Original Articles
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FOODBORNE PATHOGENS AND DISEASE Volume 11, Number 7, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2013.1717
Molecular Epidemiological Characteristics and Clonal Genetic Diversity of Staphylococcus aureus with Different Origins in China Guoxiang Chao,1,2,* Guangyu Bao,3,* Xinan Jiao1
Abstract
To understand the intrinsic links and epidemic features of Staphylococcus aureus, isolates of this bacterium were collected from different origins in China. A total of 503 isolates from different sources were tested for the presence of methicillin-resistant Staphylococcus aureus (MRSA) and types of staphylococcal cassette chromosome mec (SCCmec) elements. Of these isolates, 250 were analyzed by multilocus sequence typing (MLST). Results showed that MRSA isolates account for 21.67% and have five types. SCCmec type I, II, and III isolates were all from humans, while SCCmec type IV, V, and untypeable MRSA isolates were from clinical origin, pigs, raw milk, or environments. The results by MLST showed considerable molecular heterogeneity in 52 different sequence types (STs) including 16 novel STs, which yielded 7 clonal complexes (CCs) and 5 doublets (Ds). The results from these analyses showed that hospital-acquired MRSA (HA-MRSA), rather than community-associated MRSA (CA-MRSA), remained the major pathogenic bacterium in the clinical setting. CA-MRSA infections have been increasing and forming new pandemic clones. CC630, CC239, D9, and D398 were important clones in this study. CC630 was a new epidemic CA clone circulating among humans, environments, and animals. CC239 was a HA-MRSA-III pandemic clone of clinical origin. The D9 clone was a livestock-associated (LA) clone, which up to now, has spread only in livestock and has not been found in humans that live in Jiangsu province. In Europe and America, there are many reports of the ST398-LA-clone in pigs and in humans who are in contact with pigs. However, various studies in China, including this one, indicate that the ST398 clone could be a CA clone rather than an LA clone. This clone also has been infecting humans for about 2 decades.
Introduction
S
taphylococcus aureus has been recognized as an important zoonotic pathogen. S. aureus infection is facilitated by its propensity to develop resistance to multiple antibiotics, and methicillin-resistant S. aureus (MRSA) are the most frequently identified antibiotic-resistant pathogens. There are currently two types of recognized MRSA: hospitalacquired MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA). HA-MRSA isolates carry a large staphylococcal cassette chromosome mec (SCCmec), which is subgrouped into SCCmec type I, II, or III, whereas CAMRSA isolates carry a smaller SCCmec, such as SCCmec type IV or V (Pantosti and Venditti, 2009; David and Daum,
2010). There are distinct differences in epidemiology, microbiology, and the clinical symptoms of infections between CAMRSA and HA-MRSA (David and Daum, 2010). Arguments regarding the origins of CA-MRSA and the transmission of the MRSA between humans and animals are ongoing. In general, human carriers of MRSA are the primary natural reservoir, although mounting evidence suggests that livestock, particularly pigs, may represent important reservoirs for CA-MRSA. Generally, MRSA strains isolated from companion animals and humans are similar to human nosocomial MRSA, while MRSA strains from foods and animals appear to be specific animal-adapted clones (David and Daum, 2010; Graveland et al., 2011; Pantosti, 2012). Some special clones from livestock were designated as livestock-associated (LA)-MRSA. It
1
Jiangsu Key Laboratory of Zoonosis, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China. 2 Yangzhou Center for Disease Control and Prevention, Yangzhou, China. 3 Yangzhou No. 1 People’s Hospital, Yangzhou, China. *These two authors contributed equally to this work.
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Table 1. Methicillin-Resistant Staphylococcus aureus (MRSA) Types in 503 Isolates from Different Sources
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Sources of isolates(no.) Humans (n = 231) Animals (n = 38) Pigs (n = 56) Raw cow’s milk (n = 36) Environment (n = 67) Food (n = 75) No. of MRSA (%)b
MRSA type (no.)
No. of MSSA
I
II
III
IV
V
UT
No. of MRSA (%)a
166
1
5
40
1(d)
12
6
65/109 (59.63)
38
0
37 27
3(b)
51
1 8
15 1
16
19/109 (17.43) 9/109 (8.26) 16/109 (14.68)
75 1/109 (0.92)
5/109 (4.59)
40/109 (36.70)
4/109 (3.67)
37/109 (33.94)
22/109 (20.18)
0 109 (100)
a
The number of MRSA strains (%) in different sources. The number of MRSA strains (%) in different MRSA types. MSSA, methicillin-susceptible Staphylococcus aureus; UT, untypeable.
b
was first discovered in swine and has become an important global zoonotic pathogen in the past decade (David and Daum, 2010; Stegger et al., 2010). Multilocus sequence typing (MLST) is useful for determining genetic diversity because it indexes variations that accumulate slowly over time; thus, it can be used to measure long periods of evolution among S. aureus lineages, and this method is highly reproducible (Enright et al., 2002). A previous study using MLST and eBURST analyses showed that there are five pandemic MRSA lineages, which are the clonal complexes CC5, CC8, CC22, CC30, and CC45 (Enright et al., 2002). Along with the pandemic MRSA clones circulating in the world, new clones or subgroups (subclones) of the pandemic clones have appeared and replaced the traditional clones, becoming local pandemic clones (Enright et al., 2002; Monecke et al., 2011). The knowledge of molecular epidemiological characteristics and clonal genetic diversity of S. aureus from different origins in China is currently limited. The purpose of this study is to determine which types of SCCmec elements and local pandemic clones are present among S. aureus strains isolated from different sources, such as from the clinic, animals, food, and environments.
supermarkets (Table 1). All isolates were collected in Jiangsu province from 2006 to 2012.
Materials and Methods
Results
Bacterial isolates
MRSA and SCCmec typing
All bacterial isolates were from different origins, and were confirmed by methods described previously (Chao et al., 2013). Among the 503 isolates, 231 were from various patient tissue samples, such as blood, secretions, wounds, sputum, festers, etc., and were collected from three tertiary hospitals; 56 were from pigs located at different farms; 36 were from raw cow’s milk from distinct farms; 38 were from animals (not including pigs) such as chickens, dogs, sheep, and pigeons from animal hospitals; 67 were from environments or tools such as hair-cutting equipment, pedicure tools, bedclothes and towels in hotels or barbershops; and 75 were from retail food sources, principally from farms’ markets or
Among the 503 S. aureus isolates, 109 were MRSA because they showed resistance to cefoxitin, and they all expressed the mecA gene (CLSI, 2010). This accounts for 21.67% (109/503) of all of the isolates. The following five SCCmec types were identified: SCCmec type I (0.92%), type II (4.59%), type III (36.70%), type IV (3.67%), and type V (33.94%). About 20.18% were UT. SCCmec type I, II, and III isolates were all from humans, while SCCmec type IV, V, and UT isolates were from clinical origin, pigs, raw milk, or environments. SCCmec type III isolates were the main clinical MRSA isolates, accounting for 61.53% (40/65), followed by SCCmec type V, accounting for 18.46% (12/65). The prevalence of MRSA
SCCmec typing, antibiotic susceptibility testing
The SCCmec types (including mecA gene) were determined using a multiplex polymerase chain reaction (PCR) strategy that was developed by Oliveira et al. (Oliveira et al., 2006). Untypeable (UT) types were defined as isolates showing unexpected fragments or lacking some fragments as determined by multiplex PCR. Antibiotic susceptibility (including cefoxitin resistance) was tested as described previously (Chao et al., 2013). MLST and clone complexes
MLST was performed on 250 of 503 isolates after SCCmec typing (Table 2). Seven housekeeping genes were sequenced to determine the allelic profile. Isolates were assigned to a sequence type (ST) by the MLST database (http://saureus .mlst.net/). The program eBURST v3.0 was used to identify the diverse clonal complexes, and minimum-evolution trees were constructed from the concatenated sequences of each ST in MEGA5 (http://eburst.mlst.net).
PANDEMIC GENETIC CLONES OF S. AUREUS IN CHINA
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Table 2. Distribution of 250 Isolates for Multilocus Sequence Typing (MLST)a MRSA type
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Sources Humans Animals Pigs Raw cow’s milk Environment Foods Total
No. of isolates
I
II
III
IV
V
110 8 43 26 18 45 250
1
3
28
1(d)
14
3(b)
1 7 11
15 1
4
33
16
1 85
3
28
UT
MSSA 63 8 24 18 7 45 165
a With MLST, we are trying to find out the probable origin of methicillin-resistant Staphylococcus aureus (MRSA) and the relationship between MRSA clones and clonal complexes though analyzing S. aureus from different sources. The 250 strains were selected based on the following sources: most of MRSA strains, most of pigs (important revisors of community-acquired MRSA or livestock-associated MRSA), most milks (important media of MRSA between human and animal). See Table 3 (staphylococcal cassette chromosome mec source, methicillin-susceptible S. aureus [MSSA] source). UT, untypeable.
from clinics, pigs, raw milk and environments was 28.14% (65/231), 33.93% (19/56), 25% (9/36) and 23.88% (16/67), respectively. MRSA isolates from raw milk and environments were SCCmec type V isolates. MRSA-UT isolates were mainly from pigs. There were no MRSA isolates from food and animals (except pigs) (Table 1).
2 ST2314-MRSA-III, and 4 ST239-MSSA were isolated from the clinic (Figs. 1 and 2, Table 3).
STs, clonal complex
CC7, CC1281, CC1301, CC1920. There were two MRSA isolates in these CCs, ST1301-MRSA-V and ST2631MRSA-II, which were all isolated from humans. CC1920 included 6 STs and 33 MSSA isolates from different sources.
Among the 250 S. aureus isolates typed with MLST, 110 were from human (28 SCCmec type III, 14 SCCmec type V, 3 SCCmec type II, one SCCmec type I, one SCCmec type IVd, and 63 methicillin-susceptible S. aureus [MSSA] isolates), 8 MSSA were from animals, 43 were from pigs (3 SCCmec type IV, 1 SCCmec type V, 14 MRSA-UT, and 24 MSSA isolates), 26 were from raw cow’s milk (7 SCCmec type V, 1 SCCmecUT, and 18 MSSA), 18 were from environments (11 SCCmec type V, 7 MSSA), and 45 MSSA were from food (Table 2). Fifty-two STs were identified by MLST, of which 16 STs were new: ST2307-ST2317, ST2327, ST2630, ST2631, ST2710, and ST2711. S. aureus isolates are grouped within a single CC when five of the seven housekeeping genes have identical sequences. The ancestor of each CC is the ST with the largest number of single-locus variants. Subgroup founders can be described as single-locus variants or double-locus variants (DLV) of a founder of a CC that has become prevalent in a population (Cooper and Feil, 2004). The 52 STs generated in this data set were separated by eBURSTv3.0 and MEGA5 into 7 clonal complexes (CCs), 5 doublets (Ds), and 16 singletons (Ss) (Figs. 1 and 2, Table 3).
CC5. There were 22 isolates in CC5. Thirteen isolates belonged to ST5 including 1 ST5-MRSA-II and 3 ST5MRSA-V (Figs. 1 and 2, Table 3).
D9. There were 27 isolates, all from pigs, in D9 including 11 ST9-MRSA-UT, 1 ST9-MRSA-V, 3 ST9-MRSA-IVb, 4 ST2630-MRSA-UT, and 8 ST9-MSSA. D398. Thirteen of the 16 isolates in D398 were of clinical origin, including 14 ST398-MSSA, 1 ST398-MRSA-II, and 1 ST2199-MSSA. Three other ST398-MSSA isolates were from food. D59. Two isolates were in D59 including one ST59MRSA-IVd from the clinic and one ST338-MSSA from food. D88 and D30. Ten of the isolates were in two of the Ds. All were MSSA isolates from different sources. Ss. There were 12 Ss including 32 isolates, of which 10 isolates belonged to ST2316 from pigs. Discussion
Relationship between MRSA and CCs, Ds, STs
There were 49 isolates and 9 STs in CC630 including 29 ST630-MRSA (10 MRSA-V from humans, 7 MRSA-V from raw cow’s milk, 11 MRSA-V from environments, 1 MRSAUT from raw cow’s milk) and 11 ST630-MSSA. One ST247MRSA-I was from the clinic (Fig. 1, Table 3). CC239. CC239 is a subgroup derived from CC630. All 32 isolates including 24 ST239-MRSA-III, 2 ST1283-MRSA-III,
HA-MRSA rather than CA-MRSA remains the major pathogenic bacterium in the clinical setting; however, infections caused by CA-MRSA have increased in recent years in China (Chao et al., 2013). In this study, the prevalence of MRSA in humans, pigs, raw cow’s milk, and environments was high. All HA-MRSA isolates, most of which were SCCmec type III, came from clinical origins while CAMRSA isolates including LA-MRSA were from patients, pigs, raw cow’s milk (cows), and environments.
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FIG. 1. The ‘‘population snapshot’’ from 52 Staphylococcus aureus sequence types (STs) by eBURST v3. Seven clonal complexes (CCs) (CC630, CC239, CC1920, CC5, CC1301, CC1281, CC7) and 5 doublets (Ds) (D9, D398, D88, D30, D59) were defined using less stringent criteria (5/7 shared alleles). ST630 was the predicted clonal ancestor of CC630. CC239 is a subgroup deriving from CC630. ST239 is the founder of CC239. The sizes of the circles are relative to the number of strains in the ST.
By MLST, S. aureus populations consist of about 10 dominant human lineages including CC1, CC5, CC8, CC12, CC15, CC22, CC25, CC30, CC45, and CC51 (Lindsay, 2010). The most common MRSA isolates from hospitals belong to CC1, CC5, CC8, CC22, CC30, and CC45, and some uncommon MRSA lineages are CC59, CC80, and CC239. Each MRSA has a unique geographical distribution (Lindsay, 2010). Our results showed that S. aureus had great clonal genetic diversity. Among the 250 strains, there were 52 STs including 12 new STs, 7 CCs, and 5 Ds. Except for CC239, CC5, and the LA-clones, CC9 (D9) and CC398 (D398), a new local epidemic CC630 clone and new CC1920, CC1281, CC1301, and CC7 clones were identified. CC630 and CC239 are divergent branches of the international clone CC8, which is a Brazilian clone (Ashley and Enright, 2003). D9 and D398 clones demonstrated new characterizations that were different from other reports. The eBURST analysis revealed that the ST239, ST630, ST5, ST9, and ST398 clones coexisted in both MRSA and MSSA, which suggested that these MRSA clones might have evolved from MSSA through the acquisition of SCCmec. The ST239-MRSA clone was the predominant clone in Asian countries except for Japan and South Korea (Ko et al., 2005). In this study, all HA-MRSA-III isolates were from the same CC239 clone. Our results and other reports support the finding that CC239-MRSA-III is still the most prevalent MRSA clone throughout hospitals in China (Wang et al., 2007; Xu et al., 2009; Yan et al., 2009; Yao et al., 2010; Shen et al., 2010; Li et al., 2011; Xie et al., 2011; Liu et al., 2012; Yu et al., 2012; Chao et al., 2013; He et al., 2013). Nosocomial environments facilitate the development of HA-MRSA through the selective pressure of antibiotics. All HA-MRSA isolates in our study were of clinical origins. To the best of our knowledge, this is the first study documenting a CC630 clone including nine STs. Previously, only a few rare reports of ST630 in Hong Kong, Zhejiang, Guangdong, and Beijing in China, and in the world have been documented, indicating that all isolates were of clinical origin including the majority of MSSA and, more rarely,
MRSA (a MRSA-V, a MRSA-III, and two MRSA unknown types) (Yan et al., 2009; Li et al., 2011; Xie et al., 2011; Liu et al., 2012; Yu et al., 2012; Ho et al., 2012b; Zhao et al., 2012; He et al., 2013; Huang et al., 2013). In this study, all ST630-MRSA isolates belonging to CA-MRSA were MRSA-V except for one MRSA-UT. Isolates in this group were all from clinical origin, raw cow’s milk, or environments (hair-cutting equipment, pedicure tools, bedclothes, and towels in hotels or barbershop). The results demonstrated that humans, animals and other environments have formed an epidemic circle of CA-MRSA/MSSA. We cannot determine whether CA-MRSA originated from humans or animals. Additionally, it cannot be ignored that the environment plays an important part in the transmission and repository of CA-MRSA because S. aureus can survive for a long time on inanimate objects (Simoes et al., 2011; Uhlemann et al., 2011). One clinical isolate, ST247MRSA-I (ST247, a DLV of ST630), belongs to the pandemic ‘‘Iberian’’ clone (Robinson and Enright, 2004), while few isolates were reported in China. CC5 is another common and widespread clonal complex, next to CC8, which comprises a large number of different MRSA isolates that are both HA- and CA-MRSA, some of which have attained pandemic status (Ko et al., 2005; Monecke et al., 2011). In this study, two types of MRSA, ST5-MRSA-II (HA-MRSA) and ST5-MRSA-V (CA-MRSA), were isolated from the clinic. ST5-MRSA-II is known as the pandemic New York/Japan clone (Enright et al., 2002), while ST5-MRSA-V has been described in Australia, Ireland, Germany, and Abu Dhabi (Monecke et al., 2011). In China, ST5-MRSA-II was also a predominant clone, next to ST239-MRSA-III (Wang et al., 2007; Yan et al., 2009; Shen et al., 2010; Li et al., 2011; Xie et al., 2011; Liu et al., 2012), but few ST5-MRSA-V have been reported. MRSA-colonized animals, especially livestock, have provided and expanded reservoirs for MRSA. In the last decade, two major animal-adapted clones, ST398 in Europe and ST9 in Asia, have been reported continuously since they were originally identified (Lo et al., 2012).
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PANDEMIC GENETIC CLONES OF S. AUREUS IN CHINA
FIG. 2. A minimum-evolution tree was constructed using the concatenated sequences of seven loci from 52 sequence types by MEGA5. The clonal complexes (CCs) and doublets (Ds) in Fig. 1 basically corresponded to the CCs and Ds in the tree. Bootstrap values over 50% are shown on the branches. There have been many reports of CC9 (ST9)-MRSA isolated from pigs, other animals, and humans in China. CC9 (ST9)-MRSA-IVb and CC9 (ST9)-MRSA-V were isolated from pigs and raw meat (slaughtered pigs) in Taiwan and Hong Kong, and ST9- MRSA-IVb was isolated from butchers in Hong Kong (Guardabassi et al., 2009; Boost et al., 2013a; Ho et al., 2012a; Boost et al., 2013b). CC9 (ST9, ST97)-MRSA-IVb was isolated from bovine in five provinces on the mainland (Wang et al., 2011). ST9-MRSA was isolated from patients in Tianjin on the mainland (Zhang et al., 2003). CC9 (ST9, ST912, ST1297)-MRSA-III was isolated from swine and swine workers from four provinces
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on the mainland (Cui et al., 2009). CC9 (ST9, ST1376)MRSA and MSSA were isolated from pig farms in Sichan (Wagenaar et al., 2009). ST9-MRSA-V and IV were isolated from healthy swine and human clinical samples in Taiwan (Pantosti, 2012; Wan et al., 2013). In this study, all MRSA in D9 (ST9, ST2360) were types IVb, V, and UT and were isolated from pigs from distinct farms in Jiangsu. The clones ST9-MRSA-IVb, and -V were similar to the main CC9 (ST9)-MRSA clones reported in China. ST9-MSSA isolates also existed in D9. One characteristic of CC9 in this study was the presence of most of the MRSA-UT isolates; therefore, we assumed that special dimensional molecular structures could exist on SCCmec. Another finding is that none of the CC9 isolates were isolated from the clinic. We could regard CC9 as the only LA clone that has not spread to humans in Jiangsu yet. Even so, it is a potential zoonotic infectious agent for humans. The CC398-MRSA clone is the first LA-MRSA clone to originate from pigs. All MRSA isolates of this clone were found to share similar characteristics, such as nontypeability by pulsed-field gel electrophoresis and identical spa type. Although there is a high prevalence of CC398 in pigs and other animals, it seems that the CC398 clone is a poorly persistent colonizer in humans and that invasive infection caused by clone ST398 occurs rarely (David and Daum, 2010). In sharp contrast, in China, there are a number of reports regarding isolation of the ST398 clone from humans, not from pigs (animals), based on a search in PubMed and WanFang (in Chinese) for related articles (Ip et al., 2005; Yu et al., 2008; Chen et al., 2010; Stegger et al., 2010; Yao et al., 2010; Deng et al., 2012; Li et al., 2013). Chen et al. describe ST398-MSSA from humans appearing early in 1994 (Chen et al., 2010). Zhao et al. described 4 hospitals in Beijing that a high prevalence of ST398-MSSA clone (17.1% of 164 infections), which was found with no apparent association with animal contact during 2009–2010 (Zhao et al., 2012). In contrast to studies in Europe and the Americas, the ST398 isolate does not appear to be the dominant MRSA strain in Asian pigs/animals (David and Daum, 2010; Stegger et al., 2010; Graveland et al., 2011; Pantosti, 2012). In China, only one ST398-MRSA-V was reported from diseased animals (Zhang et al., 2011). In this study, most of the ST398-MSSA were clinical isolates, others were from foods, and none were from pigs. One ST398-MRSA-II isolate, from a clinic, could be the first reported in mainland China. We did not know whether the patients infected with ST398-MSSA/MRSA had contact with pigs or other animals. It is remarkable that a number of data have demonstrated that ST398-MSSA/MRSA infects humans, whereas few isolates were recovered from pigs or other animals. There were no signs indicating that the ST398 isolate colonized animals in China. The clone infected humans long before the first report of infection of European pigs; therefore, we have good reason to presume that ST398 may not be an LA clone, but a CA clone in China. The ST398 clone has colonized and caused infections for a long time in humans (not animals). CC59-MRSA-IV/V has been reported in Australia, the United Kingdom (Monecke et al., 2011), and in Hong Kong, Taiwan, and mainland China (Wang et al., 2004; Ip et al., 2005; Wang et al., 2007; Chen et al., 2010; Shen et al., 2010; Xie et al., 2011; Zhao et al., 2012). The results from this
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Table 3. Sequence Types (STs), Clonal Complexes (CCs), Doublets (Ds), Variants (Vs), Methicillin-Resistant Staphylococcus aureus, and Methicillin-Susceptible S. aureus (MSSA), as Well as the Sources of the 250 Isolates
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Multilocus sequence typing alleles Clone
ST
arcc
aroe
glpf
Gmk
Pta
tpi
yqil
No.
SCCmec: source (no.)
MSSA source (no.)
CC239
239 1283 2314 630
2 2 2 12
3 3 3 3
1 1 1 1
1 88 1 1
4 4 4 4
4 4 4 4
3 3 257 3
28 2 2 40
III:Ha(24) III:H(2) III:H(2) V:H(10)M(7)E(11); UT:M(1)
H(4)
8 3 770 1 2711 12 2205 12 1821 12 2309 248 247 3 2710b 18 1920 1 2312 4 2310 1 1 1 188 3 2315 1 5 1
3 3 3 3 3 3 3 95 1 1 1 1 1 1 4
1 1 1 1 1 1 1 45 1 1 1 1 1 1 1
1 1 1 1 1 1 12 206 157 157 157 1 8 177 4
4 4 297 4 40 40 4 7 1 1 1 1 1 12 12
4 4 4 1 4 4 4 15 1 1 258 1 1 1 1
3 3 3 3 3 3 16 5 1 1 1 1 1 1 10
1 1 1 1 2 1 1 1 11 1 1 9 10 1 13
CC630
CC1920
CC5
CC1301 CC1281 CC7 D9 (CC9) D398 (CC398) D88 (CC88) D30 (CC30) D59 (CC59) S S S S S S S S S S S
965 2313 6 1301 2311 121 1281 20 2631 7 943 2327 9
1 1 12 158 158 6 4 4 4 5 5 5 3
4 4 4 5 5 5 168 9 168 4 4 4 3
1 258 1 6 14 6 1 1 1 1 1 1 1
4 4 4 2 2 2 8 8 8 4 4 179 1
119 12 12 7 7 7 1 1 290 4 116 4 1
1 1 1 14 14 14 10 10 10 6 6 6 1
10 10 3 5 5 5 8 8 8 3 3 3 10
2 1 6 1 1 1 6 1 1 15 1 1 23
2630 398 2199 88 2148 243 30 59 338 2316 2317 25 15 97 479 2307 2308 12 22 1207
3 3 3 22 3 2 2 19 19 10 1 4 13 3 52 14 18 1 7 8
3 35 35 1 4 2 2 23 23 14 115 1 13 1 79 57 330 3 6 167
1 19 19 14 14 5 2 15 15 8 4 4 1 1 54 1 46 1 1 135
201 2 2 23 23 2 2 2 48 26 2 1 1 1 18 155 155 8 5 2
1 20 20 12 12 6 6 19 19 10 13 5 12 1 56 109 7 11 8 13
1 26 5 4 4 3 3 20 20 3 259 5 11 5 32 50 50 5 8 127
10 39 39 31 31 2 2 15 15 255 11 4 13 3 65 2 253 11 6 7
4 15 1 6 1 1 2 1 1 10 3 5 5 2 1 1 1 1 1 1
H(6)E(1)M(1)F(3) E(1) H(1) E(1) E(1) H(1)E(1) F(1)
I:H(1)
II:H(1); SV:H(3)
A(1) M(7)F(4) H(1) F(1) H(1)A(1)M(2)F(5) H(5)F(5) H(1) H(3)A(4)M(1)F(1) H(1)F(1) H(1) H(6)
V:H(1) H(1) H(1) H(1)M(2)F(3) F(1) II:H(1)
IVb:P(3);V:P(1); UT:P(11) UT:P(4) II:H(1)
H(4)M(4)F(7) H(1) F(1) P(8) H(11)F(3) A(1) H(4) F(2) F(1) P(1) H(2)
IVd:H(1) F(1) P(10) P(3) H(5) H(1)E(2)F(2) M(1)F(1) F(1) A(1) A(1) F(1) H(1) P(1)
a H, human origin; A, animal origin (not including pigs); M, raw cow’s milk origin; P, pig origin; E, environment origin; F, food origin; UT, untypeable. b ST2710 belonged to CC630 by MEGA5 (Fig. 2).
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PANDEMIC GENETIC CLONES OF S. AUREUS IN CHINA
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study show that the ST59 clone was not the main epidemic clone in Jiangsu province. In conclusion, this study revealed the molecular and epidemiological characteristics and clonal genetic diversity of S. aureus in China. There were 52 STs including 16 novel STs, forming 7 CCs and 5 Ds. There were also four important clones including CC239, CC630, D398, and D9 that were identified in this study. CC239 was the major HA-MRSA-III clone. CC630 was a new epidemic group, and all CA-MRSAV/MSSA isolates in this clone were from different sources, such as from the clinic, environment, or raw cow’s milk. Animals and environments played an important part in the repositories and transmission of CA-MRSA/MSSA. The CC9 clone was a LA-MRSA/MSSA clone and has spread only in livestock rather than in humans in Jiangsu province. Despite many reports of the ST398-LA-MRSA/MSSA clone originating in pigs and humans in Europe and the United States, there are many lines of evidence, including this study, showing that ST398 could be a CA clone rather than an LA clone in China.
Cui S, Li J, Hu C, Jin S, Li F, et al. Isolation and characterization of methicillin-resistant Staphylococcus aureus from swine and workers in China. J Antimicrob Chemother 2009; 64:680–683. David MZ, Daum RS. Community-associated methicillinresistant Staphylococcus aureus: Epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev 2010;23:616–687. Deng JJ, Wan CM, Mu DZ, Fan J, Xu AL, et al. Molecular epidemiological study of Staphylococcus aureus isolates in children in Chengdu. J Clin Pediatr 2012;30:507–510. (in Chinese). Enright MC, Robinson DA, Randle G, Feil EJ, Grundmann H, et al. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc Natl Acad Sci USA 2002; 99:7687–7692. Graveland H, Duim B, Van DE, Heederik D, Wageraar JA. Livestock-associated methicillin-resistant Staphylococcus aureus in animals and humans. Int J Med Microbiol 2011;301:630–634. Guardabassi L, O’Donoghue M, Moodley A, Ho J, Boost M. Novel lineage of methicillin-resistant Staphylococcus aureus, Hong Kong. Emerg Infect Dis 2009;15:1998– 2000. He WQ, Chen HB, Zhao CJ, Zhang F, Li H, et al. Population structure and characterisation of Staphylococcus aureus from bacteraemia at multiple hospitals in China: Association between antimicrobial resistance, toxin genes and genotypes. Int J Antimicrob Agents 2013;doi: 10.1016/j.ijantimicag .2013.04.031. Ho J, O’Donoghue M, Guardbassi L, Moodley A, Boost M. Characterization of methicillin-resistant Staphylococcus aureus isolates from pig carcasses in Hong Kong. Zoonoses Public Health 2012a;59:416–423. Ho PL, Chiu SS, Chan MY, Gan Y, Chow KH, et al. Molecular epidemiology and nasal carriage of Staphylococcus aureus and methicillin-resistant S. aureus among young children attending day care centers and kindergartens in Hong Kong. J Infect 2012b;64:500–506. Huang J, Ye M, Ding H, Guo Q, Ding B, et al. Prevalence of fusB in Staphylococcus aureus clinical isolates. J Med Microbiol 2013;62:1199–1203. Ip M, Yung RW, Ng TK, Luk WK, Tse C, et al. Contemporary methicillin-resistant Staphylococcus aureus clones in Hong Kong. J Clin Microbiol 2005;43:5069–5073. Ko KS, Lee JY, Suh JY, Oh WS, Peck KR, et al. Distribution of major genotypes among methicillin-resistant Staphylococcus aureus clones in Asian countries. J Clin Microbiol 2005; 43:421–426. Li DZ, Chen YS, Yang JP, Zhang W, Hu CP, et al. Preliminary molecular epidemiology of the Staphylococcus aureus in lower respiratory tract infections: A multicenter study in China. Chin Med J (Engl) 2011;124:687–692. Li SP, Li J, Wang LJ, Sun J, Sun MJ, et al. Molecular and clinical characteristics of community-acquired methicillinresistant Staphylococcus aureus isolated from Chinese children with pneumonia. J Clin Pediatr 2013;31:514–518. (in Chinese). Lindsay JA. Genomic variation and evolution of Staphylococcus aureus. Int J Med Microbiol 2010;300:98–103. Liu Q, Han L, Li B, Sun J, Ni Y. Virulence characteristic and MLST-agr genetic background of high-level mupirocinresistant, MRSA isolates from Shanghai and Wenzhou, China. PLoS ONE 2012;7:e37005.
Acknowledgments
We would like to thank all individuals who kindly provided us with the strains used in this study. This work was supported by grants from the National High Technology Research and Development Program of China (863 Program) (2012AA101601-6) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Disclosure Statement
No competing financial interests exist. References
Ashley RD, Enright MC. Evolutionary models of the emergence of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2003;47:3926–3934. Boost M, Ho J, Guardabassi L, O’Donoghue M. Colonization of butchers with livestock-associated methicillin-resistant Staphylococcus aureus. Zoonoses Public Health 2013a;60:572– 576. Boost M, Wong A, Ho J, O’Donoghue M. Isolation of methicillin-resistant Staphylococcus aureus (MRSA) from retail meats in Hong Kong. Foodborne Pathog Dis 2013b; 10:705–710. Chao GX, Zhang XP, Huang Y, Huang Y, Xu L, et al. Phenotypic and genotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA) from different sources in China. Foodborne Pathog Dis 2013;10:214–221. Chen HB, Liu YD, Jiang XH, Chen M, Wang H. Rapid change of methicillin-resistant Staphylococcus aureus clones in a Chinese tertiary care hospital over a 15-year period. Antimicrob Agents Chemother 2010;54:1842–1847. [CLSI] Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing, 19th informational supplement. CLSI Document M100-S19. Wayne, PA: CLSI, 2010. Cooper JE, Feil EJ. Multilocus sequence typing—What is resolved? Trends Microbiol 2004;12:373–377.
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Lo YP, Wan MT, Chen MM, Su HY, Lauderdale TL, et al. Molecular characterization and clonal genetic diversity of methicillin-resistant Staphylococcus aureus of pig origin in Taiwan. Comp Immunol Microbiol Infect Dis 2012;35:513– 521. Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, et al. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS ONE 2011;6:e17936. Oliveira DC, Milheirico C, de Lencastre H. Redefining a structural variant of staphylococcal cassette chromosome mec, SCCmec type VI. Antimicrob Agents Chemother 2006; 50:3457–3459. Pantosti A, Venditti M. What is MRSA? Eur Respir J 2009;34: 1190–1196. Pantosti A. Methicillin-resistant Staphylococcus aureus associated with animals and its relevance to human health. Front Microbiol 2012;3:127. Robinson DA, Enright MC. Multilocus sequence typing and the evolution of methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect 2004;10:92–97. Shen EH, Wang LH, Wang H, Sun HL, Chen MJ, et al. Research on genotyping of methicillin-resistant Staphylococcus aureus in China. Chin J Epidemiol 2010;31:308–311. (in Chinese). Simoes RR, Aires-de-Sousa M, Conceicao T, Antunes F, da Costa PM, et al. High prevalence of EMRSA-15 in Portuguese public buses: A worrisome finding. PLoS ONE 2011;6: e17630. Stegger M, Lindsay JA, Sorum M, Gould KA, Skov R. Genetic diversity in CC398 methicillin-resistant Staphylococcus aureus isolates of different geographical origin. Clin Microbiol Infect 2010;16:1017–1019. Uhlemann AC, Knox J, Miller M, Hafer C, Vasquez G, et al. The environment as an unrecognized reservoir for communityassociated methicillin resistant Staphylococcus aureus USA300: A case-control study. PLoS ONE 2011;6:e22407. Wagenaar JA, Yue H, Pritchard J, Broekhuizen-Stins M, Huijsdens X, et al. Unexpected sequence types in livestock associated methicillin-resistant Staphylococcus aureus (MRSA): MRSA ST9 and a single locus variant of ST9 in pig farming in China. Vet Microbiol 2009;139:405–409. Wan MT, Lauderdale TL, Chou CC. Characteristics and virulence factors of livestock associated ST9 methicillin-resistant Staphylococcus aureus with a novel recombinant staphylocoagulase type. Vet Microbiol 2013;23:779–784. Wang CC, Lo WT, Chu ML, Siu LK. Epidemiological typing of community-acquired methicillin-resistant Staphylococcus aureus isolates from children in Taiwan. Clin Infect Dis 2004; 39:481–487. Wang DF, Duan XH, Wang ZC. The current status of the drug resistance and evolutionary relationship of MSSA and MRSA
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isolates from bovine of China. Acta Vet Zootech Sin 2011;42:1416–1425. (in Chinese). Wang H, Liu Y, Du N, Na SL, Xu YC, et al. The molecular epidemiology of methicillin-resistant Staphylococcus aureus in China in 2005. Chin J Lab Med 2007;30:1354–1359. (in Chinese). Xie Y, He Y, Gehring A, Hu Y, Li Q, et al. Genotypes and toxin gene profiles of Staphylococcus aureus clinical isolates from China. PLoS ONE 2011;6:e28276. Xu BL, Zhang G, Ye HF, Feil EJ, Chen GR, et al. Predominance of the Hungarian clone (ST239-III) among hospital-acquired methicillin-resistant Staphylococcus aureus isolates recovered throughout mainland China. J Hosp Infect 2009;71:245–255. Yan XM, Gu YX, He LH, Zhang HF, Zhang JZ. Multilocus sequence typing of methicillin-resistant Staphylococcus aureus. Chin J Prev Med 2009;43:137–140. (in Chinese). Yao D, Yu FY, Qin ZQ, Chen C, He SS, et al. Molecular characterization of Staphylococcus aureus isolates causing skin and soft tissue infections (SSTIs). BMC Infect Dis 2010;10:133. Yu F, Chen C, Liu X, Zhang X, Lin X, et al. Prevalence of Staphylococcus aureus carrying Panton-Valentine leukocidin genes among isolates from hospitalized patients in China. Clin Microbiol Infect 2008;14:381–384. Yu F, Li T, Huang X, Xie J, Xu Y, et al. Virulence gene profiling and molecular characterization of hospital-acquired Staphylococcus aureus isolates associated with bloodstream infection. Diagn Microbiol Infect Dis 2012;74:363–368. Zhang WJ, Hao ZH, Wang Y, Cao X, Logue CM, et al. Molecular characterization of methicillin-resistant Staphylococcus aureus strains from pet animals and veterinary staff in China. Vet J 2011;190:e125–e129. Zhang YB, Zong SD, Qi W, Wu SW. Multilocus sequence typing to four clinical Staphylococcus aureus. Chin J Microbiol Immunol 2003;23:512. (in Chinese). Zhao C, Liu Y, Zhao M, Liu Y, Yu Y, et al. Characterization of community acquired Staphylococcus aureus associated with skin and soft tissue infection in Beijing: High prevalence of PVL + ST398. PLoS ONE 2012;7:e38577.
Address correspondence to: Xinan Jiao, PhD Jiangsu Key Laboratory of Zoonosis Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses Yangzhou University 88 South University Avenue Yangzhou 225009, China E-mail: [email protected]
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FOODBORNE PATHOGENS AND DISEASE Volume 11, Number 7, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2013.1717
Molecular Epidemiological Characteristics and Clonal Genetic Diversity of Staphylococcus aureus with Different Origins in China Guoxiang Chao,1,2,* Guangyu Bao,3,* Xinan Jiao1
Abstract
To understand the intrinsic links and epidemic features of Staphylococcus aureus, isolates of this bacterium were collected from different origins in China. A total of 503 isolates from different sources were tested for the presence of methicillin-resistant Staphylococcus aureus (MRSA) and types of staphylococcal cassette chromosome mec (SCCmec) elements. Of these isolates, 250 were analyzed by multilocus sequence typing (MLST). Results showed that MRSA isolates account for 21.67% and have five types. SCCmec type I, II, and III isolates were all from humans, while SCCmec type IV, V, and untypeable MRSA isolates were from clinical origin, pigs, raw milk, or environments. The results by MLST showed considerable molecular heterogeneity in 52 different sequence types (STs) including 16 novel STs, which yielded 7 clonal complexes (CCs) and 5 doublets (Ds). The results from these analyses showed that hospital-acquired MRSA (HA-MRSA), rather than community-associated MRSA (CA-MRSA), remained the major pathogenic bacterium in the clinical setting. CA-MRSA infections have been increasing and forming new pandemic clones. CC630, CC239, D9, and D398 were important clones in this study. CC630 was a new epidemic CA clone circulating among humans, environments, and animals. CC239 was a HA-MRSA-III pandemic clone of clinical origin. The D9 clone was a livestock-associated (LA) clone, which up to now, has spread only in livestock and has not been found in humans that live in Jiangsu province. In Europe and America, there are many reports of the ST398-LA-clone in pigs and in humans who are in contact with pigs. However, various studies in China, including this one, indicate that the ST398 clone could be a CA clone rather than an LA clone. This clone also has been infecting humans for about 2 decades.
Introduction
S
taphylococcus aureus has been recognized as an important zoonotic pathogen. S. aureus infection is facilitated by its propensity to develop resistance to multiple antibiotics, and methicillin-resistant S. aureus (MRSA) are the most frequently identified antibiotic-resistant pathogens. There are currently two types of recognized MRSA: hospitalacquired MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA). HA-MRSA isolates carry a large staphylococcal cassette chromosome mec (SCCmec), which is subgrouped into SCCmec type I, II, or III, whereas CAMRSA isolates carry a smaller SCCmec, such as SCCmec type IV or V (Pantosti and Venditti, 2009; David and Daum,
2010). There are distinct differences in epidemiology, microbiology, and the clinical symptoms of infections between CAMRSA and HA-MRSA (David and Daum, 2010). Arguments regarding the origins of CA-MRSA and the transmission of the MRSA between humans and animals are ongoing. In general, human carriers of MRSA are the primary natural reservoir, although mounting evidence suggests that livestock, particularly pigs, may represent important reservoirs for CA-MRSA. Generally, MRSA strains isolated from companion animals and humans are similar to human nosocomial MRSA, while MRSA strains from foods and animals appear to be specific animal-adapted clones (David and Daum, 2010; Graveland et al., 2011; Pantosti, 2012). Some special clones from livestock were designated as livestock-associated (LA)-MRSA. It
1
Jiangsu Key Laboratory of Zoonosis, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China. 2 Yangzhou Center for Disease Control and Prevention, Yangzhou, China. 3 Yangzhou No. 1 People’s Hospital, Yangzhou, China. *These two authors contributed equally to this work.
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Table 1. Methicillin-Resistant Staphylococcus aureus (MRSA) Types in 503 Isolates from Different Sources
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Sources of isolates(no.) Humans (n = 231) Animals (n = 38) Pigs (n = 56) Raw cow’s milk (n = 36) Environment (n = 67) Food (n = 75) No. of MRSA (%)b
MRSA type (no.)
No. of MSSA
I
II
III
IV
V
UT
No. of MRSA (%)a
166
1
5
40
1(d)
12
6
65/109 (59.63)
38
0
37 27
3(b)
51
1 8
15 1
16
19/109 (17.43) 9/109 (8.26) 16/109 (14.68)
75 1/109 (0.92)
5/109 (4.59)
40/109 (36.70)
4/109 (3.67)
37/109 (33.94)
22/109 (20.18)
0 109 (100)
a
The number of MRSA strains (%) in different sources. The number of MRSA strains (%) in different MRSA types. MSSA, methicillin-susceptible Staphylococcus aureus; UT, untypeable.
b
was first discovered in swine and has become an important global zoonotic pathogen in the past decade (David and Daum, 2010; Stegger et al., 2010). Multilocus sequence typing (MLST) is useful for determining genetic diversity because it indexes variations that accumulate slowly over time; thus, it can be used to measure long periods of evolution among S. aureus lineages, and this method is highly reproducible (Enright et al., 2002). A previous study using MLST and eBURST analyses showed that there are five pandemic MRSA lineages, which are the clonal complexes CC5, CC8, CC22, CC30, and CC45 (Enright et al., 2002). Along with the pandemic MRSA clones circulating in the world, new clones or subgroups (subclones) of the pandemic clones have appeared and replaced the traditional clones, becoming local pandemic clones (Enright et al., 2002; Monecke et al., 2011). The knowledge of molecular epidemiological characteristics and clonal genetic diversity of S. aureus from different origins in China is currently limited. The purpose of this study is to determine which types of SCCmec elements and local pandemic clones are present among S. aureus strains isolated from different sources, such as from the clinic, animals, food, and environments.
supermarkets (Table 1). All isolates were collected in Jiangsu province from 2006 to 2012.
Materials and Methods
Results
Bacterial isolates
MRSA and SCCmec typing
All bacterial isolates were from different origins, and were confirmed by methods described previously (Chao et al., 2013). Among the 503 isolates, 231 were from various patient tissue samples, such as blood, secretions, wounds, sputum, festers, etc., and were collected from three tertiary hospitals; 56 were from pigs located at different farms; 36 were from raw cow’s milk from distinct farms; 38 were from animals (not including pigs) such as chickens, dogs, sheep, and pigeons from animal hospitals; 67 were from environments or tools such as hair-cutting equipment, pedicure tools, bedclothes and towels in hotels or barbershops; and 75 were from retail food sources, principally from farms’ markets or
Among the 503 S. aureus isolates, 109 were MRSA because they showed resistance to cefoxitin, and they all expressed the mecA gene (CLSI, 2010). This accounts for 21.67% (109/503) of all of the isolates. The following five SCCmec types were identified: SCCmec type I (0.92%), type II (4.59%), type III (36.70%), type IV (3.67%), and type V (33.94%). About 20.18% were UT. SCCmec type I, II, and III isolates were all from humans, while SCCmec type IV, V, and UT isolates were from clinical origin, pigs, raw milk, or environments. SCCmec type III isolates were the main clinical MRSA isolates, accounting for 61.53% (40/65), followed by SCCmec type V, accounting for 18.46% (12/65). The prevalence of MRSA
SCCmec typing, antibiotic susceptibility testing
The SCCmec types (including mecA gene) were determined using a multiplex polymerase chain reaction (PCR) strategy that was developed by Oliveira et al. (Oliveira et al., 2006). Untypeable (UT) types were defined as isolates showing unexpected fragments or lacking some fragments as determined by multiplex PCR. Antibiotic susceptibility (including cefoxitin resistance) was tested as described previously (Chao et al., 2013). MLST and clone complexes
MLST was performed on 250 of 503 isolates after SCCmec typing (Table 2). Seven housekeeping genes were sequenced to determine the allelic profile. Isolates were assigned to a sequence type (ST) by the MLST database (http://saureus .mlst.net/). The program eBURST v3.0 was used to identify the diverse clonal complexes, and minimum-evolution trees were constructed from the concatenated sequences of each ST in MEGA5 (http://eburst.mlst.net).
PANDEMIC GENETIC CLONES OF S. AUREUS IN CHINA
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Table 2. Distribution of 250 Isolates for Multilocus Sequence Typing (MLST)a MRSA type
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Sources Humans Animals Pigs Raw cow’s milk Environment Foods Total
No. of isolates
I
II
III
IV
V
110 8 43 26 18 45 250
1
3
28
1(d)
14
3(b)
1 7 11
15 1
4
33
16
1 85
3
28
UT
MSSA 63 8 24 18 7 45 165
a With MLST, we are trying to find out the probable origin of methicillin-resistant Staphylococcus aureus (MRSA) and the relationship between MRSA clones and clonal complexes though analyzing S. aureus from different sources. The 250 strains were selected based on the following sources: most of MRSA strains, most of pigs (important revisors of community-acquired MRSA or livestock-associated MRSA), most milks (important media of MRSA between human and animal). See Table 3 (staphylococcal cassette chromosome mec source, methicillin-susceptible S. aureus [MSSA] source). UT, untypeable.
from clinics, pigs, raw milk and environments was 28.14% (65/231), 33.93% (19/56), 25% (9/36) and 23.88% (16/67), respectively. MRSA isolates from raw milk and environments were SCCmec type V isolates. MRSA-UT isolates were mainly from pigs. There were no MRSA isolates from food and animals (except pigs) (Table 1).
2 ST2314-MRSA-III, and 4 ST239-MSSA were isolated from the clinic (Figs. 1 and 2, Table 3).
STs, clonal complex
CC7, CC1281, CC1301, CC1920. There were two MRSA isolates in these CCs, ST1301-MRSA-V and ST2631MRSA-II, which were all isolated from humans. CC1920 included 6 STs and 33 MSSA isolates from different sources.
Among the 250 S. aureus isolates typed with MLST, 110 were from human (28 SCCmec type III, 14 SCCmec type V, 3 SCCmec type II, one SCCmec type I, one SCCmec type IVd, and 63 methicillin-susceptible S. aureus [MSSA] isolates), 8 MSSA were from animals, 43 were from pigs (3 SCCmec type IV, 1 SCCmec type V, 14 MRSA-UT, and 24 MSSA isolates), 26 were from raw cow’s milk (7 SCCmec type V, 1 SCCmecUT, and 18 MSSA), 18 were from environments (11 SCCmec type V, 7 MSSA), and 45 MSSA were from food (Table 2). Fifty-two STs were identified by MLST, of which 16 STs were new: ST2307-ST2317, ST2327, ST2630, ST2631, ST2710, and ST2711. S. aureus isolates are grouped within a single CC when five of the seven housekeeping genes have identical sequences. The ancestor of each CC is the ST with the largest number of single-locus variants. Subgroup founders can be described as single-locus variants or double-locus variants (DLV) of a founder of a CC that has become prevalent in a population (Cooper and Feil, 2004). The 52 STs generated in this data set were separated by eBURSTv3.0 and MEGA5 into 7 clonal complexes (CCs), 5 doublets (Ds), and 16 singletons (Ss) (Figs. 1 and 2, Table 3).
CC5. There were 22 isolates in CC5. Thirteen isolates belonged to ST5 including 1 ST5-MRSA-II and 3 ST5MRSA-V (Figs. 1 and 2, Table 3).
D9. There were 27 isolates, all from pigs, in D9 including 11 ST9-MRSA-UT, 1 ST9-MRSA-V, 3 ST9-MRSA-IVb, 4 ST2630-MRSA-UT, and 8 ST9-MSSA. D398. Thirteen of the 16 isolates in D398 were of clinical origin, including 14 ST398-MSSA, 1 ST398-MRSA-II, and 1 ST2199-MSSA. Three other ST398-MSSA isolates were from food. D59. Two isolates were in D59 including one ST59MRSA-IVd from the clinic and one ST338-MSSA from food. D88 and D30. Ten of the isolates were in two of the Ds. All were MSSA isolates from different sources. Ss. There were 12 Ss including 32 isolates, of which 10 isolates belonged to ST2316 from pigs. Discussion
Relationship between MRSA and CCs, Ds, STs
There were 49 isolates and 9 STs in CC630 including 29 ST630-MRSA (10 MRSA-V from humans, 7 MRSA-V from raw cow’s milk, 11 MRSA-V from environments, 1 MRSAUT from raw cow’s milk) and 11 ST630-MSSA. One ST247MRSA-I was from the clinic (Fig. 1, Table 3). CC239. CC239 is a subgroup derived from CC630. All 32 isolates including 24 ST239-MRSA-III, 2 ST1283-MRSA-III,
HA-MRSA rather than CA-MRSA remains the major pathogenic bacterium in the clinical setting; however, infections caused by CA-MRSA have increased in recent years in China (Chao et al., 2013). In this study, the prevalence of MRSA in humans, pigs, raw cow’s milk, and environments was high. All HA-MRSA isolates, most of which were SCCmec type III, came from clinical origins while CAMRSA isolates including LA-MRSA were from patients, pigs, raw cow’s milk (cows), and environments.
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FIG. 1. The ‘‘population snapshot’’ from 52 Staphylococcus aureus sequence types (STs) by eBURST v3. Seven clonal complexes (CCs) (CC630, CC239, CC1920, CC5, CC1301, CC1281, CC7) and 5 doublets (Ds) (D9, D398, D88, D30, D59) were defined using less stringent criteria (5/7 shared alleles). ST630 was the predicted clonal ancestor of CC630. CC239 is a subgroup deriving from CC630. ST239 is the founder of CC239. The sizes of the circles are relative to the number of strains in the ST.
By MLST, S. aureus populations consist of about 10 dominant human lineages including CC1, CC5, CC8, CC12, CC15, CC22, CC25, CC30, CC45, and CC51 (Lindsay, 2010). The most common MRSA isolates from hospitals belong to CC1, CC5, CC8, CC22, CC30, and CC45, and some uncommon MRSA lineages are CC59, CC80, and CC239. Each MRSA has a unique geographical distribution (Lindsay, 2010). Our results showed that S. aureus had great clonal genetic diversity. Among the 250 strains, there were 52 STs including 12 new STs, 7 CCs, and 5 Ds. Except for CC239, CC5, and the LA-clones, CC9 (D9) and CC398 (D398), a new local epidemic CC630 clone and new CC1920, CC1281, CC1301, and CC7 clones were identified. CC630 and CC239 are divergent branches of the international clone CC8, which is a Brazilian clone (Ashley and Enright, 2003). D9 and D398 clones demonstrated new characterizations that were different from other reports. The eBURST analysis revealed that the ST239, ST630, ST5, ST9, and ST398 clones coexisted in both MRSA and MSSA, which suggested that these MRSA clones might have evolved from MSSA through the acquisition of SCCmec. The ST239-MRSA clone was the predominant clone in Asian countries except for Japan and South Korea (Ko et al., 2005). In this study, all HA-MRSA-III isolates were from the same CC239 clone. Our results and other reports support the finding that CC239-MRSA-III is still the most prevalent MRSA clone throughout hospitals in China (Wang et al., 2007; Xu et al., 2009; Yan et al., 2009; Yao et al., 2010; Shen et al., 2010; Li et al., 2011; Xie et al., 2011; Liu et al., 2012; Yu et al., 2012; Chao et al., 2013; He et al., 2013). Nosocomial environments facilitate the development of HA-MRSA through the selective pressure of antibiotics. All HA-MRSA isolates in our study were of clinical origins. To the best of our knowledge, this is the first study documenting a CC630 clone including nine STs. Previously, only a few rare reports of ST630 in Hong Kong, Zhejiang, Guangdong, and Beijing in China, and in the world have been documented, indicating that all isolates were of clinical origin including the majority of MSSA and, more rarely,
MRSA (a MRSA-V, a MRSA-III, and two MRSA unknown types) (Yan et al., 2009; Li et al., 2011; Xie et al., 2011; Liu et al., 2012; Yu et al., 2012; Ho et al., 2012b; Zhao et al., 2012; He et al., 2013; Huang et al., 2013). In this study, all ST630-MRSA isolates belonging to CA-MRSA were MRSA-V except for one MRSA-UT. Isolates in this group were all from clinical origin, raw cow’s milk, or environments (hair-cutting equipment, pedicure tools, bedclothes, and towels in hotels or barbershop). The results demonstrated that humans, animals and other environments have formed an epidemic circle of CA-MRSA/MSSA. We cannot determine whether CA-MRSA originated from humans or animals. Additionally, it cannot be ignored that the environment plays an important part in the transmission and repository of CA-MRSA because S. aureus can survive for a long time on inanimate objects (Simoes et al., 2011; Uhlemann et al., 2011). One clinical isolate, ST247MRSA-I (ST247, a DLV of ST630), belongs to the pandemic ‘‘Iberian’’ clone (Robinson and Enright, 2004), while few isolates were reported in China. CC5 is another common and widespread clonal complex, next to CC8, which comprises a large number of different MRSA isolates that are both HA- and CA-MRSA, some of which have attained pandemic status (Ko et al., 2005; Monecke et al., 2011). In this study, two types of MRSA, ST5-MRSA-II (HA-MRSA) and ST5-MRSA-V (CA-MRSA), were isolated from the clinic. ST5-MRSA-II is known as the pandemic New York/Japan clone (Enright et al., 2002), while ST5-MRSA-V has been described in Australia, Ireland, Germany, and Abu Dhabi (Monecke et al., 2011). In China, ST5-MRSA-II was also a predominant clone, next to ST239-MRSA-III (Wang et al., 2007; Yan et al., 2009; Shen et al., 2010; Li et al., 2011; Xie et al., 2011; Liu et al., 2012), but few ST5-MRSA-V have been reported. MRSA-colonized animals, especially livestock, have provided and expanded reservoirs for MRSA. In the last decade, two major animal-adapted clones, ST398 in Europe and ST9 in Asia, have been reported continuously since they were originally identified (Lo et al., 2012).
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PANDEMIC GENETIC CLONES OF S. AUREUS IN CHINA
FIG. 2. A minimum-evolution tree was constructed using the concatenated sequences of seven loci from 52 sequence types by MEGA5. The clonal complexes (CCs) and doublets (Ds) in Fig. 1 basically corresponded to the CCs and Ds in the tree. Bootstrap values over 50% are shown on the branches. There have been many reports of CC9 (ST9)-MRSA isolated from pigs, other animals, and humans in China. CC9 (ST9)-MRSA-IVb and CC9 (ST9)-MRSA-V were isolated from pigs and raw meat (slaughtered pigs) in Taiwan and Hong Kong, and ST9- MRSA-IVb was isolated from butchers in Hong Kong (Guardabassi et al., 2009; Boost et al., 2013a; Ho et al., 2012a; Boost et al., 2013b). CC9 (ST9, ST97)-MRSA-IVb was isolated from bovine in five provinces on the mainland (Wang et al., 2011). ST9-MRSA was isolated from patients in Tianjin on the mainland (Zhang et al., 2003). CC9 (ST9, ST912, ST1297)-MRSA-III was isolated from swine and swine workers from four provinces
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on the mainland (Cui et al., 2009). CC9 (ST9, ST1376)MRSA and MSSA were isolated from pig farms in Sichan (Wagenaar et al., 2009). ST9-MRSA-V and IV were isolated from healthy swine and human clinical samples in Taiwan (Pantosti, 2012; Wan et al., 2013). In this study, all MRSA in D9 (ST9, ST2360) were types IVb, V, and UT and were isolated from pigs from distinct farms in Jiangsu. The clones ST9-MRSA-IVb, and -V were similar to the main CC9 (ST9)-MRSA clones reported in China. ST9-MSSA isolates also existed in D9. One characteristic of CC9 in this study was the presence of most of the MRSA-UT isolates; therefore, we assumed that special dimensional molecular structures could exist on SCCmec. Another finding is that none of the CC9 isolates were isolated from the clinic. We could regard CC9 as the only LA clone that has not spread to humans in Jiangsu yet. Even so, it is a potential zoonotic infectious agent for humans. The CC398-MRSA clone is the first LA-MRSA clone to originate from pigs. All MRSA isolates of this clone were found to share similar characteristics, such as nontypeability by pulsed-field gel electrophoresis and identical spa type. Although there is a high prevalence of CC398 in pigs and other animals, it seems that the CC398 clone is a poorly persistent colonizer in humans and that invasive infection caused by clone ST398 occurs rarely (David and Daum, 2010). In sharp contrast, in China, there are a number of reports regarding isolation of the ST398 clone from humans, not from pigs (animals), based on a search in PubMed and WanFang (in Chinese) for related articles (Ip et al., 2005; Yu et al., 2008; Chen et al., 2010; Stegger et al., 2010; Yao et al., 2010; Deng et al., 2012; Li et al., 2013). Chen et al. describe ST398-MSSA from humans appearing early in 1994 (Chen et al., 2010). Zhao et al. described 4 hospitals in Beijing that a high prevalence of ST398-MSSA clone (17.1% of 164 infections), which was found with no apparent association with animal contact during 2009–2010 (Zhao et al., 2012). In contrast to studies in Europe and the Americas, the ST398 isolate does not appear to be the dominant MRSA strain in Asian pigs/animals (David and Daum, 2010; Stegger et al., 2010; Graveland et al., 2011; Pantosti, 2012). In China, only one ST398-MRSA-V was reported from diseased animals (Zhang et al., 2011). In this study, most of the ST398-MSSA were clinical isolates, others were from foods, and none were from pigs. One ST398-MRSA-II isolate, from a clinic, could be the first reported in mainland China. We did not know whether the patients infected with ST398-MSSA/MRSA had contact with pigs or other animals. It is remarkable that a number of data have demonstrated that ST398-MSSA/MRSA infects humans, whereas few isolates were recovered from pigs or other animals. There were no signs indicating that the ST398 isolate colonized animals in China. The clone infected humans long before the first report of infection of European pigs; therefore, we have good reason to presume that ST398 may not be an LA clone, but a CA clone in China. The ST398 clone has colonized and caused infections for a long time in humans (not animals). CC59-MRSA-IV/V has been reported in Australia, the United Kingdom (Monecke et al., 2011), and in Hong Kong, Taiwan, and mainland China (Wang et al., 2004; Ip et al., 2005; Wang et al., 2007; Chen et al., 2010; Shen et al., 2010; Xie et al., 2011; Zhao et al., 2012). The results from this
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Table 3. Sequence Types (STs), Clonal Complexes (CCs), Doublets (Ds), Variants (Vs), Methicillin-Resistant Staphylococcus aureus, and Methicillin-Susceptible S. aureus (MSSA), as Well as the Sources of the 250 Isolates
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Multilocus sequence typing alleles Clone
ST
arcc
aroe
glpf
Gmk
Pta
tpi
yqil
No.
SCCmec: source (no.)
MSSA source (no.)
CC239
239 1283 2314 630
2 2 2 12
3 3 3 3
1 1 1 1
1 88 1 1
4 4 4 4
4 4 4 4
3 3 257 3
28 2 2 40
III:Ha(24) III:H(2) III:H(2) V:H(10)M(7)E(11); UT:M(1)
H(4)
8 3 770 1 2711 12 2205 12 1821 12 2309 248 247 3 2710b 18 1920 1 2312 4 2310 1 1 1 188 3 2315 1 5 1
3 3 3 3 3 3 3 95 1 1 1 1 1 1 4
1 1 1 1 1 1 1 45 1 1 1 1 1 1 1
1 1 1 1 1 1 12 206 157 157 157 1 8 177 4
4 4 297 4 40 40 4 7 1 1 1 1 1 12 12
4 4 4 1 4 4 4 15 1 1 258 1 1 1 1
3 3 3 3 3 3 16 5 1 1 1 1 1 1 10
1 1 1 1 2 1 1 1 11 1 1 9 10 1 13
CC630
CC1920
CC5
CC1301 CC1281 CC7 D9 (CC9) D398 (CC398) D88 (CC88) D30 (CC30) D59 (CC59) S S S S S S S S S S S
965 2313 6 1301 2311 121 1281 20 2631 7 943 2327 9
1 1 12 158 158 6 4 4 4 5 5 5 3
4 4 4 5 5 5 168 9 168 4 4 4 3
1 258 1 6 14 6 1 1 1 1 1 1 1
4 4 4 2 2 2 8 8 8 4 4 179 1
119 12 12 7 7 7 1 1 290 4 116 4 1
1 1 1 14 14 14 10 10 10 6 6 6 1
10 10 3 5 5 5 8 8 8 3 3 3 10
2 1 6 1 1 1 6 1 1 15 1 1 23
2630 398 2199 88 2148 243 30 59 338 2316 2317 25 15 97 479 2307 2308 12 22 1207
3 3 3 22 3 2 2 19 19 10 1 4 13 3 52 14 18 1 7 8
3 35 35 1 4 2 2 23 23 14 115 1 13 1 79 57 330 3 6 167
1 19 19 14 14 5 2 15 15 8 4 4 1 1 54 1 46 1 1 135
201 2 2 23 23 2 2 2 48 26 2 1 1 1 18 155 155 8 5 2
1 20 20 12 12 6 6 19 19 10 13 5 12 1 56 109 7 11 8 13
1 26 5 4 4 3 3 20 20 3 259 5 11 5 32 50 50 5 8 127
10 39 39 31 31 2 2 15 15 255 11 4 13 3 65 2 253 11 6 7
4 15 1 6 1 1 2 1 1 10 3 5 5 2 1 1 1 1 1 1
H(6)E(1)M(1)F(3) E(1) H(1) E(1) E(1) H(1)E(1) F(1)
I:H(1)
II:H(1); SV:H(3)
A(1) M(7)F(4) H(1) F(1) H(1)A(1)M(2)F(5) H(5)F(5) H(1) H(3)A(4)M(1)F(1) H(1)F(1) H(1) H(6)
V:H(1) H(1) H(1) H(1)M(2)F(3) F(1) II:H(1)
IVb:P(3);V:P(1); UT:P(11) UT:P(4) II:H(1)
H(4)M(4)F(7) H(1) F(1) P(8) H(11)F(3) A(1) H(4) F(2) F(1) P(1) H(2)
IVd:H(1) F(1) P(10) P(3) H(5) H(1)E(2)F(2) M(1)F(1) F(1) A(1) A(1) F(1) H(1) P(1)
a H, human origin; A, animal origin (not including pigs); M, raw cow’s milk origin; P, pig origin; E, environment origin; F, food origin; UT, untypeable. b ST2710 belonged to CC630 by MEGA5 (Fig. 2).
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study show that the ST59 clone was not the main epidemic clone in Jiangsu province. In conclusion, this study revealed the molecular and epidemiological characteristics and clonal genetic diversity of S. aureus in China. There were 52 STs including 16 novel STs, forming 7 CCs and 5 Ds. There were also four important clones including CC239, CC630, D398, and D9 that were identified in this study. CC239 was the major HA-MRSA-III clone. CC630 was a new epidemic group, and all CA-MRSAV/MSSA isolates in this clone were from different sources, such as from the clinic, environment, or raw cow’s milk. Animals and environments played an important part in the repositories and transmission of CA-MRSA/MSSA. The CC9 clone was a LA-MRSA/MSSA clone and has spread only in livestock rather than in humans in Jiangsu province. Despite many reports of the ST398-LA-MRSA/MSSA clone originating in pigs and humans in Europe and the United States, there are many lines of evidence, including this study, showing that ST398 could be a CA clone rather than an LA clone in China.
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Acknowledgments
We would like to thank all individuals who kindly provided us with the strains used in this study. This work was supported by grants from the National High Technology Research and Development Program of China (863 Program) (2012AA101601-6) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Xinan Jiao, PhD Jiangsu Key Laboratory of Zoonosis Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses Yangzhou University 88 South University Avenue Yangzhou 225009, China E-mail: [email protected]
