ACCEPTED. Division of pediatric infectious diseases, Chang Gung Children s Hospital and Chang

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1 JCM Accepts, published online ahead of print on 1 October 00 J. Clin. Microbiol. doi:./jcm.0-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Prevalence of methicillin-resistant Staphylococcus aureus nasal colonization among Taiwanese children, Yhu-Chering Huang 1,, MD, PhD, Kao-Pin Hwang,, MD, PhD, Po-Yen Chen, MD, Chih-Jung Chen 1,, MD, Tzou-Yien Lin 1,, MD Division of pediatric infectious diseases, Chang Gung Children s Hospital and Chang Gung Memorial Hospital at Linko 1 and Kaohsiung, Taiwan College of Medicine, Chang Gung University, Taoyuan. Taiwan Department of Pediatrics, Taichung Veterans General Hospital, Taichung, Taiwan Running title: prevalence of MRSA carriage in Taiwan Correspondence and reprints request: Dr. Yhu-Chering Huang, Department of Pediatrics, Chang Gung Children s Hospital,, Fu-Shin Street, Kweishan, Taoyuan, Taiwan Tel: --0 ext 0 1 Fax: ychuang@adm.cgmh.org.tw The authors have indicated they have no financial relationships relevant to this article to disclose. 1

2 Abstract From July 00 to October 00, a total of,0 children, aged between months and years, presented for a well-child healthcare visit to one of three medical centers, which are located in the northern, central and southern parts of Taiwan, respectively, were surveyed for the nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA). The overall prevalence of S. aureus and MRSA nasal carriage among the children was % and.%, respectively (1% and.% in central, % and.% in southern, and % and.% in northern regions). Of the 1 MRSA isolates (%) available for analysis, a total of pulsed-field gel electrophoresis (PFGE) patterns with two major patterns (C, 1% and D, %) were identified. 1 isolates (0%) contained type IV staphylococcal cassette chromosome mec (SCCmec) DNA and isolates (%) contained SCCmec V T. The presence of Panton-Valentine Leukocidin (PVL) genes was detected in 0 isolates (%). Most MRSA isolates belonged to one of two major clones characterized as sequence type /PFGE C/SCCmec IV/absence of PVL genes (%) and ST /PFGE D/SCCmec V T /presence of PVL genes (%). 1 We concluded that between 00 and 00,.% of healthy Taiwanese children were 1 colonized with MRSA in nares. MRSA harbored in healthy children indicates 1 accelerated spread in the community. 1 Key words: community-acquired, methicillin-resistant Staphylococcus aureus, 0 colonization, Taiwan

3 Introduction Recent reports indicate that community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are increasing world-wide and may now involve persons without risk factors predisposing for acquisition [,-1,0,]. Asymptomatic CA-MRSA colonization has been documented in healthy children attending the emergent department and outpatient clinic of the children s hospital [,1,,,]. S. aureus, including MRSA, carriage is well known to be a significant risk factor for subsequent infection [,] and the anterior nares are the most consistent sites of colonization. The presence of S. aureus nasal colonization can provide an indication of a high risk for subsequent infection. In Taiwan, previous reports [1,,,1,1,,1] have indicated that during the period from 1 to 00, MRSA accounted for.% to % of CA S. aureus infections in children without risk factors and the MRSA colonization rate in the general population ranged from 1.% ~.% for school children,.% for healthy 1 children presented for healthcare visits, to.% for health care workers and 1.% 1 for contacts of CA-MRSA infection. It is noteworthy, however, that most of these 1 studies were conducted in the northern part of Taiwan and no island-wide survey has 1 yet been conducted to elucidate this issue. To estimate the extent of MRSA in the 0 community in Taiwan and to assess if there is an increasing trend of MRSA nasal

4 colonization in the healthy children during the past years, we conducted this island-wide survey between 00 and 00. All collected MRSA isolates were also further characterized by molecular methods.

5 Materials and Methods This study was approved by the Institutional Review Board of the Chang Gung Memorial Hospital. From July 00 to October 00, all children aged between months and years who presented for a well-child healthcare visit to any one of three medical centers in Taiwan were invited to participate in this study. The three medical centers involved were the Chang Gung Children s Hospitals at Linko (Hospital A) and Kaohsiung (Hospital C), and Taichung Veterans General Hospital (Hospital B), which are situated,respectively, in northern, southern and central parts of Taiwan. In each hospital, around 0 subjects were recruited for study for each month and the age of the subjects were evenly distributed in separate age ranges, which included >-months, > to 1 months, >1 to 1 months, >1 to months, > to years, > to years and > to years. A culture of the anterior nares for the detection of MRSA was obtained from each subject after a written consent was obtained from their parents/guardians. Survey specimens for culture were obtained with a cotton swab, placed in the 1 transport medium (Venturi Transystem, Copan Innovation Ltd., Limmerick, Ireland), 1 and then brought to and processed in the microbiologic laboratories within four hours 1 of the sample taken. All S. aureus isolates were sent to Chang Gung Memorial 1 Hospital at Linko for microbiologic characterization. Identification of MRSA was

6 confirmed according to Clinical and Laboratory Standards Institute (CLSI) guidelines, []. Pulsed-field gel electrophoresis (PFGE) with SmaI digestion was used in this study to fingerprint the MRSA isolates, and was performed according to the procedures described previously [,1,1]. The genotypes were designated, as in our previous studies [,1-1], in alphabetical order; any new genotype, if identified, was designated consecutively. PFGE patterns with fewer than -band differences from an existing genotype were defined as subtypes of that genotype. The SCCmec typing for isolates was determined by a multiplex PCR strategy described previously []. Control strains for SCCmec types I, II, III and IVa, kindly provided by Dr Keiichi Hiramatsu, were as follows: type I, NCTC; type II, N1; type III, /0; and type IVa, JCSC. SCCmec typing for type V T was determined by a particular primer described elsewhere [1] and the strain TSGH-1, kindly provided by Dr Chi-Chien Wang, was used as control. However, SCCmec typing method for type V T yielded inconsistent results, thus an alternative method was used. An isolate with only two bands (1 bp and bp) appearing in the multiplex 1 PCR analysis may have indicated that the isolate contained SCCmec V T. To confirm 1 their identities, a novel pair of primers, ccrc-f ( -CAC TTA ATC CAT TGA CAC 1 AG- ) and ccrc-r ( -AAA GAT TGA GGG ATA AGA CT- ), were designed 1 according to the published sequence (GenBank accession no. AY1) of the ccrc

7 gene of a Taiwanese strain, S. aureus TSGH-1. Amplification of a specific 1-bp DNA fragment, which was subjected to further sequence analysis for some representative isolates in preliminary experiments, confirmed that the isolates contained SCCmec V T. The presence of Panton-Valentine Leukocidin (PVL) genes was determined by a PCR strategy described previously []. Some isolates of representative PFGE patterns were selected and underwent multilocus sequence typing (MLST) as described elsewhere []. The allelic profiles were assigned through comparison of the sequences at each locus with those of the known alleles in the S. aureus MLST database and were defined as sequence types (STs) accordingly.

8 Results During the study period, 1, subjects were recruited from Hospital A, 1,0 subjects from Hospital B (from July 00 to June 00) and subjects from Hospital C (from October 00 to June 00). All the children enrolled are Taiwanese. The subjects enrolled in each age group ranged from 0 in children aged >-1 months to in children aged >- years. Of the total of,0 subjects enrolled in this study, 1 (%) were colonized with S. aureus. Of the 1 isolates 1 (1%) were demonstrated to be MRSA. The details of the nasal MRSA colonization prevalence of the subjects in the different parts of Taiwan are shown in Table 1. The MRSA colonization rate in northern Taiwan was significantly higher than that in central (p < 0.001) and southern (p < 0.0) parts of Taiwan. The nasal MRSA colonization prevalence of the subjects in each age group was.% for the children aged >- months, and.%,.%,.%,.0%,.%, and.1% for the children aged >-1 months, >1-1 months, >1- months, >- years, >- years and >- years, respectively. In those less than 1 months of age, the carriage rate decreased 1 with increasing age (p = 0.00 by Mantel-Haenszel test for trend), while in those 1 older than 1 months of age, the carriage rate increased with increasing age (p < ). 1 Of the 1 MRSA isolates, 1 isolates were available for analysis. All of these

9 1 isolates were sensitive to vancomycin and teicoplanin. All but two of the isolates identified from Hospital A were resistant to penicillin. Most isolates were resistant to erythromycin and clindamycin but sensitive to SXT and doxycycline. The detailed susceptibility distribution of various antibiotics to the isolates is shown in Table. No significant difference in antibiotic susceptibility patterns was noted among the isolates from the three different regions of Taiwan. Table illustrates the detailed distribution of PFGE patterns, SCCmec types and the presence/absence of PVL genes in these isolates. A total of ten PFGE patterns were identified. Patterns C and D were the two most common patterns and accounted for % and % of the isolates analyze, respectively. The distribution of PFGE patterns among the three regions showed a trend for a difference (p = 0.0 by a log-likelihood contingency test). Four types (types II, III, IV, and V T ) of SCCmec genes were identified among the isolates, with type IV (0%) being the predominant type, followed by type V T (%). The distribution of SCCmec types among the three regions was significantly different (p = 0.0). Four isolates of the AF PFGE pattern 1 were untypable by the methods used in this study. PVL genes were present in 0 1 isolates (%). isolates underwent MLST and eight sequence types (ST) were 1 identified. ST was the most common sequence type and accounted for of 1 PFGE type C isolates, of PFGE type D isolates and the isolate of PFGE type AN.

10 The other two isolates of PFGE type D were ST, which is a single locus variant of ST (a single nucleotide difference in gmk locus). The remaining one isolate of PFGE type C belonged to a new sequence type, which is a single locus variant of ST (a single nucleotide difference in pta locus). One isolate of PFGE type F also belonged to a new sequence type, which is also a single locus variant of ST (a single nucleotide difference in gmk locus).the detailed association of PFGE patterns with sequence types, SCCmec types and the presence of PVL gene of these isolates are shown in Table. The MRSA isolates characterized by ST/PFGE type C/SCCmec IV/absence of PVL genes and ST/PFGE type D/SCCmec V T /presence of PVL genes were the two most common clones and accounted for % and % of the isolates analyzed, respectively. Discussion Results from this study indicate that the national prevalence of nasal MRSA colonization among otherwise healthy children in Taiwan was.% during the period from July 00 to October 00 inclusively, the value ranging from.% in the 1 central region of Taiwan to.% in the northern region of Taiwan. Compared with 1 those among the healthy children during the period [1,1,] (Table ), 1 though the study population were different for these studies, nasal MRSA colonization 1 prevalence among healthy children in Taiwan increased significantly from 1.% in

11 001 to.% (p < by chi-square test) during the period 00 to 00 for northern Taiwan and significantly from.% to.% for southern Taiwan (p < by chi-square test). This increasing trend of nasal MRSA colonization prevalence might account for the increasing incidence of CA-MRSA infection in children in Taiwan [,,1]. In the United States, where CA-MRSA is also being increasingly reported, MRSA colonization prevalence for the general population appeared to have been relatively low until the year 00 [1,1,,,]. In a survey [1] involving, persons conducted between 001 and 00, national S. aureus and MRSA nasal colonization prevalence estimates were.% and 0.%, respectively. For healthy children the nasal colonization rates ranged from 0.% to.% [1,,,] as reported in several pediatric studies, however, an increasing trend in this regard has been noted in certain areas of the US recently []. Creech II et al [] reported that nasal MRSA colonization rate among healthy children in Nashville, Tennessee increased significantly from 0.% in 001 to.% in 00, a picture not dissimilar to what we showed in the present study from Taiwan. 1 In the United States, CA-MRSA strains have been recognized as a novel 1 pathogen which was genetically different from the nosocomial MRSA strains [1,]. 1 They have limited antibiotic resistance (except to β-lactams), have two common 1 pulsed-gel electrophoresis (PFGE) patterns (USA 00 and USA 00), possess

12 different exotoxin gene profiles (e.g. Panton-Valentine leukocidin, PVL) and contain type IV staphylococcal cassette chromosome mec (SCCmec) DNA []. In contrast, the CA-MRSA clinical isolates in Taiwan were multi-resistant and shared two common PFGE patterns (as patterns D and C in this study) [1,,,0]. In the current study, more than 0% of the colonized MRSA isolates were multi-resistant to erythromycin and clindamycin but sensitive to SXT and doxycycline. In addition, most colonized isolates shared common molecular characteristics and more than 0% of the isolates belonged to one of two major clones characterized by ST/PFGE type C/SCCmec IV/absence of PVL genes and ST/PFGE type D/SCCmec V T /presence of PVL genes. However, among the clinical isolates, the clone characterized by ST/PFGE type D/SCCmec V T /presence of PVL genes was the dominant clone [1,,0], while among the colonized isolates, the clone characterized by ST/PFGE type C/SCCmec IV/absence of PVL genes was dominant. It seemed that PVL genes, which is reported to be a virulent factor associated with necrotizing pneumonia, and abscesses [], may be associated with the ability of PVL (+) clone to cause infection. 1 There existed several limitations in the current study. Firstly, the demographic 1 characteristics and the risk factors associated with MRSA acquisition were not 1 analyzed and compared between the children with and without CA-MRSA 1 colonization, though all the children were healthy and presented for healthcare visits. 1

13 Living with a family member who works in a hospital or clinic and demographic characteristics (eg. age, gender) were reported to be associated with an increased risk of MRSA colonization [,1,]. Secondly, the persistence of MRSA carriage in the subjects could not be determined and the incidence of subsequent MRSA infection in the subjects could not be measured in this cross-sectional analysis of MRSA nasal colonization prevalence. In summary,.% of healthy children in Taiwan were colonized with MRSA in the nares during the period from 00 to 00. MRSA carriage in the children may accelerate the spread in the community. Two major CA-MRSA clones were identified CCEPTED and would appear to have spread island-wide. Further studies are needed to determine the host factors of colonization, and to develop strategies to disrupt transmission of 1ACA-MRSA to susceptible hosts. 1

14 Acknowledgement This study was supported by a grant from National Science Counseling of Executive Yuan of Taiwan. (NSC-1-B1A-1) 1

15 References Boyle-Vavra S, Ereshefsky B, Wang CC, Daum RS. Successful multiresistant community-associated methicillin-resistant Staphylococcus aureus lineage from Taipei, Taiwan, that carries either the novel Staphylococcal chromosome cassette mec (SCCmec) type V T or SCCmec type IV. J Clin Microbiol 00;:1-0.. Chambers HF. The changing epidemiology of Staphylococcus aureus? Emerg Infect Dis 001;:1-.. Chen CJ, Huang YC, Chiu CH, Su LH, Lin TY. Clinical features and genotyping analysis of community-acquired methicillin-resistant Staphylococcus aureus infections in Taiwanese children. Pediatr Infect Dis J 00;:0-.. Chen FJ, Lauderdale TL, Huang IW, et al. Methicillin-resistant Staphylococcus aureus in Taiwan. Emerg Infect Dis 00; : -.. Clinical and Laboratory Standards Institutes. Performance standards for antimicrobial disk diffusion susceptibility testings: 1 th informational supplement. Wayne, Pa: Clinical and Laboratory Standards Institutes, Creech II CB, Kernodle DS, Alsentzer A, Wilson C, Edwards KM. Increasing 1 rates of nasal carriage of methicillin-resistant Staphylococcus aureus in healthy 1 children. Pediatr Infect Dis J 00;: Ellis MW, Hospenthal DR, Dooley DP, Gray PJ, Murray CK. Natural history of 1

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19 Prevalence of methicillin-resistant Staphylococcus aureus nasal carriage in the community pediatric population. Pediatr Infect Dis J 00;1:1-.. Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 00;:1-1.. Shopsin B, Mathema B, Martinez J, et al. Prevalence of methicillin-resistant and methicillin-susceptible Staphylococcus aureus in the community. J Infect Dis 000;1:-.. Suggs AH, Maranan MC, Boyle-Vavra S, Daum RS. Methicillin-resistant and borderline methicillin-resistant asymptomatic Staphylococcus aureus colonization in children without identified risk factors. Pediatr Infect Dis 1;1:-.. von Eiff C, Becker K, Machka K, Stammer H, Peters G. Nasal carriage as a source of Staphylococcus aureus bacteremia. N Engl J Med 001;:-. 0. Wang CC, Lo WT, Chu ML, Siu LK. Epidemiological typing of 1 community-acquired methicillin-resistant Staphylococcus aureus isolates from 1 children in Taiwan. Clin Infect Dis 00;: Wu KC, Chiu HH, Wang JH, Lee NS, Lin HC, Hsieh CC, et al. Characteristics of 1 community-acquired methicillin-resistant Staphylococcus aureus in infants and 1

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21 Table 1. Nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) among infants and children presented for a well-child healthcare visit in Taiwan Area of Taiwan Subject No.(%) No. with S. aureus (%) No. with MRSA Northern 1, (.) (.) Central 1,0 (1.) (.) Southern 1 () 1 (.) Total,0 1 (.) 1 (.) *The carriage of S. aureus among children in central region was significantly lower than those among children in northern and southern regions (p < 0.001) **The carriage of MRSA among children in northern region was significantly higher than that among children in central (p < 0.001) and southern (p = 0.0) regions. (%) 1

22 Table. Antibiotic susceptibility rates of 1 colonized methiciliin-resistant Staphylococcus aureus isolates from children in Taiwan Area of Taiwan Penicillin Erythromycin Clindamycin Doxycycline SXT Vancomycin Teicoplanin No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) Northern (1.) (.) 1 () () () (0) (0) (n=) Central (n=) 0 1 (.) (.1) () () (0) (0) Southern 0 () () 0 (0) () 0 (0) 0 (0) (n=0) Total (n=1) (0.) 1 (.) 1 (.0) 0 () 0 () 1 (0) 1 (0) SXT, trimethoprim/sulfamethoxazole

23 Table. Distribution of pulsed-field gel electrophoresis (PFGE) patterns, staphylococcal chromosomal cassette (SCCmec) types and presence of Panton-Valentine leukocidin (PVL) genes among 1 colonized methicillin-resistant Staphylococcus aureus isolates Area of Taiwan PFGE patterns, No. (%) SCCmec types, No. (%) PVL genes, No (%) A C D Other II III IV V T Present Absent Northern (n=) 1 (0.) () 0 () 1 () () (1) 0 () 0 () Central (n=) (.) () () (.) 0 (.) () (1) () 1 () Southern (n=0) 0 () (1) () () 0 () (0) (1) 1 () Total (n=1) (1.) () () 1 (.0) (0.) (0.) 1 (0) () 0 () 1 () *SCCmec types were untypable for isolates **The distribution of SCCmec types was significantly different among the three regions (p = 0.0).

24 Table. Association of pulsed-field gel electrophoresis (PFGE) patterns with multilocus sequencing types (MLST), staphylococcal chromosomal cassette (SCCmec) types and presence of Panton-Valentine leukocidin (PVL) genes of 1 methicillin-resistant Staphylococcus aureus isolates Characteristics PFGE patterns (isolates number) A (n=) C (n=) D (n=) F (n=) AF (n=) AK (n=) AN (n=) AQ (n=) AR (n=1) BA (n=1) Subtype No SCCmec type III(),IV(1), IV(1), V T (), II(), V T () PVL genes Absent Absent(1), IV() V T (1) untypable IV IV IV IV IV Present(), Absent Absent Absent Absent Absent Absent Absent present() absent() Sequence type, new*, *, new* *All sequence types are a single locus variant of ST but differ from each other

25 Table. Reported prevalence rates of methicillin-resistant Staphylococcus aureus nasal colonization for healthy Taiwanese children between 001 and 00 Period Area of Taiwan Subject Subject age Subject No. No. with MRSA 001 Southern School children years ~ 1 years (.) Lu [0] (%) References Northern School children years ~1 years (1.) Huang [1] 00 Northern School children, healthcare visits < 1 years 0 (.)* Boyle-Vara [1] Northern Children for months ~ 1, (.) This study Central healthcare visits years 1,0 (.) Southern 1 (.) *if restricted to those without risk factors, colonized subjects would be (.%)

26 #The prevalence increased significantly in the northern (p < by chi-square test) and southern (p < 0.001) regions of Taiwan during the period 001 to 00.