, fusidic

430 acid and/or retapamulin resistance. BMC Microbiol 2020;20(1):183. DOI:

Pr

431 10.1186/s12866-020-01862-z.

21

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4584919
432 [32] Gu F, He W, Xiao S, Wang S, Li X, Zeng Q et al. Antimicrobial Resistance and

ed
433 Molecular Epidemiology of Staphylococcus aureus Causing Bloodstream Infections at

434 Ruijin Hospital in Shanghai from 2013 to 2018. Sci Rep 2020;10(1):6019. DOI:

iew
435 10.1038/s41598-020-63248-5.

436 [33] Li V, Chui L, Louie L, Simor A, Golding GR, Louie M. Cost-effectiveness and efficacy

437 of spa, SCCmec, and PVL genotyping of methicillin-resistant Staphylococcus aureus as

ev
438 compared to pulsed-field gel Electrophoresis. PLoS One 2013;8(11):e79149. DOI:

439 10.1371/journal.pone.0079149.

r
440 [34] Tsao FY, Kou HW, Huang YC. Dissemination of methicillin-resistant Staphylococcus

441

442
er
aureus sequence type 45 among nursing home residents and staff in Taiwan. Clin

Microbiol Infect 2015;21(5):451-8. DOI: 10.1016/j.cmi.2014.12.019.

pe
443 [35] Bens CC, Voss A, Klaassen CH. Presence of a novel DNA methylation enzyme in

444 methicillin-resistant Staphylococcus aureus isolates associated with pig farming leads to

445 uninterpretable results in standard pulsed-field gel electrophoresis analysis. J Clin

ot

446 Microbiol 2006;44(5):1875-6. DOI: 10.1128/jcm.44.5.1875-1876.2006.

447 [36] Chlebowicz MA, Nganou K, Kozytska S, Arends JP, Engelmann S, Grundmann H et al.
tn

448 Recombination between ccrC genes in a type V (5C2&5) staphylococcal cassette

449 chromosome mec (SCCmec) of Staphylococcus aureus ST398 leads to conversion from
rin

450 methicillin resistance to methicillin susceptibility in vivo. Antimicrob Agents Chemother

451 2010;54(2):783-91. DOI: 10.1128/aac.00696-09.

ep

452 [37] Cheung GYC, Bae JS, Otto M. Pathogenicity and virulence of Staphylococcus aureus.

453 Virulence 2021;12(1):547-569. DOI: 10.1080/21505594.2021.1878688.

Pr

22

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4584919
454 [38] Chongtrakool P, Ito T, Ma XX, Kondo Y, Trakulsomboon S, Tiensasitorn C et al.

ed
455 Staphylococcal cassette chromosome mec (SCCmec) typing of methicillin-resistant

456 Staphylococcus aureus strains isolated in 11 Asian countries: a proposal for a new

iew
457 nomenclature for SCCmec elements. Antimicrob Agents Chemother 2006;50(3):1001-12.

458 DOI: 10.1128/aac.50.3.1001-1012.2006.

459 [39] Song JH, Hsueh PR, Chung DR, Ko KS, Kang CI, Peck KR et al. Spread of methicillin-

ev
460 resistant Staphylococcus aureus between the community and the hospitals in Asian

461 countries: an ANSORP study. J Antimicrob Chemother 2011;66(5):1061-9. DOI:

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462 10.1093/jac/dkr024.

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464 Table 1: Distribution of antimicrobial resistance profiles and the presence of PVL among

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465 reported MRSA and MSSA isolated from Queen Sirikit Navel Hospital, Chonburi, Thailand

466 between April 2017 and May 2018

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Number
of
PVL
resistant Antibiotic classes MSSA MRSA
positive
antibiotic
classes

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1 β-lactam (AMP and PEN only) 2 - -
β-lactam (AMP and PEN only), Folate pathway inhibitors 1 - 1
2
β-lactam, Fluoroquinolones - 1 -
β-lactam (AMP and PEN only), Lincosamides, Macrolides 1 - 1

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3 β-lactam, Fluoroquinolones, Tetracyclines - 1 -
β-lactam, Lincosamides, Macrolides - 2 2

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β-lactam, Lincosamides, Macrolides, Folate pathway inhibitors
β-lactam, Fluoroquinolones, Aminoglycosides, Tetracyclines
β-lactam, Fluoroquinolones, Lincosamides, Macrolides
β-lactam, Fluoroquinolones, Lincosamides, Macrolides,
-
-
-
2
1
2
2
-
-
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- 2 -
Tetracyclines
5
β-lactam, Fluoroquinolones, Lincosamides, Macrolides,
- 4 -
Aminoglycosides
β-lactam, Fluoroquinolones, Lincosamides, Macrolides,
- 9 -
Aminoglycosides, Tetracyclines
6
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β-lactam, Fluoroquinolones, Lincosamides, Macrolides,

- 1 -
Aminoglycosides, Monoxycarbolic acid
β-lactam, Fluoroquinolones, Lincosamides, Macrolides,
- 1 -
Aminoglycosides, Ansamycins, Tetracyclines
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7
β-lactam, Fluoroquinolones, Lincosamides, Macrolides,
- 1 -
Ansamycins, Tetracyclines, Folate pathway inhibitors
467 MRSA: methicillin-resistant Staphylococcus aureus; MSSA: methicillin-susceptible Staphylococcus
468 aureus; PVL: Panton-Valentine leucocidin; β-lactams group (AMP – ampicillin, PEN – penicillin, and
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469 oxacillin), Aminoglycosides (gentamicin), Macrolides (erythromycin), Lincosamides (clindamycin),

470 Fluoroquinolones (ciprofloxacin), Tetracyclines (tetracycline), Ansamycins (rifampin), Monoxycarbolic
471 acid (mupirocin), and Folate pathway inhibitors group (trimethoprim-sulfamethoxazole)
472
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473

474

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477
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Fig. 1. PCR amplification products obtained with SCCmec types I-V by multiplex PCR. Lanes
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478 on the left and right of the gel contain 100-bp (GeneRulerTM) ladder molecular size markers
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 ed
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479
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480 Fig. 2. A dendrogram of PFGE clusters and genotypic relationships of reported S. aureus isolates. SmaI
481 patterns were analyzed using the Dice coefficient method, using the unweighted pair group method with
482 averages (UPGMA) at 1.5% tolerance and 1.5% optimization settings. PFGE groups determined by
483 cluster analysis are numbered from 1 to 7. The MLST ST types, spa types, SCCmec types, mecA, and
484 antibiotic resistance profile are also included. Abbreviations for the antibacterial agents: amikacin
485 (AMK), ampicillin (AMP), ciprofloxacin (CIP), clindamycin (CLI), erythromycin (ERY), gentamicin
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486 (GEN), linezolid (LZD), mupirocin (MUP), oxacillin (OXA), penicillin (PEN), rifampin (RIF),
487 teicoplanin (TEC), tetracycline (TET), trimethoprim-sulfamethoxazole (SXT), vancomycin (VAN). R –
488 resistant, S – sensitive, and X – uninterpretable.
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 Molecular Characterization of Methicillin-Resistant Staphylococcus aureus

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Strains Isolated from Chonburi, Thailand
Patcharawalai Wassanarungroj1, Panida Nobthai1, Sirigade Ruekit1, Apichai Srijan1, Theerasak
pimsawat2, Rosarin Kormanee2, Suthisak Nakornchai2, Chaiwat Sakdinava2, Prawet Sukhchat2,

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Oralak Serichantalergs1, Paphavee Lertsethtakarn1,*, John M. Crawford3, and Brett E.
Swierczewski4
1Department of Bacteria and Parasitic Diseases, Armed Forces Research Institute of Medical
Sciences (AFRIMS), Bangkok, 10400, Thailand,
2Queen Sirikit Naval Hospital, Chonburi, Thailand,

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3US Army Medical Research Institute of Chemical Defense, Gunpowder, Maryland, USA
4US Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA.

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*Corresponding author: Department of Bacteria and Parasitic Diseases, Armed Forces Research

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Institute of Medical Sciences (AFRIMS) 315/6 Rajvithi Rd. Bangkok, 10400, Thailand. E-mail
address: PaphaveeL@afrims.org Telephone: +6626962700 ext.4530
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