- Email: [email protected]
Emergence, transmission and phylogeny of meticillin-resistant Staphylococcus aureus sequence type 8 (USA300) in Taiwan
Accepted Manuscript Emergence, transmission and phylogeny of meticillin-resistant Staphylococcus aureus sequence type 8 (USA300) in Taiwan Yhu-Chering Huang, MD, PhD, Chih-Jung Chen, MD, PhD, Ching-Chia Kuo, MD, Min-Chi Lu, MD, PhD PII:
S0195-6701(18)30107-5
DOI:
10.1016/j.jhin.2018.02.014
Reference:
YJHIN 5351
To appear in:
Journal of Hospital Infection
Received Date: 15 December 2017 Accepted Date: 14 February 2018
Please cite this article as: Huang Y-C, Chen C-J, Kuo C-C, Lu M-C, Emergence, transmission and phylogeny of meticillin-resistant Staphylococcus aureus sequence type 8 (USA300) in Taiwan, Journal of Hospital Infection (2018), doi: 10.1016/j.jhin.2018.02.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Format: Short report
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Title: Emergence, transmission and phylogeny of meticillin-resistant
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Staphylococcus aureus sequence type 8 (USA300) in Taiwan
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Yhu-Chering Huang1, MD, PhD, Chih-Jung Chen1, MD, PhD, Ching-Chia Kuo1, MD,
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Min-Chi Lu2, MD, PhD,
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Taiwan, and Chang Gung University College of Medicine, Taoyuan, Taiwan
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2
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Department of Microbiology and Immunology, School of Medicine, China Medical
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Division of Pediatric Infectious Diseases, Chang Gung Memorial Hospital, Taoyuan,
Department of Medicine, China Medical University Hospital, Taichung, Taiwan, and
University, Taichung, Taiwan
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Running Title: Identification of MRSA USA300 in Taiwan
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Address for correspondence: Yhu-Chering Huang, Department of Pediatrics, Chang
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Gung Memorial Hospital, No. 5, Fu-Shin Street, Gueishan 333, Taoyuan, Taiwan.
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TEL: +886-3-3281200, Fax: +886-3-3288957, E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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We identified 27 (0.51%) USA300 isolates from a pool of 5308 meticillin-resistant
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Staphylococcus aureus (MRSA) isolates collected in Taiwan between 1995 and
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October 2015, including 12 infecting isolates from 10 patients. The first two isolates
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were identified in 2005 and 23 isolates since 2010. Phylogenetic analysis revealed all
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the local isolates were closely related to those in North America and there was a clade
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consisting of 13 local isolates from 10 patients. MRSA USA300 existed in Taiwan in
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2005 or earlier, with increasing identification since 2010. Local transmission of
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USA300 strains have occurred in Taiwan after imported from North America.
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Keywords: meticillin-resistant Staphylococcus aureus, sequence type 8, USA300,
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pulsotype, Taiwan
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Introduction In Taiwan, meticillin-resistant Staphylococcus aureus (MRSA) was first documented in early 1980s and rapidly increased in 1990s [1]. While a declining trend
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of healthcare-associated (HA)-MRSA incidence was observed in 2000s,
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community-associated (CA)-MRSA infections have been increasingly reported,
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particularly in pediatric patients [1-3]. MRSA sequence type (ST) 59 is the major
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community clone in Taiwan previously reported, while MRSA ST8 (USA300) has
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prevailed in the community in North America [1-4]. Emergence of MRSA ST 8 has
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been reported in several Asian countries [3,5-10]. Recently, we identified four MRSA
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ST 8 isolates from children with CA-MRSA infection in 2012 in our hospital [11],
41
prompting our study on this strain in Taiwan. Herein, we report the emergence,
42
transmission and phylogenetic analysis of MRSA USA300 isolates identified in our
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laboratory in Taiwan as well as the clinical features of these patients with MRSA
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USA300 infections.
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Methods:
For surveillance of the molecular epidemiology of MRSA, we collected and
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molecularly characterized a total of 5308 MRSA isolates island-wide in Taiwan
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between 1995 and October 2015 in our laboratory, which is located in Chang Gung 3
ACCEPTED MANUSCRIPT Memorial Hospital (CGMH). In addition to CGMH, the isolates were also collected
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from at least 10 hospitals in Taiwan. 1809 isolates (34%) were collected between
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1995 and 2004, 2537 isolates (48%) between 2005 and 2010 and 962 isolates (18%)
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between 2011 and 2015. The isolates included 2968 (56%) infecting isolates, 2235
54
(42%) human colonizing isolates and 105 (2.0%) non-human isolates (93 from pigs, 9
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from the environmental objects, 2 from a bus and one from the pork). Among the
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infecting isolates, 980 isolates were identified from paediatric patients, 276 from adult
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patients and the remaining 1712 isolates from all age groups for surveillance of
58
molecular epidemiology of MRSA in Taiwan (we estimate that more than 90% of
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these isolates were from adults). The infecting isolates were identified from any body
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site; 460 (15%) of these were community-associated. Among the colonizing isolates,
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1632 isolates (73%) were identified from children < 18 years of age, including infants
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hospitalized in neonatal intensive care units (NICUs), neonates in nurseries, children
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for well baby clinic visits, school children and case patients. The 603 adult isolates
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originated from parturient mothers and elderly nursing home residents. Fifty-two
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percent of the 2235 colonizing isolates were identified from the subjects without risk
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factors for MRSA acquisition.
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Identification of MRSA USA300
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To identify USA300 MRSA isolates, we examined and compared the PFGE 4
ACCEPTED MANUSCRIPT patterns of these isolates with USA300 reference isolate (control strain, LAC). For
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those with same or similar PFGE pattern as USA300, we performed further
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characterizations, including staphylococcal cassette chromosomal typing (SCCmec),
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multilocus sequence typing, spa typing, and detection of arginine catabolic metabolic
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element (ACME) and Panton-Valentine leukocidin (PVL) genes and some other toxin
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genes. All the methods were performed according to the procedures described
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elsewhere [11-13].
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Whole genome sequencing (WGS) and Phylogenetic analysis
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All the USA 300 isolates identified were subjected to whole genome sequencing
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(WGS), which was performed on an Illumina MiSeq sequencer (Illumina, San Diego,
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CA, USA) [13] and the results were deposited at DDBJ/ENA/GenBank under the
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accession PRJNA393756. Single nucleotide polymorphisms (SNPs) in the core
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genome were used to construct the phylogenies of the USA300 MRSA strains [14,15].
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To clarify the genetic relatedness of the strains in Taiwan and global isolates, 10
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published whole genome sequences data of USA300 strains were downloaded from
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the GenBank database and included for analysis.
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Clinical features of the case patients with confirmed MRSA USA300 infection were
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retrospectively reviewed. This study was approved by Chang Gung Medical 5
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Foundation Institutional Review Board (IRB No.:201600069B0).
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Results: Among the pool of 5308 MRSA isolates, we identified 27 (0.51%) USA300
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isolates. There were 12 infecting isolates from 10 patients with MRSA infection. The
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colonizing strains comprised one isolate each from a patient (Patient 7) and their
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mother’s breast milk (Patient 1); one isolate from a healthy child in the carriage
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survey (Patient 2); four isolates from human immunodeficiency virus (HIV)-infected
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patients in a carriage survey; seven isolates from elderly residents of three nursing
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homes; and one environmental isolate. Patient 7 had three infecting isolates and one
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colonizing isolate available for analysis. The details of the 27 isolates are presented in
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Table 1. The first two isolates were identified in 2005 and 23 isolates since 2010.
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Of the 10 infected patients seven were children or adolescents (age range 15 days to 15 years; median 6 years) and three were elderly (> 85 years) (Table 1). Of the
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seven children and adolescents, five had skin and soft tissue infection (SSTI) and
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recovered uneventfully. One had central venous catheter-related bacteraemia and the
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other (Patient 7) was a 15-day-old neonate with community-onset necrotizing
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pneumonia with empyema and bronchopleural fistula. He recovered after surgery and
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a 28-day course of vancomycin and linezolid. Three infecting isolates from this
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patient, and one colonizing isolate from the mother, were available for
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characterization. All 27 isolates shared the common molecular characteristics with USA 300 MRSA, including the same pulsotype, carrying type IV SCCmec, possessing type I
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ACME, PVL genes and arcA, bsaA,Sek and Seq genes and belonging to ST 8 and spa
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type t008 complex. Three subtypes were identified, one to three bands different from
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the control (LAC) strain. All 27 isolates were susceptible to vancomycin, teicoplanin,
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linezolid, fusidic acid, daptomycin, clindamycin, trimethoprim/sulfamethoxazole, and
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doxycycline, but only one isolate was susceptible to erythromycin and nine isolates
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were resistant to ciprofloxacin.
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The phylogeny based on SNPs in core genomes of 27 Taiwanese USA300 strains and 10 global USA300 reference strains is displayed in Figure 1. In this analysis, the
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greatest number of SNP was identified in a Venezuela isolate (V1859) when the
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earliest isolate TCH1516 was used as reference (451 SNPs). The strains from
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Germany (MRSA_S1) and Colombia (HUV03 and HUV07) were also
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phylogenetically distant from the strains isolated in North America in early 2000s
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(TCH1516, FPR3757 and C2406). Recombination events were more frequently
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identified in these genetically distant strains. The data suggested that the USA300
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strains present in certain regions outside the United States might have undergone
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substantial genomic evolution including point mutations and recombinations.
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ACCEPTED MANUSCRIPT However, on the contrary, most strains (including all the strains identified in Taiwan
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and those from Belgium (UA-S391) and Suriname (USA300-SUR23 and
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USA300-SUR24) remained closely related to the North American strains (Figure 1).
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Among the local USA300 strains in Taiwan, we found a clade consisting of 13 strains
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identified from 10 (43.5%) of 23 subjects of different age populations, at different
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facilities, and associated with distinct clinical syndromes (ellipse shadow, Figure 1).
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These strongly suggested that local transmission of USA300 strains had occurred in
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Taiwan after imported from North America.
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Discussion
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To our knowledge, this is the first report focusing on MRSA USA300 in Taiwan and whole genome sequencing of MRSA USA300 in Asia. In this study, the first
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USA300 isolate was identified in 2005 from a school-aged child with SSTI, which
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suggests that USA300 existed in Taiwan more than 10 years. Although our MRSA
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collection was not complete, the fact that 78% of the 27 USA300 isolates were
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identified after 2011 suggests that there has recently been a true increase in the
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prevalence of USA300 isolates in Taiwan.
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The scenario of a wide variation in disease spectrum is similar to that observed in
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North America [4]. Also of note is that USA300 strains were found in widely differing
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surveillance populations, including nursing home residents and HIV-infected patients. 8
ACCEPTED MANUSCRIPT Our findings suggest that MRSA USA300 in spreading from person-to-person in
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Taiwan. Recently, identification of MRSA USA300 has been reported from several
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Asian countries such as Japan, South Korea and Singapore. Since 2007 there have
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been two outbreaks of MRSA USA300 reported in Japan [5,6]. Nagao et al [5]
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reported an outbreak in a general hospital ward involving six health care workers and
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four patients; eight had SSTIs and one each had catheter-related bacteraemia and
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pneumonia. The other outbreak affected one patient and three nurses, all of whom had
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SSTIs [6]. Since being first identified in South Korea in 2008 [7], Jung et al [9]
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reported 10 USA300 isolates among 818 MRSA bloodstream isolates collected from
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four medical centers between 2008 and 2015. All 10 isolates were
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healthcare-associated. In Singapore Grant et al [10] reported an outbreak of 27 SSTIs
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over three months in an international school (most of whom held US passports and
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had travelled to the US in previous months), including eight confirmed MRSA
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USA300 cases. These experiences from other Asian countries suggest that once a new
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pathogen is introduced into the community it is only a matter of time before it reaches
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healthcare settings, resulting then in nosocomial spread and even outbreaks.
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Because our study was retrospective, detailed travel histories were not obtained.
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However, we retrospectively contacted the mothers of Cases 6 and 7 (both were
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neonates), who both confirmed that no household members had travelled overseas in 9
ACCEPTED MANUSCRIPT the preceding months. Moreover, the close phylogenetic relatedness between our
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strains of USA300 and those identified in North America, and the clustering in clades
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(most notably six of the eight nursing home isolates) suggest that MRSA USA300
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might have spread locally in Taiwan after imported from North America.
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In conclusion, asymptomatic carriers of MRSA USA300 may now be spreading
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this strain in the community in Taiwan. However, since all the isolates investigated in
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this study were from a research collection, more systematic surveillance studies are
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needed to fully determine the current epidemiology of MRSA USA 300 in Taiwan.
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172 Acknowledgement
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This study was supported by a grant from Ministry of Science and Technology,
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Republic of China (MOST 105－2314－B－182A－138) and a grant from Chang
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Gung Memorial Hospital (CMRPG3C1883).
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Conflicts of interest: None
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References
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1. Huang YC, Chen CJ. Community-associated methicillin-resistant Staphylococcus
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aureus in children in Taiwan, 2000s. Int J Antimicrob Agent 2011;38:2-8.
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2. Chuang YY, Huang YC. Molecular epidemiology of community-associated meticillin-resistant Staphylococcus aureus in Asia. Lancet Infect Dis
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3. Chen CJ, Huang YC. New epidemiology of Staphylococcus aureus in Asia. Clin
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4. Carrel M, Perencevich EN, David MZ. USA300 methicillin-resistant
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5. Nagao M, Iinuma Y, Suzuki M, Matsushima A, Takakura S, Ito Y, et al. First outbreak of methicillin-resistant Staphylococcus aureus USA300 harboring the
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6. Mine Y, Higuchi W, Taira K, Nakasone I, Tateyama M, Yamamoto T, et al.
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Staphylococcus aureus causing severe furuncles and carbuncles in Japan. J
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ACCEPTED MANUSCRIPT 7. Park C, Lee DG, Choi SM, Park SH, Choi J-H, Yoo J-H, et al. A case of perianal
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8. Sohn KM, Chung DR, Baek JY, Kim SH, Joo E-J, Ha YE, et al. Post-influenza
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9. Jung J, Song EH, Park SY, Lee SR, Park SJ, Sung H, et al. Emergence of Panton-Valentine leucocidin-positive ST8-methicillin-resistant Staphylococcus
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10. Grant D, Koh TH, Tan YE, Hsu LY, Kurup A, Donahue SK, et al. An outbreak of community-associated methicillin-resistant Staphylococcus aureus subtype
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USA300 at an international school in Singapore. Ann Acad Med Singap 2013; 42:
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11. Wang HK, Huang CY, Huang YC. Clinical features and molecular characteristics
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of childhood community-associated methicillin-resistant Staphylococcus aureus
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infection in a medical center in northern Taiwan, 2012. BMC Infect Dis 12
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2017;17(1): 470.. 12. Diep BA, Stone GG, Basuino L, Graber CJ, Miller A, des Etages SA, et al. The arginine catabolic mobile element and staphylococcal chromosomal cassette mec
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linkage: convergence of virulence and resistance in the USA300 clone of
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methicillin-resistant Staphylococcus aureus. J Infect Dis 2008; 197:1523-1530 13. Popovich KJ, Snitkin E, Green SJ, Aroutcheva A, Hayden MK, Hota B, et al.
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Genomic epidemiology of USA300 methicillin-resistant Staphylococcus aureus in
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an urban community. Clin Infect Dis 2016; 62:37-44.
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14. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell
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sequencing. J Comput Biol 2012; 19:455–477.
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15. Darling ACE, Mau B, Blattner FR, Perna NT. Mauve: Multiple alignment of
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16. Croucher NJ, Page AJ, Connor TR, Delaney AJ, Keane JA, Bentley SD,et al.
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Rapid phylogenetic analysis of large samples of recombinant bacterial whole
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genome sequences using Gubbins. Nucleic Acids Res 2015; 43:e15.
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Table 1. Clinical features of the 23 patients with 27 USA 300 MRSA isolates
237
identified in Taiwan Strain no.
Patient
Year
Age
Sex
Clinical diagnosis
Additional
no.
observations SSTI (I/D)
Cured
TW01_1
1-1
2005
Colonized
Household contact of patient 1
TW02
2
2008
11 months
F
Colonized
Carriage survey
TW03
3
2009
93 years
F
Pneumonia, Bacteremia
Died
TW04
4
2010
88 years
F
Bacteremia
Critical care discharge
TW05
5
2010
85 years
M
Pneumonia, Bacteremia
Cured
TW06
6
2011
1 month
M
Bacteremia (CVC)
Cured
TW07_1-4
7*
2012
15 days
M
Pneumonia, SSTI
Cured
TW08
8
2012
3 months
F
SSTI
Cured
TW09
9
2012
7 years
M
SSTI
Cured
TW10
10
2012
16 years
M
SSTI
Cured
TW11
11
2012
14 years
F
SSTI
Cured
TW12
12
2013
76 years
F
Colonized
TW13
13
2013
77 years
F
Colonized
TW14
14
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Nursing home surveillance
Environmental object
15
2013
85 years
M
Colonized
16
2013
87 years
M
Colonized
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TW16
F
2013
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TW15
7 years
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2005
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TW01
TW17
17
2013
83 years
F
Colonized
TW18
18
2013
95 years
F
Colonized
TW19
19
2013
80 years
M
Colonized
TW20
20
2015
37 years
M
colonized
TW21
21
2015
42 years
M
colonized
TW22
22
2015
41 years
M
colonized
TW23
23
2015
28 years
M
colonized
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SSTI, skin and soft tissue infection; I/D, incision and drainage; CVC, central venous
239
catheter; HIV, human immunodeficiency virus 14
HIV-infected individuals surveillance
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*Patient 7 had three clinical isolates and one colonizing isolate.
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Figure 1. Phylogenetic analysis of 27 USA300 MRSA isolates based on single
244
nucleotide polymorphisms in the core genomes. Ten global USA300 isolates were
245
included for comparison. The ellipse shadow indicates a clade of isolates with very
246
close genetic relatedness, suggesting local transmission of the USA300 strain.
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16
20 08
3 201 12
TW 18
13 20 2 TW07-4 2012 TWTW07-1 2012 TW07-2
Be lgi um
TE D
s xa Te
05 TW01 202005 01-1 TW
20
12
e am rin Su 13 20 me 23 rina Su UR -S 013 00 42 A3 R2 US 00-SU A3 US SF 2 00 2 3757 FPR a 04 Canad C2406 20
10 TW
TW06 2011 TW1 9 201 TW 3 09 201 2
EP
TW 03
50 SNV
TW 16
20
TW04 2 01 0 TW05 2010
20 TW 23 5
15
201
20
TW 22
21 TW
15
20 15
09
TW 20
AC C
TW1 1 20
02 TW
2
2
1 20 08012 32 T0W 7- 012
01
V1859 2007 Venezuela
20 1
20
olombia
A3 00
16
bia
US
15
2006 C
91
H TC
HUV07
olom
S3
TW 17
UA -
an y HUV 03 2 006 C
TW14 20 13
Ge rm
SC
00 3
TW12 2013 2013 TW13 013
12
2 15 TW
SA S
M AN U
MR
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13
S0195-6701(18)30107-5
DOI:
10.1016/j.jhin.2018.02.014
Reference:
YJHIN 5351
To appear in:
Journal of Hospital Infection
Received Date: 15 December 2017 Accepted Date: 14 February 2018
Please cite this article as: Huang Y-C, Chen C-J, Kuo C-C, Lu M-C, Emergence, transmission and phylogeny of meticillin-resistant Staphylococcus aureus sequence type 8 (USA300) in Taiwan, Journal of Hospital Infection (2018), doi: 10.1016/j.jhin.2018.02.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Format: Short report
2
Title: Emergence, transmission and phylogeny of meticillin-resistant
3
Staphylococcus aureus sequence type 8 (USA300) in Taiwan
4
Yhu-Chering Huang1, MD, PhD, Chih-Jung Chen1, MD, PhD, Ching-Chia Kuo1, MD,
5
Min-Chi Lu2, MD, PhD,
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1
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Taiwan, and Chang Gung University College of Medicine, Taoyuan, Taiwan
8
2
9
Department of Microbiology and Immunology, School of Medicine, China Medical
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Division of Pediatric Infectious Diseases, Chang Gung Memorial Hospital, Taoyuan,
Department of Medicine, China Medical University Hospital, Taichung, Taiwan, and
University, Taichung, Taiwan
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Running Title: Identification of MRSA USA300 in Taiwan
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Address for correspondence: Yhu-Chering Huang, Department of Pediatrics, Chang
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Gung Memorial Hospital, No. 5, Fu-Shin Street, Gueishan 333, Taoyuan, Taiwan.
15
TEL: +886-3-3281200, Fax: +886-3-3288957, E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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We identified 27 (0.51%) USA300 isolates from a pool of 5308 meticillin-resistant
19
Staphylococcus aureus (MRSA) isolates collected in Taiwan between 1995 and
20
October 2015, including 12 infecting isolates from 10 patients. The first two isolates
21
were identified in 2005 and 23 isolates since 2010. Phylogenetic analysis revealed all
22
the local isolates were closely related to those in North America and there was a clade
23
consisting of 13 local isolates from 10 patients. MRSA USA300 existed in Taiwan in
24
2005 or earlier, with increasing identification since 2010. Local transmission of
25
USA300 strains have occurred in Taiwan after imported from North America.
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Keywords: meticillin-resistant Staphylococcus aureus, sequence type 8, USA300,
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pulsotype, Taiwan
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Introduction In Taiwan, meticillin-resistant Staphylococcus aureus (MRSA) was first documented in early 1980s and rapidly increased in 1990s [1]. While a declining trend
34
of healthcare-associated (HA)-MRSA incidence was observed in 2000s,
35
community-associated (CA)-MRSA infections have been increasingly reported,
36
particularly in pediatric patients [1-3]. MRSA sequence type (ST) 59 is the major
37
community clone in Taiwan previously reported, while MRSA ST8 (USA300) has
38
prevailed in the community in North America [1-4]. Emergence of MRSA ST 8 has
39
been reported in several Asian countries [3,5-10]. Recently, we identified four MRSA
40
ST 8 isolates from children with CA-MRSA infection in 2012 in our hospital [11],
41
prompting our study on this strain in Taiwan. Herein, we report the emergence,
42
transmission and phylogenetic analysis of MRSA USA300 isolates identified in our
43
laboratory in Taiwan as well as the clinical features of these patients with MRSA
44
USA300 infections.
46 47
SC
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45
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Methods:
For surveillance of the molecular epidemiology of MRSA, we collected and
48
molecularly characterized a total of 5308 MRSA isolates island-wide in Taiwan
49
between 1995 and October 2015 in our laboratory, which is located in Chang Gung 3
ACCEPTED MANUSCRIPT Memorial Hospital (CGMH). In addition to CGMH, the isolates were also collected
51
from at least 10 hospitals in Taiwan. 1809 isolates (34%) were collected between
52
1995 and 2004, 2537 isolates (48%) between 2005 and 2010 and 962 isolates (18%)
53
between 2011 and 2015. The isolates included 2968 (56%) infecting isolates, 2235
54
(42%) human colonizing isolates and 105 (2.0%) non-human isolates (93 from pigs, 9
55
from the environmental objects, 2 from a bus and one from the pork). Among the
56
infecting isolates, 980 isolates were identified from paediatric patients, 276 from adult
57
patients and the remaining 1712 isolates from all age groups for surveillance of
58
molecular epidemiology of MRSA in Taiwan (we estimate that more than 90% of
59
these isolates were from adults). The infecting isolates were identified from any body
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site; 460 (15%) of these were community-associated. Among the colonizing isolates,
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1632 isolates (73%) were identified from children < 18 years of age, including infants
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hospitalized in neonatal intensive care units (NICUs), neonates in nurseries, children
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for well baby clinic visits, school children and case patients. The 603 adult isolates
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originated from parturient mothers and elderly nursing home residents. Fifty-two
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percent of the 2235 colonizing isolates were identified from the subjects without risk
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factors for MRSA acquisition.
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Identification of MRSA USA300
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To identify USA300 MRSA isolates, we examined and compared the PFGE 4
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those with same or similar PFGE pattern as USA300, we performed further
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characterizations, including staphylococcal cassette chromosomal typing (SCCmec),
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multilocus sequence typing, spa typing, and detection of arginine catabolic metabolic
73
element (ACME) and Panton-Valentine leukocidin (PVL) genes and some other toxin
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genes. All the methods were performed according to the procedures described
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elsewhere [11-13].
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Whole genome sequencing (WGS) and Phylogenetic analysis
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All the USA 300 isolates identified were subjected to whole genome sequencing
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(WGS), which was performed on an Illumina MiSeq sequencer (Illumina, San Diego,
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CA, USA) [13] and the results were deposited at DDBJ/ENA/GenBank under the
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accession PRJNA393756. Single nucleotide polymorphisms (SNPs) in the core
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genome were used to construct the phylogenies of the USA300 MRSA strains [14,15].
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To clarify the genetic relatedness of the strains in Taiwan and global isolates, 10
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published whole genome sequences data of USA300 strains were downloaded from
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the GenBank database and included for analysis.
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Clinical features of the case patients with confirmed MRSA USA300 infection were
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retrospectively reviewed. This study was approved by Chang Gung Medical 5
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Foundation Institutional Review Board (IRB No.:201600069B0).
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Results: Among the pool of 5308 MRSA isolates, we identified 27 (0.51%) USA300
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isolates. There were 12 infecting isolates from 10 patients with MRSA infection. The
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colonizing strains comprised one isolate each from a patient (Patient 7) and their
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mother’s breast milk (Patient 1); one isolate from a healthy child in the carriage
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survey (Patient 2); four isolates from human immunodeficiency virus (HIV)-infected
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patients in a carriage survey; seven isolates from elderly residents of three nursing
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homes; and one environmental isolate. Patient 7 had three infecting isolates and one
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colonizing isolate available for analysis. The details of the 27 isolates are presented in
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Table 1. The first two isolates were identified in 2005 and 23 isolates since 2010.
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Of the 10 infected patients seven were children or adolescents (age range 15 days to 15 years; median 6 years) and three were elderly (> 85 years) (Table 1). Of the
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seven children and adolescents, five had skin and soft tissue infection (SSTI) and
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recovered uneventfully. One had central venous catheter-related bacteraemia and the
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other (Patient 7) was a 15-day-old neonate with community-onset necrotizing
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pneumonia with empyema and bronchopleural fistula. He recovered after surgery and
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a 28-day course of vancomycin and linezolid. Three infecting isolates from this
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patient, and one colonizing isolate from the mother, were available for
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characterization. All 27 isolates shared the common molecular characteristics with USA 300 MRSA, including the same pulsotype, carrying type IV SCCmec, possessing type I
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ACME, PVL genes and arcA, bsaA,Sek and Seq genes and belonging to ST 8 and spa
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type t008 complex. Three subtypes were identified, one to three bands different from
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the control (LAC) strain. All 27 isolates were susceptible to vancomycin, teicoplanin,
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linezolid, fusidic acid, daptomycin, clindamycin, trimethoprim/sulfamethoxazole, and
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doxycycline, but only one isolate was susceptible to erythromycin and nine isolates
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were resistant to ciprofloxacin.
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The phylogeny based on SNPs in core genomes of 27 Taiwanese USA300 strains and 10 global USA300 reference strains is displayed in Figure 1. In this analysis, the
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greatest number of SNP was identified in a Venezuela isolate (V1859) when the
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earliest isolate TCH1516 was used as reference (451 SNPs). The strains from
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Germany (MRSA_S1) and Colombia (HUV03 and HUV07) were also
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phylogenetically distant from the strains isolated in North America in early 2000s
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(TCH1516, FPR3757 and C2406). Recombination events were more frequently
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identified in these genetically distant strains. The data suggested that the USA300
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strains present in certain regions outside the United States might have undergone
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substantial genomic evolution including point mutations and recombinations.
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and those from Belgium (UA-S391) and Suriname (USA300-SUR23 and
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USA300-SUR24) remained closely related to the North American strains (Figure 1).
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Among the local USA300 strains in Taiwan, we found a clade consisting of 13 strains
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identified from 10 (43.5%) of 23 subjects of different age populations, at different
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facilities, and associated with distinct clinical syndromes (ellipse shadow, Figure 1).
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These strongly suggested that local transmission of USA300 strains had occurred in
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Taiwan after imported from North America.
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Discussion
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To our knowledge, this is the first report focusing on MRSA USA300 in Taiwan and whole genome sequencing of MRSA USA300 in Asia. In this study, the first
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USA300 isolate was identified in 2005 from a school-aged child with SSTI, which
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suggests that USA300 existed in Taiwan more than 10 years. Although our MRSA
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collection was not complete, the fact that 78% of the 27 USA300 isolates were
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identified after 2011 suggests that there has recently been a true increase in the
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prevalence of USA300 isolates in Taiwan.
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The scenario of a wide variation in disease spectrum is similar to that observed in
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North America [4]. Also of note is that USA300 strains were found in widely differing
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surveillance populations, including nursing home residents and HIV-infected patients. 8
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Taiwan. Recently, identification of MRSA USA300 has been reported from several
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Asian countries such as Japan, South Korea and Singapore. Since 2007 there have
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been two outbreaks of MRSA USA300 reported in Japan [5,6]. Nagao et al [5]
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reported an outbreak in a general hospital ward involving six health care workers and
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four patients; eight had SSTIs and one each had catheter-related bacteraemia and
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pneumonia. The other outbreak affected one patient and three nurses, all of whom had
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SSTIs [6]. Since being first identified in South Korea in 2008 [7], Jung et al [9]
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reported 10 USA300 isolates among 818 MRSA bloodstream isolates collected from
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four medical centers between 2008 and 2015. All 10 isolates were
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healthcare-associated. In Singapore Grant et al [10] reported an outbreak of 27 SSTIs
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over three months in an international school (most of whom held US passports and
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had travelled to the US in previous months), including eight confirmed MRSA
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USA300 cases. These experiences from other Asian countries suggest that once a new
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pathogen is introduced into the community it is only a matter of time before it reaches
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healthcare settings, resulting then in nosocomial spread and even outbreaks.
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Because our study was retrospective, detailed travel histories were not obtained.
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However, we retrospectively contacted the mothers of Cases 6 and 7 (both were
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neonates), who both confirmed that no household members had travelled overseas in 9
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strains of USA300 and those identified in North America, and the clustering in clades
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(most notably six of the eight nursing home isolates) suggest that MRSA USA300
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might have spread locally in Taiwan after imported from North America.
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In conclusion, asymptomatic carriers of MRSA USA300 may now be spreading
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this strain in the community in Taiwan. However, since all the isolates investigated in
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this study were from a research collection, more systematic surveillance studies are
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needed to fully determine the current epidemiology of MRSA USA 300 in Taiwan.
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172 Acknowledgement
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This study was supported by a grant from Ministry of Science and Technology,
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Republic of China (MOST 105－2314－B－182A－138) and a grant from Chang
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Gung Memorial Hospital (CMRPG3C1883).
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Conflicts of interest: None
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Table 1. Clinical features of the 23 patients with 27 USA 300 MRSA isolates
237
identified in Taiwan Strain no.
Patient
Year
Age
Sex
Clinical diagnosis
Additional
no.
observations SSTI (I/D)
Cured
TW01_1
1-1
2005
Colonized
Household contact of patient 1
TW02
2
2008
11 months
F
Colonized
Carriage survey
TW03
3
2009
93 years
F
Pneumonia, Bacteremia
Died
TW04
4
2010
88 years
F
Bacteremia
Critical care discharge
TW05
5
2010
85 years
M
Pneumonia, Bacteremia
Cured
TW06
6
2011
1 month
M
Bacteremia (CVC)
Cured
TW07_1-4
7*
2012
15 days
M
Pneumonia, SSTI
Cured
TW08
8
2012
3 months
F
SSTI
Cured
TW09
9
2012
7 years
M
SSTI
Cured
TW10
10
2012
16 years
M
SSTI
Cured
TW11
11
2012
14 years
F
SSTI
Cured
TW12
12
2013
76 years
F
Colonized
TW13
13
2013
77 years
F
Colonized
TW14
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Nursing home surveillance
Environmental object
15
2013
85 years
M
Colonized
16
2013
87 years
M
Colonized
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TW16
F
2013
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TW15
7 years
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2005
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TW01
TW17
17
2013
83 years
F
Colonized
TW18
18
2013
95 years
F
Colonized
TW19
19
2013
80 years
M
Colonized
TW20
20
2015
37 years
M
colonized
TW21
21
2015
42 years
M
colonized
TW22
22
2015
41 years
M
colonized
TW23
23
2015
28 years
M
colonized
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SSTI, skin and soft tissue infection; I/D, incision and drainage; CVC, central venous
239
catheter; HIV, human immunodeficiency virus 14
HIV-infected individuals surveillance
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*Patient 7 had three clinical isolates and one colonizing isolate.
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Figure 1. Phylogenetic analysis of 27 USA300 MRSA isolates based on single
244
nucleotide polymorphisms in the core genomes. Ten global USA300 isolates were
245
included for comparison. The ellipse shadow indicates a clade of isolates with very
246
close genetic relatedness, suggesting local transmission of the USA300 strain.
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16
20 08
3 201 12
TW 18
13 20 2 TW07-4 2012 TWTW07-1 2012 TW07-2
Be lgi um
TE D
s xa Te
05 TW01 202005 01-1 TW
20
12
e am rin Su 13 20 me 23 rina Su UR -S 013 00 42 A3 R2 US 00-SU A3 US SF 2 00 2 3757 FPR a 04 Canad C2406 20
10 TW
TW06 2011 TW1 9 201 TW 3 09 201 2
EP
TW 03
50 SNV
TW 16
20
TW04 2 01 0 TW05 2010
20 TW 23 5
15
201
20
TW 22
21 TW
15
20 15
09
TW 20
AC C
TW1 1 20
02 TW
2
2
1 20 08012 32 T0W 7- 012
01
V1859 2007 Venezuela
20 1
20
olombia
A3 00
16
bia
US
15
2006 C
91
H TC
HUV07
olom
S3
TW 17
UA -
an y HUV 03 2 006 C
TW14 20 13
Ge rm
SC
00 3
TW12 2013 2013 TW13 013
12
2 15 TW
SA S
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MR
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20
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