Infectious Diseases, 2015; Early Online: 1–9
ORIGINAL ARTICLE
Characterization of pediatric hospital-associated infection caused by methicillin-resistant Staphylococcus aureus in mainland China
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XUE NING1,2, MINGJIAO SUN1, YANHONG QIAO1, FANG DONG2, WENQI SONG2, KAIHU YAO1, YONGHONG YANG1 & XUZHUANG SHEN1 From the 1Key Laboratory of Major Diseases in Children and National Key Discipline of Pediatrics (Capital Medical University), Ministry of Education, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Beijing, PR China, and 2Beijing Children’s Hospital, Capital Medical University, Beijing, PR China
Abstract Background: This study was conducted to investigate the clinical features of hospital-associated infections (HAIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) in Chinese children, and the molecular characteristics of the bacteria. Methods: Patients with HAIs caused by MRSA were identified retrospectively. All isolates were analyzed using molecular typing and antimicrobial susceptibility tests. Results: In total, 150 patients were identified, with a median age of 18 months. The most common infection was pneumonia (55.3%), followed by skin and soft tissue infections (46%). Invasive infections were observed in 52 patients (34.7%), and their hospital stay was longer compared with non-invasive cases (21 vs 12 days, p ⫽ 0.047). A total of 16 sequence types (STs) were identified. ST239 and ST59 were common clones, accounting for 46% and 28% of cases, respectively. Compared with cases caused by ST239-SCCmecI-III, patients infected by ST59-SCCmecIV-V had a lower median age (11 vs 41 months, p ⫽ 0.047) and more commonly developed invasive infection (50% vs 18.8%, p ⫽ 0.006). Conclusions: Invasive infections accounted for a large proportion of HAIs caused by MRSA. ST59-SCCmecIV/V, a common clone in the community, caused HAIs in Chinese children, more often infected younger children and caused invasive infections.
Keywords: Hospital-associated infections, invasive infection, methicillin-resistant Staphylococcus aureus, MRSA-ST59
Introduction Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a life-threatening multidrug-resistant bacterium, which causes various infections, including skin and soft tissue infections (SSTIs), endocarditis, toxic shock syndrome, necrotizing fasciitis, necrotizing pneumonia and sepsis [1]. Since the onset of the pandemic waves of MRSA over the past few decades, it has become a common cause of both hospital- and community-associated infections worldwide [2]. Limited clones of hospital-associated MRSA (HA-MRSA) have emerged and been disseminated worldwide, including ST5, ST239, ST22, ST36 and ST45 [3], while clones of community-associated MRSA (CA-MRSA) are more diversified. With the continuing global epidemic of CA-MRSA, the strain
types USA300 [4], ST1 [5] and ST72 [6] have emerged as a cause of hospital-associated infections (HAIs) in the USA, the UK and Korea, respectively. According to previous studies, the prevalence of MRSA in hospitals for adults varies from 13% to 80% [7,8]. In children, many studies have focused on CA-MRSA infections [9]. Although measures such as universal hand hygiene practices were introduced into hospitals in mainland China several years ago, HAIs are still common [10]. To the best of our knowledge, there is limited information characterizing the HAIs caused by MRSA in children in mainland China. We conducted this study to collect clinical information and determine the molecular characteristics of HA-MRSA in the largest children’s hospital in Beijing, China.
Correspondence: Xuzhuang Shen, Beijing Children’s Hospital affiliated with the Capital Medical University, Nan Li Shi Road, Beijing, 100045, PR China. Tel: ⫹ 86 1059616652. Fax: ⫹ 86 1068028401. E-mail: [email protected] (Received 17 July 2014 ; accepted 24 December 2014 ) ISSN 0036-5548 print/ISSN 1651-1980 online © 2015 Informa Healthcare DOI: 10.3109/00365548.2015.1006675
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Materials and methods
Molecular typing methods
Hospital setting and patient selection
Multilocus sequence typing (MLST) was performed as previously described [12]. The MLST allelic profiles and sequence types (STs) were assigned by submission to the MLST database (http://saureus.mlst. net). Related STs were grouped within a clonal complex (CC), which is determined by an algorithm called eBURST. The SCCmec types and subtypes were determined using two multiplex PCR strategies as previously described [13]. The prototypes of SCCmec types I-V for the isolates and SCCmecIV subtypes for the subtypes were used as the quality control sample. Spa typing was performed as previously described [14]. Purified spa PCR products were sequenced and compared using the spa database website (http://www.ridom.de/spaserver).
This study was conducted in Beijing Children’s Hospital, which is a teaching hospital affiliated to the Capital Medical University with 1000 beds, and which provides both primary and tertiary care. Patients with confirmed HA-MRSA infections were identified from the hospital’s database between January 2009 and June 2013. The clinical signs and course of these patients were consistent with MRSA infection and the isolates were recovered from infected sites, including pus, endotracheal aspirates, hydrothorax, synovial fluid or blood. The medical records of all identified patients were retrospectively reviewed and the following information was collected: age, gender, underlying conditions (history of immunodeficiency diseases, diabetes mellitus, cancer and congenital heart diseases), risk factors (immunosuppressant drugs, burn/scald/traumatic wounds, surgery, central venous catheter), laboratory data (including white blood cell counts, levels of C-reactive protein and coinfections with other pathogens) and clinical outcome. This study was approved by the Ethics Committee of Beijing Children’s Hospital affiliated to the Capital Medical University (2008-35).
Case definition According to the Centers for Disease Control and Prevention (CDC), HA-MRSA infections were defined as follows: cultures were taken from an inpatient more than 48 h after admission or from an outpatient with risk factors for HAI, namely, recent (within the past year) hospitalization or medical procedure (e.g. dialysis, surgery and catheters) [11]. Severe pneumonia was defined by any one of the following: requirement for intensive care unit (ICU) admission, necrotizing or cavitary infiltrates, or empyema. Invasive MRSA infection was defined as the isolation of MRSA from a normally sterile body site, including blood, pleural fluid, cerebrospinal fluid, pericardial fluid, peritoneal fluid, joint/synovial fluid, bone or internal body sites [11].
Identification of strains and DNA extraction Collection, transportation and identification of MRSA isolates were performed as previously described [9]. DNA was extracted from all isolates using a DNA extraction kit (Saibaisheng, China) and used as the template in all polymerase chain reactions (PCRs).
Antimicrobial susceptibility testing Antimicrobial susceptibility was determined using the disk-diffusion method and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute. Susceptibilities to penicillin, erythromycin, clindamycin, cefuroxime, tetracycline, chloramphenicol, gentamicin, ciprofloxacin, sulfamethoxazoletrimethoprim, rifampicin, cefoxitin, azithromycin vancomycin, linezolid, tigecycline and fusidic acid (Sigma, St Louis, MO, USA) were determined. Susceptibility testing to fusidic acid, tigecycline and mupirocin was carried out according to a previous study [15]. Staphylococcus aureus ATCC29213 was used as a control strain. Isolates resistant to three or more antimicrobial classes were defined as multidrug resistant.
Screening for 42 virulence genes All isolates of ST239 and ST59 were screened for 42 known virulence genes. the presence of the 21 known superantigen (SAg) genes (enterotoxin genes: sea, seh, sec, sed, sek, see, seb, sem, sel, seo, sen, seg, seq, sej, sei, seu, ser and sep; toxic shock syndrome toxin gene: tsst-1; and two exfoliative toxin genes: eta and etd), and the agr types was tested by six multiplex PCRs. The genes of exfoliative toxin B gene (etb), fibronectin-binding protein genes (fnbA and fnbB), clumping factor genes (clfA and clfB), bone sialoprotein gene (bbp), collagen-binding protein gene (cna), extracellular adherence protein gene (eap), elastin-binding protein gene (ebpS), genes for the serine-aspartate repeat family of proteins (sdrC, sdrD and sdrE), staphylococcal surface protein A gene (spa), hemolysin genes (hla, hlb, hld, hlg and psma) and leukocidin genes (pvl, lukD and lukE) were screened by singleplex PCRs. The primers are listed in Supplementary Table I. (available online at: http://informahealthcare.
Nosocomial infection caused by MRSA in children com/doi/abs/10.3109/00365548.2015.1006675) [16,17–22]. Statistical analysis SPSS 17.0 software was used for statistical analysis. The categorical variables were compared using the chi-squared or two-tailed Fisher’s exact test. The means of two independent samples were compared using Student’s t test. Multiple logistic regression analysis was used to analyze the risk factors of invasive HA-MRSA infection. A p values less than 0.05 was considered statistically significant.
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Results Clinical features In total, 150 patients with hospital-associated MRSA infections were identified and their clinical charac-
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teristics are summarized in Table I. Ninety-seven patients were male. The patients ranged in age from 5 days to 14 years, with a median age of 18 months. Thirty-two (21.3%) were neonates aged less than 1 month. Fever was the initial presentation in 139 patients (92.7%). Ten types of infection were identified. Single-site infections were found in 124 patients (82.7%), and 26 patients (17.3%) had two or more infective sites simultaneously. Fifty-two patients (34.7%) had invasive and 98 (65.3%) had non-invasive infections. The most common infection was pneumonia (83/150, 55.3%), followed by SSTIs (69/150, 46%), bacteremia (21/150, 14%), osteomyelitis (5/150, 3.3%) and urinary tract infection (1/150, 0.7%). Invasive infections included severe pneumonia (n ⫽ 15), SSTIs (n ⫽ 13), bacteremia (n ⫽ 2), osteomyelitis (n ⫽ 2), urinary tract infection (n ⫽ 1), bacteremia related to pneumonia (n ⫽ 7), bacteremia related to SSTIs
Table I. Demographics and clinical characteristics of patients with hospital-associated infections caused by methicillin-resistant Staphylococcus aureus.
Item Age Age ⱕ 1 month Male Type of infection Pneumonia Skin and soft tissue infection Bloodstream infection Urinary tract infection Bone/joint infection Underlying diseases Cancer Congenital heart disease Immunodeficiency diseases Type 1 diabetes mellitus Risk factors Receiving immunosuppressant drugs Burn/scald/traumatic wound Surgery Central venous catheter Fever Polymicrobial infection Complication Respiratory failure Heart failure Shock Blood coagulation disorders Laboratory data White blood cells (109/l) Neutrophil count mean (109/l) C-reactive protein (mg/l) Outcomes Intensive care unit admission Mechanical ventilation Length of hospitalization (days) Mortality Data are shown as mean (range) or n (%).
Total cases (n ⫽ 150)
Invasive cases (n ⫽ 52)
Non-invasive cases (n ⫽ 98)
p
18 (5 days-14 years) 43 (28.7) 97 (64.7)
25 (12 days-8 years) 16 (30.8) 37 (71.2)
17 (5 days-14 years) 27 (27.6) 60 (61.2)
0.826 0.678 0.226
83 69 21 1 5 42 32 5 3 2 109 23 48 23 15 136 28 32 18 3 4 7
(55.3) (46) (14) (0.7) (3.3) (28) (21.3) (3.3) (2) (1.3) (72.7) (15.3) (32) (15.3) (10) (90.7) (18.7) (21.3) (12) (2) (2.7) (4.7)
15.8 (1.4–76.3) 11.9 (0.75–65.4) 50.7 (0.9–256.1) 45 21 15 4
(30) (14) (2–59) (2.7)
25 22 21 1 5 19 14 3 1 1 38 10 9 7 12 50 12 15 9 1 2 3
(48.1) (42.3) (40.4) (1.9) (9.6) (36.5) (26.9) (5.8) (1.9) (1.9) (73.1) (19.2) (17.3) (13.5) (23.1) (96.2) (23.1) (28.8) (17.3) (1.9) (3.8) (5.8)
16.7 (1.4–76.3) 12.5 (0.75–65.4) 62.4 (0.9–256.1) 25 12 21 3
(48.1) (23.1) (4–59) (5.8)
58 47 0 0 0 23 18 2 2 1 71 13 39 16 3 82 16 17 9 2 2 4
(59.2) (48) (0) (0) (0) (23.5) (18.4) (2) (2) (1) (72.4) (13.3) (39.8) (16.3) (3.1) (83.7) (16.3) (17.3) (9.2) (2) (2) (4.1)
15.6 (4.1–56.5) 10.7 (1.3–41.3) 43 (1.2–132) 20 9 12 1
(20.4) (9.2) (2–43) (1)
0.193 0.509 ⬍ 0.001 0.351 0.004 0.09 0.223 0.342 1.000 1.000 0.935 0.335 0.005 0.643 ⬍ 0.001 0.139 0.313 0.102 0.145 1.000 0.610 0.694 0.792 0.831 0.634 ⬍ 0.001 0.020 0.047 0.120
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(n ⫽ 6), bacteremia related to osteomyelitis (n ⫽ 3) and bacteremia complicated with pneumonia and SSTIs (n ⫽ 3). Non-invasive infections included pneumonia (n ⫽ 51), SSTIs (n ⫽ 40) and pneumonia complicated with SSTIs (n ⫽ 7). Underlying conditions including cancer, immunodeficiency diseases, type 1 diabetes mellitus and congenital heart disease were found in 42 patients (28%). Patients with comorbidity accounted for 36.5% of invasive infections. Risk factors were found in 109 patients. Central venous catheters were more common in invasive infection [95% confidence interval (CI) 0.028–0.393, p ⬍ 0.001]. Burns/scald/traumatic wounds were more common in non-invasive cases (95% CI 0.139–0.722, p ⫽ 0.005). Gender, underlying disease, immunosuppressant drugs, burns/scalds/traumatic wounds, surgery and central venous catheters were analyzed using multiple logistic regression. The results show that central venous catheter was a risk factor for invasive HAMRSA infection (OR 0.293, 95% CI 0.121–0.711, p ⫽ 0.007). Among the patients who had polymicrobial infections, cytomegalovirus was the most common copathogen (n ⫽ 8), followed by Klebsiella pneumoniae (n ⫽ 4), Epstein-Barr virus (n ⫽ 3), Mycoplasma pneumonia (n ⫽ 3), Pseudomonas aeruginosa (n ⫽ 2), Staphylococcus epidermidis (n ⫽ 2), Chlamydia pneumoniae (n ⫽ 1), Acinetobacter baumanii (n ⫽ 1), Haemophilus parainfluenzae (n ⫽ 1), measles virus (n ⫽ 1) and respiratory syncytial virus (n ⫽ 1). All patients were treated empirically with antibiotics. Cephalosporins (90%, 135/150), including first generation (3.7%, 4/108), second generation (29.6%, 32/108), third generation (66.7%, 72/108) and fourth generation (18%, 27/150), and vancomycin (4%, 6/150) were intravenously administered before pathogen detection. Sixteen patients received mupirocin for SSTIs. One-hundred and seventeen of the 150 patients (78%) were switched to other drugs after pathogens were detected in blood cultures. Vancomycin, linezolid and teicoplanin were given to 97, 14 and six patients, respectively. Clinical outcomes, including rates of ICU admission, respiratory failure and mortality related to HAMRSA infections were 32.3% (49/150), 9.3% (14/150) and 2.7% (4/150), respectively. Patients with invasive MRSA infection had higher rates of ICU admission (95% CI 0.133–0.576, p ⬍ 0.001) and mechanical ventilation (95% CI 0.131–0.864, p ⫽ 0.020) than non-invasive cases. Molecular characteristics of MRSA isolates Among the 150 HA-MRSA strains, 16 STs were detected, belonging to 12 CCs (Table II). The
most prevalent ST was ST239 (69/150, 46%), followed by ST59 (42/150, 28%). Other STs varied from 0.7% to 6%. The most common SCCmec type was SCCmecIII (79/150, 52.7%), followed by SCCmecIV (45/150, 30%) and SCCmecII (12/150, 8%). In addition, MRSA with SCCmecIV/V accounted for 32.7% (49/150) of all isolates. Seven isolates could not be SCCmec typed. Three SCCmecIV subtypes were detected: SCCmecIVa (43/45, 95.6%), SCCmecIVb (1/45, 2.2%) and SCCmecIVc (1/45, 2.2%). Thirty-two spa types were identified. The most common was t030 (34/150, 22.7%), followed by t437 (29/150, 19.3%), t037 (19/150, 12.7%) and t459 (14/150, 9.3%). Two isolates could not be spa typed, as shown in Table II. The most prevalent clone of MRSA was ST239III (65/150, 43.3%), including ST239-III-t030 (34/65, 55.4%), ST239-III-t037 (17/65, 26.2%) and ST239-III-t459 (12/65, 18.5%), followed by ST59IVa (34/150, 22.7%), including ST59-IVa-t437 (27/42, 64.3%), ST59-IVa-t441 (4/42, 9.5%) and ST59-IVa-t3523 (2/42, 4.8%). ST59-V and ST5-II accounted for 2.7% and 5.3%, respectively. ST1801 and ST960, which were both single-locus variants of ST239, and livestock-associated-MRSA ST9 and ST398, were also detected. Thirty-four isolates (22.7%) were Panton-Valentine leukocidin (PVL) positive. The positive rates in clones ST239 and ST59 were 14.5% (10/69) and 40.5% (17/42), respectively (p ⫽ 0.002). Among pvl-positive strains, the most prevalent clone was ST59-SCCmecIVat437 (13/34, 38.2%). Antibiotic susceptibility All isolates were susceptible to vancomycin, tigecycline and linezolid (Table III). Resistance to other classes of antibiotics was highly variable. The resistance rates to penicillin, cefuroxime, azithromycin, clindamycin, erythromycin, tetracycline, ciprofloxacin, gentamicin, rifampin, chloramphenicol, trimethoprim/sulfamethoxazole, fusidic acid and mupirocin were 97.3%, 92%, 86.7%, 82%, 77.3%, 70.7%, 61.3%, 56%, 49.3%, 15.3%, 8%, 0.7% and 0.7%, respectively. The resistance rates to gentamicin, rifampin, tetracycline and ciprofloxacin differed between MRSA-ST59 and ST239 (p ⬍ 0.01). The multidrug resistance rates of all isolates, MRSA-ST59 isolates and MRSA-ST239 isolates were 84.0%, 78.6% and 81.2%, respectively. Moreover, 72.5% (50/69) of the ST239 strains were included in two resistance profiles, which were PEN-CEF-GEN-TET-RIF-ERY-CLI-CIP (36/69, 52.2%) and PEN-CEF-GEN-TET-RIF (13/69, 18.8%), whereas the predominant antibiotic
Nosocomial infection caused by MRSA in children
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Table II. Molecular characteristics of methicillin-resistant Staphylococcus aureus isolated from Chinese children with hospital-associated infections. MLST (n ⫽ 150) CC 8 (48)
ST
pvl
SCCmec (n ⫽ 150)
Spa (n ⫽ 150)
239 (46)
10 (6.7)
III (43.3)
t030 (20), t037 (11.3), t459 (8), t969 (1.3), t2270 (1.3), t7221 (0.7), t2760 (0.7) t030 (2) t008 (0.7) t037 (0.7) t459 (0.7) t459 (0.7) t437 (0.7), NT (0.7) t437 (18.7), t441 (2.7), t3523 (1.3) t437 (2), t441 (0.7) t441 (1.3) t437 (0.7) t2270 (0.7) t002 (4), t686 (0.7), t601 (0.7) NT (0.7) t1376 (1.3), t127 (0.7), t4431 (0.7) t8723 (0.7) t1246 (0.7), t3078 (0.7), t3523 (0.7) t078 (0.7), t081 (1.3) t796 (1.3), t037 (0.7) t375 (0.7) t375 (0.7) t664 (0.7) t10555 (0.7) t664 (0.7) t899 (0.7) t437 (1.3) t034 (0.7) t127 (0.7) t202 (0.7)
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1801 (1.3)
59 (29.3)
960 (0.7) 59 (28)
1 (0.7)
5 (6)
338 (0.7) 375 (0.7) 5 (6)
88 (3.3)
88 (3.3)
2 (1.3)
15 (4)
25 (4)
2 (1.3)
7 (2) 509 (1.3)
(0.7) (8.7) (1.3) (0.7)
7 (2) 509 (1.3)
72 (2)
72 (2)
9 (0.7) 398 (2)
9 (0.7) 398 (2)
1 (0.7) Singleton (0.7)
1 13 2 1
1 (0.7) 93 (0.7)
1 (0.7) 1 (0.7)
II IVa I III III III IVa V NT III IVa II IVa III I IVa NT III II III IVc IVa NT IVb IVa I NT III
(2) (0.7) (0.7) (0.7) (0.7) (1.3) (22.7) (2.7) (1.3) (0.7) (0.7) (5.3) (0.7) (2.7) (0.7) (2) (2) (2) (0.7) (0.7) (0.7) (0.7) (0.7) (0.7) (1.3) (0.7) (0.7) (0.7)
Data are shown as n (%). MLST, multilocus sequence typing; CC, clonal complex; ST, sequence type; SCCmec, Staphylococcus aureus chromosome typing; Spa, Staphylococcus protein A typing; NT, non-typeable.
resistance profile PEN-CEF-TET-ERY-CLI-AZI accounted for only 23.8% (10/42) of ST59 strains. Distributions of 42 virulence genes and agr types in two major sequence types Strains of ST59 and ST239 were selected to study virulence characteristics. All 111 strains contained SAg genes. The prevalent SAg genes were sek (104/111, 93.7%), seq (98/111, 88.3%), sea (73/111, 65.8%) and seb (35/111, 31.5%). Genes sed, ser, eta, etb and etd were not detected. All isolates carried the adhesion genes fnbA, clfA, clfB, ebpS, sdrC and spa. A majority harbored bbp (108/111, 97.3%), sdrE (107/111, 96.4%) and lukE (92/111, 82.9%) genes. All strains carried hla and hlb. Genes hld (97.3%) and hlg (94.6%) were carried by a majority of the strains. However, the leukocidin gene pvl (19.8%) was not common and lukM was not detected. The gene encoding α-type phenol-soluble modulin was
detected in all strains. A total of 10 SAg gene combinations was observed. The most prevalent one was sea-sek-seq, carried by 71% (49/69) of ST239, whereas seb-sek-seq was observed in 76.2% (32/42) of ST59. Virulence genes were associated with specific molecular types. The sea (61/69) and lukE (65/69) genes were more often identified in ST239, whereas seb (42/42) and pvl (20/42) were more often identified in ST59 (p ⬍ 0.05). lukM, can and sdrC genes were not common in ST239 and ST59. Moreover, 99.1% (110/111) of isolates were agr type I and one isolate of ST239 was negative for agr typing. Comparison of hospital-associated infections caused by MRSA ST59, ST239 and ST5 In this study, 69 (46%) and 42 (28%) patients were infected by MRSA ST239 and ST59, respectively. The median age of children infected by ST59 and ST239 was 11 and 41 months, respectively
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X. Ning et al. Table III. Antimicrobial resistance of 150 methicillin-resistant Staphylococcus aureus isolates based on multilocus sequence typing.
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STs ST239 ST59 ST5 ST25 ST88 ST72 ST7 ST398 ST1801 ST509 ST338 ST375 ST93 ST960 ST1 Total
n
PEN
CEF
GEN CHL TET
69 42 9a 6 5 3 3 3 2 1 1 1 1 1 1 150
100 95.2 8 5 5 3 3 2 2 1 1 1 1 1 1 146
100 84.1* 97.6 19 9 8 5 2 4 1 3 0 2 1 3 1 2 1 1 1 1 0 1 0 1 1 1 1 1 1 138 84
RIF
11.6 94.2* 88.4* 19 45.2 9.5 1 8 6 2 3 1 2 2 1 1 1 0 1 2 0 0 1 0 0 1 1 0 1 0 0 0 0 0 0 0 0 1 0 0 1 0 0 1 0 23 106 74
ERY
CLI
AZI
CIP
TRI
76.8 85.7 7 5 4 2 2 3 1 0 1 1 0 1 0 116
81.2 90.5 7 5 4 2 2 3 1 0 1 1 1 1 1 123
10.1 94.2* 10.1 23.8 21.4 4.8 1 8 2 1 2 1 1 1 0 2 1 0 1 1 0 0 0 0 0 1 0 0 1 0 1 0 0 0 0 0 1 1 0 1 1 0 0 1 0 130 92 12
FUS MUP 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1
0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1
PEN, penicillin; CEF, cefuroxime; GEN, gentamicin; CHL, chloramphenicol; TET, tetracycline; RIF, rifampin; ERY, erythromycin; CLI, clindamycin; AZI, azithromycin; CIP, ciprofloxacin; TRI, trimethoprim; FUS, fusidic acid; MUP, mupirocin. aSequence types (STs) with fewer than 10 isolates were not calculated in the percentage of antibiotic resistance. Comparison between MRSA-ST239 and ST59 isolates: *p ⬍ 0.01.
(p ⫽ 0.047). In addition, 26.2% (11/42) and 10.1% (7/69) of the patients infected by ST59 and ST239 were neonates (95% CI 0.112–0.901, p ⫽ 0.026). Fifty percent (21/42) of patients infected with ST59 developed invasive infections compared with 24.6% (17/69) of those with ST239 infections (95% CI 0.145–0.739, p ⫽ 0.006). A comparison of clinical information among the major MRSA clones is shown in Supplementary Table II available online at: http:// informahealthcare.com/doi/abs/10.3109/00365548. 2015.1006675. There was no difference in outcome, underlying diseases and risk factors among patients infected with ST59 and ST239 isolates.
Discussion Studies have demonstrated that most HAIs caused by MRSA can be attributed to a relatively small number of epidemic clones. In Asia, the most frequent clones are Brazil/Hungary (ST239-III) and New York/Japan (ST5-II) [23]. Data from Wang’s study confirmed that these two clones have spread across China among adults [24]. Similarly, in the present study, the most prevalent clone in HA-MRSA in Chinese children was ST239-III, followed by ST59-IV, while ST5-II accounted for only 5.3%. The data from this study suggest that the genetic background of HA-MRSA in pediatric patients in mainland China is different from that found in other reports and varies in different populations even in the same region. Recent reports suggested that MRSA with CA genotypes are beginning to supplant or overtake
HA-MRSA clones as a cause of HAIs [25]. USA300 is an increasingly common community pathogen in the USA with a parallel tendency to cause HAIs. In Korea, 24% of healthcare-associated bacteremias were caused by the major ST72-IV CA-MRSA clone [6]. The ST59-IV and ST59-V clones were reported to cause HAIs in Taiwan and accounted for 25% of nosocomial bloodstream infections [26]. In the present study, nearly one-third of MRSA had CA genotypes, including ST59-IV/V, ST88-IV, ST25-IV and ST72-IV, which were previously reported to be common clones in CA-MRSA infections in China, Madagascar, Algeria and Korea, respectively [27]. Among isolates with CA genotypes, ST59-IV/V was a major clone. The influx of CA-MRSA into hospitals may present several challenges. CA-MRSA can cause infections in previously healthy individuals, and exposure of CA-MRSA to in-hospital antibiotic pressure encourages the emergence of multiple resistance. The control of MRSA in hospitals is hampered by the constant reintroductions of CA-MRSA from an expanding community reservoir. The pvl gene is an important virulence factor reported to cause SSTIs and necrotizing pneumonia [28]. In the present study, the carriage rate of the pvl gene in HA-MRSA strains (22.7%) was higher than in previous reports from European countries, Australia and Asia, which varied from 0.7% to 13.2% [26,29–32]. An increased prevalence of PVLproducing MRSA strains with CA genotypes in hospitals may result in more virulent nosocomial strains. The carriage rate of ST59 was higher than that of
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Nosocomial infection caused by MRSA in children ST239 in our study. An increased prevalence of PVL-producing CA-MRSA strains in hospitals may give rise to more severe infections. In this study, sea-sek-seq was carried by 71% of ST239, whereas seb-sek-seq was observed in 76.2% of ST59. These strains may be a repertoire of enterotoxins and may have the potential to spread widely. Adhesins have important functions in helping S. aureus to avoid host defenses and promote colonization. No significant difference was observed between the STs in the presence of fnbA, clfA, clfB, bbp, eap, ebpS, sdrC, sdrE and spa. cna was more frequently observed in ST239, which is in agreement with the study by Deurenberg et al. [33]. Previous studies described the clinical spectrum of HAIs caused by MRSA in the neonatal intensive care unit and in adult patients [6,11]. MRSA usually causes HAIs in populations with risk factors. In this study, infections were more common at a young age, with a median of 1.5 years. The proportion of invasive infections was 34.7%, and the most common types were severe pneumonia, SSTIs and bacteremia. Invasive infections were more common in patients with certain risk factors and led to a higher rate of ICU admission, medical ventilation and a longer hospital stay than non-invasive infections. Compared with patients infected by ST239, cases with ST59 were more common in children less than 1 year old, especially in neonates. Nearly half of infections caused by MRSA-ST59 were invasive, including severe pneumonia and bacteremia, whereas less than one-fifth of infections with ST239 were invasive. These findings may be related to the immature immune system of young children and the virulence of ST59 strains. In addition, two variants (ST1801 and ST960) of ST239 were detected, and both caused invasive infections. However, no significant differences were found for gender, infection site, fever at diagnosis, complication, outcomes or polymicrobial isolation between the two major clones. ST9 and ST398, which were the most prevalent livestock-associated MRSA clones in Asia and Europe, were first found in children. No patients infected by strains of ST9 or ST398 had contact with animals. Further investigations into the changes in virulence of these strains are necessary. In addition, the antibiotic resistance rate among STs differed. For example, the resistance rates to gentamicin, rifampin, tetracycline and ciprofloxacin were higher in ST239 than in ST59. This study provides information on the clinical features of HAI and the molecular characteristics HA-MRSA in children in mainland China. Invasive infections accounted for a substantial portion of HAIs caused by MRSA and were more common in patients with risk factors, and half were multisite
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infections. MRSA with CA genotype accounted for more than one-third of HAIs, with ST59 being the dominant clone. In addition, patients infected by MRSA-ST59-IV/V were younger and more often developed invasive infections. More effective infection control measures are needed to curtail the colonization and dissemination of MRSA in hospitalized patients.
Acknowledgements Special thanks to Professor Teruyo Ito of Juntendo University, Japan, for the reference MRSA strains used as the control for SCCmec typing. This study was supported by the National Natural Science Foundation of China [grant no. 81171648] and the National Natural Science Foundation of China and the Research Grants Council of Hong Kong Joint Research Scheme [no. 81061160509]. Declaration of interest: All the authors listed have read through the manuscript and approved it for publication, and declare no conflicts of interest.
References [1] Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998;339:520–32. [2] Chambers HF, Deleo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 2009; 7:629–41. [3] Aires-de-Sousa M, Correia B, de Lencastre H. Multilaboratory Project Collaborators. Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period. J Clin Microbiol 2008;46:2912–7. [4] Saiman L, O’Keefe M, Graham PL III, Wu F, Saïd-Salim B, Kreiswirth B, et al. Hospital transmission of community acquired methicillin-resistant Staphylococcus aureus among postpartum women. Clin Infect Dis 2003;37:1313–9. [5] David MD, Kearns AM, Gossain S, Ganner M, Holmes A. Community-associated meticillin-resistant Staphylococcus aureus: nosocomial transmission in a neonatal unit. J Hosp Infect 2006;64:244–50. [6] Park SH, Park C, Yoo JH, Choi SM, Choi JH, Shin HH, et al. Emergence of community-associated methicillin-resistant Staphylococcus aureus strains as a cause of healthcareassociated bloodstream infections in Korea. Infect Control Hosp Epidemiol 2009;30:146–55. [7] Wang H, Liu Y, Sun H, Xu Y, Xie X, Chen M. In vitro activity of ceftobiprole, linezolid, tigecycline, and 23 other antimicrobial agents against Staphylococcus aureus isolates in China. Diagn Microbiol Infect Dis 2008;62:226–9. [8] Kawamura I, Ohmagari N, Noda S, Sugiyama T, Kurai H. Preventing the transmission of methicillin-resistant Staphylococcus aureus at a tertiary care cancer center in Japan: quality improvement report. Am J Infect Control 2013; 41:1105–6. [9] Geng W, Yang Y, Wu D, Huang G, Wang C, Deng L, et al. Molecular characteristics of community-acquired,
8
[10]
[11]
[12]
[13]
Infect Dis Downloaded from informahealthcare.com by Gazi Univ. on 05/01/15 For personal use only.
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
X. Ning et al. methicillin-resistant Staphylococcus aureus isolated from Chinese children. FEMS Immunol Med Microbiol 2010; 58:356–62. Li T, Song Y, Zhu Y, Du X, Li M. Current status of Staphylococcus aureus infection in a central teaching hospital in Shanghai, China. BMC Microbiol 2013;8;13:153. Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, Ray S, et al. Invasive methicillin-resistant Staphylococcus aureus infections in the United States. JAMA 2007;298:1763–71. Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin susceptible clones of Staphylococcus aureus. J Clin Microbiol 2000;38:1008–15. Milheirico C, Oliveira DC, de Lencastre H. Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus. Antimicrob Agents Chemother 2007;51:3374–7. Koreen L, Ramaswamy SV, Graviss EA, Naidich S, Musser JM, Kreiswirth BN. Spa typing method for discriminating among Staphylococcus aureus isolates: implications for use of a single marker to detect genetic micro- and macrovariation. J Clin Microbiol 2004;42:792–9. Li J, Wang L, Ip M, Sun M, Sun J, Huang G, et al. Molecular and clinical characteristics of clonal complex 59 methicillin-resistant Staphylococcus aureus infections in mainland China. PLoS One 2013;8:e70602. Li M, Diep BA, Villaruz AE, Braughton KR, Jiang X, DeLeo FR, et al. Evolution of virulence in epidemic communityassociated methicillin-resistant Staphylococcus aureus. Proc Natl Acad Sci U S A 2009;106:5883–8. Jarraud S, Mougel C, Thioulouse J, Lina G, Meugnier H, Forey F, et al. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect Immun 2002;70:631–41. Zhang S, Iandolo JJ, Stewart GC. The enterotoxin D plasmid of Staphylococcus aureus encodes a second enterotoxin determinant (sej). FEMS Microbiol Lett 1998;168: 227–33. Holtfreter S, Bauer K, Thomas D, Feig C, Lorenz V, Roschack K, et al. egc-Encoded superantigens from Staphylococcus aureus are neutralized by human sera much less efficiently than are classical staphylococcal enterotoxins or toxic shock syndrome toxin. Infect Immun 2004;72:4061–71. Lina G, Boutite F, Tristan A, Bes M, Etienne J, Vandenesch F. Bacterial competition for human nasal cavity colonization: role of Staphylococcal agr alleles. Appl Environ Microbiol 2003;69:18–23. Shukla SK, Karow ME, Brady JM, Stemper ME, Kislow J, Moore N, et al. Virulence genes and genotypic associations in nasal carriage, community-associated methicillin-susceptible and methicillin-resistant USA400 Staphylococcus aureus isolates. J Clin Microbiol 2010;48:3582–92.
Supplementary material available online Supplementary Tables I and II
[22] Campbell SJ, Deshmukh HS, Nelson CL, Bae IG, Stryjewski ME, Federspiel JJ, et al. Genotypic characteristics of Staphylococcus aureus isolates from a multinational trial of complicated skin and skin structure infections. J Clin Microbiol 2008;46:678–84. [23] Chen CJ, Huang YC, Su LH, Wu TL, Huang SH, Chien CC, et al. Molecular epidemiology and antimicrobial resistance of methicillin-resistant Staphylococcus aureus bloodstream isolates in Taiwan, 2010. PLoS One 2014;26;9:e101184. [24] Liu Y, Wang H, Du N, Shen E, Chen H, Niu J, et al. Molecular evidence for spread of two major methicillin-resistant Staphylococcus aureus clones with a unique geographic distribution in Chinese hospitals. Antimicrob Agents Chemother 2009;53:512–8. [25] Boyce JM. Community-associated methicillin-resistant Staphylococcus aureus as a cause of health care-associated infection. Clin Infect Dis 2008;46:795–8. [26] Huang YC, Su LH, Wu TL, Lin TY. Changing molecular epidemiology of methicillin-resistant Staphylococcus aureus bloodstream isolates from a teaching hospital in northern Taiwan. J Clin Microbiol 2006;44:2268–70. [27] DeLeo FR, Otto M, Kreiswirth BN, Chambers HF. Community-associated meticillin-resistant Staphylococcus aureus. Lancet 2010;375:1557–68. [28] Chi CY, Lin CC, Liao IC, Yao YC, Shen FC, Liu CC, et al. Panton-Valentine leukocidin facilitates the escape of Staphylococcus aureus from human keratinocyte endosomes and induces apoptosis. J Infect Dis 2014;209:224–35. [29] Chini V, Petinaki E, Foka A, Paratiras S, Dimitracopoulos G, Spiliopoulou I. Spread of Staphylococcus aureus clinical isolates carrying Pantone Valentine leukocidin genes during a 3-year period in Greece. Clin Microbiol Infect 2006;12:29–34. [30] Wannet WJ, Heck ME, Pluister GN, Spalburg E, van Santen MG, Huijsdans XW, et al. Panton-Valentine leukocidin positive MRSA in 2003: the Dutch situation. Euro Surveill 2004;9:28–9. [31] Raab U, Kahlau D, Wagenlehner F, Reischl U, Ehrenstein V, Lehn N, et al. Prevalence of and risk factors for carriage of Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus among residents and staff of a German nursing home. Infect Control Hosp Epidemiol 2006;27:208–11. [32] Köck R, Brakensiek L, Mellmann A, Kipp F, Henderikx M, Harmsen D, et al. Cross-border comparison of the admission prevalence and clonal structure of meticillin-resistant Staphylococcus aureus. J Hosp Infect 2009;71:320–6. [33] Deurenberg RH, Rijnders MI, Sebastian S, Welling MA, Beisser PS, Stobberingh EE. The Staphylococcus aureus lineage-specific markers collagen adhesin and toxic shock syndrome toxin 1 distinguish multilocus sequence typing clonal complexes within spa clonal complexes. Diagn Microbiol Infect Dis 2009;65:116–122.
ORIGINAL ARTICLE
Characterization of pediatric hospital-associated infection caused by methicillin-resistant Staphylococcus aureus in mainland China
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XUE NING1,2, MINGJIAO SUN1, YANHONG QIAO1, FANG DONG2, WENQI SONG2, KAIHU YAO1, YONGHONG YANG1 & XUZHUANG SHEN1 From the 1Key Laboratory of Major Diseases in Children and National Key Discipline of Pediatrics (Capital Medical University), Ministry of Education, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Beijing, PR China, and 2Beijing Children’s Hospital, Capital Medical University, Beijing, PR China
Abstract Background: This study was conducted to investigate the clinical features of hospital-associated infections (HAIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) in Chinese children, and the molecular characteristics of the bacteria. Methods: Patients with HAIs caused by MRSA were identified retrospectively. All isolates were analyzed using molecular typing and antimicrobial susceptibility tests. Results: In total, 150 patients were identified, with a median age of 18 months. The most common infection was pneumonia (55.3%), followed by skin and soft tissue infections (46%). Invasive infections were observed in 52 patients (34.7%), and their hospital stay was longer compared with non-invasive cases (21 vs 12 days, p ⫽ 0.047). A total of 16 sequence types (STs) were identified. ST239 and ST59 were common clones, accounting for 46% and 28% of cases, respectively. Compared with cases caused by ST239-SCCmecI-III, patients infected by ST59-SCCmecIV-V had a lower median age (11 vs 41 months, p ⫽ 0.047) and more commonly developed invasive infection (50% vs 18.8%, p ⫽ 0.006). Conclusions: Invasive infections accounted for a large proportion of HAIs caused by MRSA. ST59-SCCmecIV/V, a common clone in the community, caused HAIs in Chinese children, more often infected younger children and caused invasive infections.
Keywords: Hospital-associated infections, invasive infection, methicillin-resistant Staphylococcus aureus, MRSA-ST59
Introduction Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a life-threatening multidrug-resistant bacterium, which causes various infections, including skin and soft tissue infections (SSTIs), endocarditis, toxic shock syndrome, necrotizing fasciitis, necrotizing pneumonia and sepsis [1]. Since the onset of the pandemic waves of MRSA over the past few decades, it has become a common cause of both hospital- and community-associated infections worldwide [2]. Limited clones of hospital-associated MRSA (HA-MRSA) have emerged and been disseminated worldwide, including ST5, ST239, ST22, ST36 and ST45 [3], while clones of community-associated MRSA (CA-MRSA) are more diversified. With the continuing global epidemic of CA-MRSA, the strain
types USA300 [4], ST1 [5] and ST72 [6] have emerged as a cause of hospital-associated infections (HAIs) in the USA, the UK and Korea, respectively. According to previous studies, the prevalence of MRSA in hospitals for adults varies from 13% to 80% [7,8]. In children, many studies have focused on CA-MRSA infections [9]. Although measures such as universal hand hygiene practices were introduced into hospitals in mainland China several years ago, HAIs are still common [10]. To the best of our knowledge, there is limited information characterizing the HAIs caused by MRSA in children in mainland China. We conducted this study to collect clinical information and determine the molecular characteristics of HA-MRSA in the largest children’s hospital in Beijing, China.
Correspondence: Xuzhuang Shen, Beijing Children’s Hospital affiliated with the Capital Medical University, Nan Li Shi Road, Beijing, 100045, PR China. Tel: ⫹ 86 1059616652. Fax: ⫹ 86 1068028401. E-mail: [email protected] (Received 17 July 2014 ; accepted 24 December 2014 ) ISSN 0036-5548 print/ISSN 1651-1980 online © 2015 Informa Healthcare DOI: 10.3109/00365548.2015.1006675
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Materials and methods
Molecular typing methods
Hospital setting and patient selection
Multilocus sequence typing (MLST) was performed as previously described [12]. The MLST allelic profiles and sequence types (STs) were assigned by submission to the MLST database (http://saureus.mlst. net). Related STs were grouped within a clonal complex (CC), which is determined by an algorithm called eBURST. The SCCmec types and subtypes were determined using two multiplex PCR strategies as previously described [13]. The prototypes of SCCmec types I-V for the isolates and SCCmecIV subtypes for the subtypes were used as the quality control sample. Spa typing was performed as previously described [14]. Purified spa PCR products were sequenced and compared using the spa database website (http://www.ridom.de/spaserver).
This study was conducted in Beijing Children’s Hospital, which is a teaching hospital affiliated to the Capital Medical University with 1000 beds, and which provides both primary and tertiary care. Patients with confirmed HA-MRSA infections were identified from the hospital’s database between January 2009 and June 2013. The clinical signs and course of these patients were consistent with MRSA infection and the isolates were recovered from infected sites, including pus, endotracheal aspirates, hydrothorax, synovial fluid or blood. The medical records of all identified patients were retrospectively reviewed and the following information was collected: age, gender, underlying conditions (history of immunodeficiency diseases, diabetes mellitus, cancer and congenital heart diseases), risk factors (immunosuppressant drugs, burn/scald/traumatic wounds, surgery, central venous catheter), laboratory data (including white blood cell counts, levels of C-reactive protein and coinfections with other pathogens) and clinical outcome. This study was approved by the Ethics Committee of Beijing Children’s Hospital affiliated to the Capital Medical University (2008-35).
Case definition According to the Centers for Disease Control and Prevention (CDC), HA-MRSA infections were defined as follows: cultures were taken from an inpatient more than 48 h after admission or from an outpatient with risk factors for HAI, namely, recent (within the past year) hospitalization or medical procedure (e.g. dialysis, surgery and catheters) [11]. Severe pneumonia was defined by any one of the following: requirement for intensive care unit (ICU) admission, necrotizing or cavitary infiltrates, or empyema. Invasive MRSA infection was defined as the isolation of MRSA from a normally sterile body site, including blood, pleural fluid, cerebrospinal fluid, pericardial fluid, peritoneal fluid, joint/synovial fluid, bone or internal body sites [11].
Identification of strains and DNA extraction Collection, transportation and identification of MRSA isolates were performed as previously described [9]. DNA was extracted from all isolates using a DNA extraction kit (Saibaisheng, China) and used as the template in all polymerase chain reactions (PCRs).
Antimicrobial susceptibility testing Antimicrobial susceptibility was determined using the disk-diffusion method and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute. Susceptibilities to penicillin, erythromycin, clindamycin, cefuroxime, tetracycline, chloramphenicol, gentamicin, ciprofloxacin, sulfamethoxazoletrimethoprim, rifampicin, cefoxitin, azithromycin vancomycin, linezolid, tigecycline and fusidic acid (Sigma, St Louis, MO, USA) were determined. Susceptibility testing to fusidic acid, tigecycline and mupirocin was carried out according to a previous study [15]. Staphylococcus aureus ATCC29213 was used as a control strain. Isolates resistant to three or more antimicrobial classes were defined as multidrug resistant.
Screening for 42 virulence genes All isolates of ST239 and ST59 were screened for 42 known virulence genes. the presence of the 21 known superantigen (SAg) genes (enterotoxin genes: sea, seh, sec, sed, sek, see, seb, sem, sel, seo, sen, seg, seq, sej, sei, seu, ser and sep; toxic shock syndrome toxin gene: tsst-1; and two exfoliative toxin genes: eta and etd), and the agr types was tested by six multiplex PCRs. The genes of exfoliative toxin B gene (etb), fibronectin-binding protein genes (fnbA and fnbB), clumping factor genes (clfA and clfB), bone sialoprotein gene (bbp), collagen-binding protein gene (cna), extracellular adherence protein gene (eap), elastin-binding protein gene (ebpS), genes for the serine-aspartate repeat family of proteins (sdrC, sdrD and sdrE), staphylococcal surface protein A gene (spa), hemolysin genes (hla, hlb, hld, hlg and psma) and leukocidin genes (pvl, lukD and lukE) were screened by singleplex PCRs. The primers are listed in Supplementary Table I. (available online at: http://informahealthcare.
Nosocomial infection caused by MRSA in children com/doi/abs/10.3109/00365548.2015.1006675) [16,17–22]. Statistical analysis SPSS 17.0 software was used for statistical analysis. The categorical variables were compared using the chi-squared or two-tailed Fisher’s exact test. The means of two independent samples were compared using Student’s t test. Multiple logistic regression analysis was used to analyze the risk factors of invasive HA-MRSA infection. A p values less than 0.05 was considered statistically significant.
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Results Clinical features In total, 150 patients with hospital-associated MRSA infections were identified and their clinical charac-
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teristics are summarized in Table I. Ninety-seven patients were male. The patients ranged in age from 5 days to 14 years, with a median age of 18 months. Thirty-two (21.3%) were neonates aged less than 1 month. Fever was the initial presentation in 139 patients (92.7%). Ten types of infection were identified. Single-site infections were found in 124 patients (82.7%), and 26 patients (17.3%) had two or more infective sites simultaneously. Fifty-two patients (34.7%) had invasive and 98 (65.3%) had non-invasive infections. The most common infection was pneumonia (83/150, 55.3%), followed by SSTIs (69/150, 46%), bacteremia (21/150, 14%), osteomyelitis (5/150, 3.3%) and urinary tract infection (1/150, 0.7%). Invasive infections included severe pneumonia (n ⫽ 15), SSTIs (n ⫽ 13), bacteremia (n ⫽ 2), osteomyelitis (n ⫽ 2), urinary tract infection (n ⫽ 1), bacteremia related to pneumonia (n ⫽ 7), bacteremia related to SSTIs
Table I. Demographics and clinical characteristics of patients with hospital-associated infections caused by methicillin-resistant Staphylococcus aureus.
Item Age Age ⱕ 1 month Male Type of infection Pneumonia Skin and soft tissue infection Bloodstream infection Urinary tract infection Bone/joint infection Underlying diseases Cancer Congenital heart disease Immunodeficiency diseases Type 1 diabetes mellitus Risk factors Receiving immunosuppressant drugs Burn/scald/traumatic wound Surgery Central venous catheter Fever Polymicrobial infection Complication Respiratory failure Heart failure Shock Blood coagulation disorders Laboratory data White blood cells (109/l) Neutrophil count mean (109/l) C-reactive protein (mg/l) Outcomes Intensive care unit admission Mechanical ventilation Length of hospitalization (days) Mortality Data are shown as mean (range) or n (%).
Total cases (n ⫽ 150)
Invasive cases (n ⫽ 52)
Non-invasive cases (n ⫽ 98)
p
18 (5 days-14 years) 43 (28.7) 97 (64.7)
25 (12 days-8 years) 16 (30.8) 37 (71.2)
17 (5 days-14 years) 27 (27.6) 60 (61.2)
0.826 0.678 0.226
83 69 21 1 5 42 32 5 3 2 109 23 48 23 15 136 28 32 18 3 4 7
(55.3) (46) (14) (0.7) (3.3) (28) (21.3) (3.3) (2) (1.3) (72.7) (15.3) (32) (15.3) (10) (90.7) (18.7) (21.3) (12) (2) (2.7) (4.7)
15.8 (1.4–76.3) 11.9 (0.75–65.4) 50.7 (0.9–256.1) 45 21 15 4
(30) (14) (2–59) (2.7)
25 22 21 1 5 19 14 3 1 1 38 10 9 7 12 50 12 15 9 1 2 3
(48.1) (42.3) (40.4) (1.9) (9.6) (36.5) (26.9) (5.8) (1.9) (1.9) (73.1) (19.2) (17.3) (13.5) (23.1) (96.2) (23.1) (28.8) (17.3) (1.9) (3.8) (5.8)
16.7 (1.4–76.3) 12.5 (0.75–65.4) 62.4 (0.9–256.1) 25 12 21 3
(48.1) (23.1) (4–59) (5.8)
58 47 0 0 0 23 18 2 2 1 71 13 39 16 3 82 16 17 9 2 2 4
(59.2) (48) (0) (0) (0) (23.5) (18.4) (2) (2) (1) (72.4) (13.3) (39.8) (16.3) (3.1) (83.7) (16.3) (17.3) (9.2) (2) (2) (4.1)
15.6 (4.1–56.5) 10.7 (1.3–41.3) 43 (1.2–132) 20 9 12 1
(20.4) (9.2) (2–43) (1)
0.193 0.509 ⬍ 0.001 0.351 0.004 0.09 0.223 0.342 1.000 1.000 0.935 0.335 0.005 0.643 ⬍ 0.001 0.139 0.313 0.102 0.145 1.000 0.610 0.694 0.792 0.831 0.634 ⬍ 0.001 0.020 0.047 0.120
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X. Ning et al.
(n ⫽ 6), bacteremia related to osteomyelitis (n ⫽ 3) and bacteremia complicated with pneumonia and SSTIs (n ⫽ 3). Non-invasive infections included pneumonia (n ⫽ 51), SSTIs (n ⫽ 40) and pneumonia complicated with SSTIs (n ⫽ 7). Underlying conditions including cancer, immunodeficiency diseases, type 1 diabetes mellitus and congenital heart disease were found in 42 patients (28%). Patients with comorbidity accounted for 36.5% of invasive infections. Risk factors were found in 109 patients. Central venous catheters were more common in invasive infection [95% confidence interval (CI) 0.028–0.393, p ⬍ 0.001]. Burns/scald/traumatic wounds were more common in non-invasive cases (95% CI 0.139–0.722, p ⫽ 0.005). Gender, underlying disease, immunosuppressant drugs, burns/scalds/traumatic wounds, surgery and central venous catheters were analyzed using multiple logistic regression. The results show that central venous catheter was a risk factor for invasive HAMRSA infection (OR 0.293, 95% CI 0.121–0.711, p ⫽ 0.007). Among the patients who had polymicrobial infections, cytomegalovirus was the most common copathogen (n ⫽ 8), followed by Klebsiella pneumoniae (n ⫽ 4), Epstein-Barr virus (n ⫽ 3), Mycoplasma pneumonia (n ⫽ 3), Pseudomonas aeruginosa (n ⫽ 2), Staphylococcus epidermidis (n ⫽ 2), Chlamydia pneumoniae (n ⫽ 1), Acinetobacter baumanii (n ⫽ 1), Haemophilus parainfluenzae (n ⫽ 1), measles virus (n ⫽ 1) and respiratory syncytial virus (n ⫽ 1). All patients were treated empirically with antibiotics. Cephalosporins (90%, 135/150), including first generation (3.7%, 4/108), second generation (29.6%, 32/108), third generation (66.7%, 72/108) and fourth generation (18%, 27/150), and vancomycin (4%, 6/150) were intravenously administered before pathogen detection. Sixteen patients received mupirocin for SSTIs. One-hundred and seventeen of the 150 patients (78%) were switched to other drugs after pathogens were detected in blood cultures. Vancomycin, linezolid and teicoplanin were given to 97, 14 and six patients, respectively. Clinical outcomes, including rates of ICU admission, respiratory failure and mortality related to HAMRSA infections were 32.3% (49/150), 9.3% (14/150) and 2.7% (4/150), respectively. Patients with invasive MRSA infection had higher rates of ICU admission (95% CI 0.133–0.576, p ⬍ 0.001) and mechanical ventilation (95% CI 0.131–0.864, p ⫽ 0.020) than non-invasive cases. Molecular characteristics of MRSA isolates Among the 150 HA-MRSA strains, 16 STs were detected, belonging to 12 CCs (Table II). The
most prevalent ST was ST239 (69/150, 46%), followed by ST59 (42/150, 28%). Other STs varied from 0.7% to 6%. The most common SCCmec type was SCCmecIII (79/150, 52.7%), followed by SCCmecIV (45/150, 30%) and SCCmecII (12/150, 8%). In addition, MRSA with SCCmecIV/V accounted for 32.7% (49/150) of all isolates. Seven isolates could not be SCCmec typed. Three SCCmecIV subtypes were detected: SCCmecIVa (43/45, 95.6%), SCCmecIVb (1/45, 2.2%) and SCCmecIVc (1/45, 2.2%). Thirty-two spa types were identified. The most common was t030 (34/150, 22.7%), followed by t437 (29/150, 19.3%), t037 (19/150, 12.7%) and t459 (14/150, 9.3%). Two isolates could not be spa typed, as shown in Table II. The most prevalent clone of MRSA was ST239III (65/150, 43.3%), including ST239-III-t030 (34/65, 55.4%), ST239-III-t037 (17/65, 26.2%) and ST239-III-t459 (12/65, 18.5%), followed by ST59IVa (34/150, 22.7%), including ST59-IVa-t437 (27/42, 64.3%), ST59-IVa-t441 (4/42, 9.5%) and ST59-IVa-t3523 (2/42, 4.8%). ST59-V and ST5-II accounted for 2.7% and 5.3%, respectively. ST1801 and ST960, which were both single-locus variants of ST239, and livestock-associated-MRSA ST9 and ST398, were also detected. Thirty-four isolates (22.7%) were Panton-Valentine leukocidin (PVL) positive. The positive rates in clones ST239 and ST59 were 14.5% (10/69) and 40.5% (17/42), respectively (p ⫽ 0.002). Among pvl-positive strains, the most prevalent clone was ST59-SCCmecIVat437 (13/34, 38.2%). Antibiotic susceptibility All isolates were susceptible to vancomycin, tigecycline and linezolid (Table III). Resistance to other classes of antibiotics was highly variable. The resistance rates to penicillin, cefuroxime, azithromycin, clindamycin, erythromycin, tetracycline, ciprofloxacin, gentamicin, rifampin, chloramphenicol, trimethoprim/sulfamethoxazole, fusidic acid and mupirocin were 97.3%, 92%, 86.7%, 82%, 77.3%, 70.7%, 61.3%, 56%, 49.3%, 15.3%, 8%, 0.7% and 0.7%, respectively. The resistance rates to gentamicin, rifampin, tetracycline and ciprofloxacin differed between MRSA-ST59 and ST239 (p ⬍ 0.01). The multidrug resistance rates of all isolates, MRSA-ST59 isolates and MRSA-ST239 isolates were 84.0%, 78.6% and 81.2%, respectively. Moreover, 72.5% (50/69) of the ST239 strains were included in two resistance profiles, which were PEN-CEF-GEN-TET-RIF-ERY-CLI-CIP (36/69, 52.2%) and PEN-CEF-GEN-TET-RIF (13/69, 18.8%), whereas the predominant antibiotic
Nosocomial infection caused by MRSA in children
5
Table II. Molecular characteristics of methicillin-resistant Staphylococcus aureus isolated from Chinese children with hospital-associated infections. MLST (n ⫽ 150) CC 8 (48)
ST
pvl
SCCmec (n ⫽ 150)
Spa (n ⫽ 150)
239 (46)
10 (6.7)
III (43.3)
t030 (20), t037 (11.3), t459 (8), t969 (1.3), t2270 (1.3), t7221 (0.7), t2760 (0.7) t030 (2) t008 (0.7) t037 (0.7) t459 (0.7) t459 (0.7) t437 (0.7), NT (0.7) t437 (18.7), t441 (2.7), t3523 (1.3) t437 (2), t441 (0.7) t441 (1.3) t437 (0.7) t2270 (0.7) t002 (4), t686 (0.7), t601 (0.7) NT (0.7) t1376 (1.3), t127 (0.7), t4431 (0.7) t8723 (0.7) t1246 (0.7), t3078 (0.7), t3523 (0.7) t078 (0.7), t081 (1.3) t796 (1.3), t037 (0.7) t375 (0.7) t375 (0.7) t664 (0.7) t10555 (0.7) t664 (0.7) t899 (0.7) t437 (1.3) t034 (0.7) t127 (0.7) t202 (0.7)
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1801 (1.3)
59 (29.3)
960 (0.7) 59 (28)
1 (0.7)
5 (6)
338 (0.7) 375 (0.7) 5 (6)
88 (3.3)
88 (3.3)
2 (1.3)
15 (4)
25 (4)
2 (1.3)
7 (2) 509 (1.3)
(0.7) (8.7) (1.3) (0.7)
7 (2) 509 (1.3)
72 (2)
72 (2)
9 (0.7) 398 (2)
9 (0.7) 398 (2)
1 (0.7) Singleton (0.7)
1 13 2 1
1 (0.7) 93 (0.7)
1 (0.7) 1 (0.7)
II IVa I III III III IVa V NT III IVa II IVa III I IVa NT III II III IVc IVa NT IVb IVa I NT III
(2) (0.7) (0.7) (0.7) (0.7) (1.3) (22.7) (2.7) (1.3) (0.7) (0.7) (5.3) (0.7) (2.7) (0.7) (2) (2) (2) (0.7) (0.7) (0.7) (0.7) (0.7) (0.7) (1.3) (0.7) (0.7) (0.7)
Data are shown as n (%). MLST, multilocus sequence typing; CC, clonal complex; ST, sequence type; SCCmec, Staphylococcus aureus chromosome typing; Spa, Staphylococcus protein A typing; NT, non-typeable.
resistance profile PEN-CEF-TET-ERY-CLI-AZI accounted for only 23.8% (10/42) of ST59 strains. Distributions of 42 virulence genes and agr types in two major sequence types Strains of ST59 and ST239 were selected to study virulence characteristics. All 111 strains contained SAg genes. The prevalent SAg genes were sek (104/111, 93.7%), seq (98/111, 88.3%), sea (73/111, 65.8%) and seb (35/111, 31.5%). Genes sed, ser, eta, etb and etd were not detected. All isolates carried the adhesion genes fnbA, clfA, clfB, ebpS, sdrC and spa. A majority harbored bbp (108/111, 97.3%), sdrE (107/111, 96.4%) and lukE (92/111, 82.9%) genes. All strains carried hla and hlb. Genes hld (97.3%) and hlg (94.6%) were carried by a majority of the strains. However, the leukocidin gene pvl (19.8%) was not common and lukM was not detected. The gene encoding α-type phenol-soluble modulin was
detected in all strains. A total of 10 SAg gene combinations was observed. The most prevalent one was sea-sek-seq, carried by 71% (49/69) of ST239, whereas seb-sek-seq was observed in 76.2% (32/42) of ST59. Virulence genes were associated with specific molecular types. The sea (61/69) and lukE (65/69) genes were more often identified in ST239, whereas seb (42/42) and pvl (20/42) were more often identified in ST59 (p ⬍ 0.05). lukM, can and sdrC genes were not common in ST239 and ST59. Moreover, 99.1% (110/111) of isolates were agr type I and one isolate of ST239 was negative for agr typing. Comparison of hospital-associated infections caused by MRSA ST59, ST239 and ST5 In this study, 69 (46%) and 42 (28%) patients were infected by MRSA ST239 and ST59, respectively. The median age of children infected by ST59 and ST239 was 11 and 41 months, respectively
6
X. Ning et al. Table III. Antimicrobial resistance of 150 methicillin-resistant Staphylococcus aureus isolates based on multilocus sequence typing.
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STs ST239 ST59 ST5 ST25 ST88 ST72 ST7 ST398 ST1801 ST509 ST338 ST375 ST93 ST960 ST1 Total
n
PEN
CEF
GEN CHL TET
69 42 9a 6 5 3 3 3 2 1 1 1 1 1 1 150
100 95.2 8 5 5 3 3 2 2 1 1 1 1 1 1 146
100 84.1* 97.6 19 9 8 5 2 4 1 3 0 2 1 3 1 2 1 1 1 1 0 1 0 1 1 1 1 1 1 138 84
RIF
11.6 94.2* 88.4* 19 45.2 9.5 1 8 6 2 3 1 2 2 1 1 1 0 1 2 0 0 1 0 0 1 1 0 1 0 0 0 0 0 0 0 0 1 0 0 1 0 0 1 0 23 106 74
ERY
CLI
AZI
CIP
TRI
76.8 85.7 7 5 4 2 2 3 1 0 1 1 0 1 0 116
81.2 90.5 7 5 4 2 2 3 1 0 1 1 1 1 1 123
10.1 94.2* 10.1 23.8 21.4 4.8 1 8 2 1 2 1 1 1 0 2 1 0 1 1 0 0 0 0 0 1 0 0 1 0 1 0 0 0 0 0 1 1 0 1 1 0 0 1 0 130 92 12
FUS MUP 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1
0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1
PEN, penicillin; CEF, cefuroxime; GEN, gentamicin; CHL, chloramphenicol; TET, tetracycline; RIF, rifampin; ERY, erythromycin; CLI, clindamycin; AZI, azithromycin; CIP, ciprofloxacin; TRI, trimethoprim; FUS, fusidic acid; MUP, mupirocin. aSequence types (STs) with fewer than 10 isolates were not calculated in the percentage of antibiotic resistance. Comparison between MRSA-ST239 and ST59 isolates: *p ⬍ 0.01.
(p ⫽ 0.047). In addition, 26.2% (11/42) and 10.1% (7/69) of the patients infected by ST59 and ST239 were neonates (95% CI 0.112–0.901, p ⫽ 0.026). Fifty percent (21/42) of patients infected with ST59 developed invasive infections compared with 24.6% (17/69) of those with ST239 infections (95% CI 0.145–0.739, p ⫽ 0.006). A comparison of clinical information among the major MRSA clones is shown in Supplementary Table II available online at: http:// informahealthcare.com/doi/abs/10.3109/00365548. 2015.1006675. There was no difference in outcome, underlying diseases and risk factors among patients infected with ST59 and ST239 isolates.
Discussion Studies have demonstrated that most HAIs caused by MRSA can be attributed to a relatively small number of epidemic clones. In Asia, the most frequent clones are Brazil/Hungary (ST239-III) and New York/Japan (ST5-II) [23]. Data from Wang’s study confirmed that these two clones have spread across China among adults [24]. Similarly, in the present study, the most prevalent clone in HA-MRSA in Chinese children was ST239-III, followed by ST59-IV, while ST5-II accounted for only 5.3%. The data from this study suggest that the genetic background of HA-MRSA in pediatric patients in mainland China is different from that found in other reports and varies in different populations even in the same region. Recent reports suggested that MRSA with CA genotypes are beginning to supplant or overtake
HA-MRSA clones as a cause of HAIs [25]. USA300 is an increasingly common community pathogen in the USA with a parallel tendency to cause HAIs. In Korea, 24% of healthcare-associated bacteremias were caused by the major ST72-IV CA-MRSA clone [6]. The ST59-IV and ST59-V clones were reported to cause HAIs in Taiwan and accounted for 25% of nosocomial bloodstream infections [26]. In the present study, nearly one-third of MRSA had CA genotypes, including ST59-IV/V, ST88-IV, ST25-IV and ST72-IV, which were previously reported to be common clones in CA-MRSA infections in China, Madagascar, Algeria and Korea, respectively [27]. Among isolates with CA genotypes, ST59-IV/V was a major clone. The influx of CA-MRSA into hospitals may present several challenges. CA-MRSA can cause infections in previously healthy individuals, and exposure of CA-MRSA to in-hospital antibiotic pressure encourages the emergence of multiple resistance. The control of MRSA in hospitals is hampered by the constant reintroductions of CA-MRSA from an expanding community reservoir. The pvl gene is an important virulence factor reported to cause SSTIs and necrotizing pneumonia [28]. In the present study, the carriage rate of the pvl gene in HA-MRSA strains (22.7%) was higher than in previous reports from European countries, Australia and Asia, which varied from 0.7% to 13.2% [26,29–32]. An increased prevalence of PVLproducing MRSA strains with CA genotypes in hospitals may result in more virulent nosocomial strains. The carriage rate of ST59 was higher than that of
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Nosocomial infection caused by MRSA in children ST239 in our study. An increased prevalence of PVL-producing CA-MRSA strains in hospitals may give rise to more severe infections. In this study, sea-sek-seq was carried by 71% of ST239, whereas seb-sek-seq was observed in 76.2% of ST59. These strains may be a repertoire of enterotoxins and may have the potential to spread widely. Adhesins have important functions in helping S. aureus to avoid host defenses and promote colonization. No significant difference was observed between the STs in the presence of fnbA, clfA, clfB, bbp, eap, ebpS, sdrC, sdrE and spa. cna was more frequently observed in ST239, which is in agreement with the study by Deurenberg et al. [33]. Previous studies described the clinical spectrum of HAIs caused by MRSA in the neonatal intensive care unit and in adult patients [6,11]. MRSA usually causes HAIs in populations with risk factors. In this study, infections were more common at a young age, with a median of 1.5 years. The proportion of invasive infections was 34.7%, and the most common types were severe pneumonia, SSTIs and bacteremia. Invasive infections were more common in patients with certain risk factors and led to a higher rate of ICU admission, medical ventilation and a longer hospital stay than non-invasive infections. Compared with patients infected by ST239, cases with ST59 were more common in children less than 1 year old, especially in neonates. Nearly half of infections caused by MRSA-ST59 were invasive, including severe pneumonia and bacteremia, whereas less than one-fifth of infections with ST239 were invasive. These findings may be related to the immature immune system of young children and the virulence of ST59 strains. In addition, two variants (ST1801 and ST960) of ST239 were detected, and both caused invasive infections. However, no significant differences were found for gender, infection site, fever at diagnosis, complication, outcomes or polymicrobial isolation between the two major clones. ST9 and ST398, which were the most prevalent livestock-associated MRSA clones in Asia and Europe, were first found in children. No patients infected by strains of ST9 or ST398 had contact with animals. Further investigations into the changes in virulence of these strains are necessary. In addition, the antibiotic resistance rate among STs differed. For example, the resistance rates to gentamicin, rifampin, tetracycline and ciprofloxacin were higher in ST239 than in ST59. This study provides information on the clinical features of HAI and the molecular characteristics HA-MRSA in children in mainland China. Invasive infections accounted for a substantial portion of HAIs caused by MRSA and were more common in patients with risk factors, and half were multisite
7
infections. MRSA with CA genotype accounted for more than one-third of HAIs, with ST59 being the dominant clone. In addition, patients infected by MRSA-ST59-IV/V were younger and more often developed invasive infections. More effective infection control measures are needed to curtail the colonization and dissemination of MRSA in hospitalized patients.
Acknowledgements Special thanks to Professor Teruyo Ito of Juntendo University, Japan, for the reference MRSA strains used as the control for SCCmec typing. This study was supported by the National Natural Science Foundation of China [grant no. 81171648] and the National Natural Science Foundation of China and the Research Grants Council of Hong Kong Joint Research Scheme [no. 81061160509]. Declaration of interest: All the authors listed have read through the manuscript and approved it for publication, and declare no conflicts of interest.
References [1] Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998;339:520–32. [2] Chambers HF, Deleo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 2009; 7:629–41. [3] Aires-de-Sousa M, Correia B, de Lencastre H. Multilaboratory Project Collaborators. Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period. J Clin Microbiol 2008;46:2912–7. [4] Saiman L, O’Keefe M, Graham PL III, Wu F, Saïd-Salim B, Kreiswirth B, et al. Hospital transmission of community acquired methicillin-resistant Staphylococcus aureus among postpartum women. Clin Infect Dis 2003;37:1313–9. [5] David MD, Kearns AM, Gossain S, Ganner M, Holmes A. Community-associated meticillin-resistant Staphylococcus aureus: nosocomial transmission in a neonatal unit. J Hosp Infect 2006;64:244–50. [6] Park SH, Park C, Yoo JH, Choi SM, Choi JH, Shin HH, et al. Emergence of community-associated methicillin-resistant Staphylococcus aureus strains as a cause of healthcareassociated bloodstream infections in Korea. Infect Control Hosp Epidemiol 2009;30:146–55. [7] Wang H, Liu Y, Sun H, Xu Y, Xie X, Chen M. In vitro activity of ceftobiprole, linezolid, tigecycline, and 23 other antimicrobial agents against Staphylococcus aureus isolates in China. Diagn Microbiol Infect Dis 2008;62:226–9. [8] Kawamura I, Ohmagari N, Noda S, Sugiyama T, Kurai H. Preventing the transmission of methicillin-resistant Staphylococcus aureus at a tertiary care cancer center in Japan: quality improvement report. Am J Infect Control 2013; 41:1105–6. [9] Geng W, Yang Y, Wu D, Huang G, Wang C, Deng L, et al. Molecular characteristics of community-acquired,
8
[10]
[11]
[12]
[13]
Infect Dis Downloaded from informahealthcare.com by Gazi Univ. on 05/01/15 For personal use only.
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
X. Ning et al. methicillin-resistant Staphylococcus aureus isolated from Chinese children. FEMS Immunol Med Microbiol 2010; 58:356–62. Li T, Song Y, Zhu Y, Du X, Li M. Current status of Staphylococcus aureus infection in a central teaching hospital in Shanghai, China. BMC Microbiol 2013;8;13:153. Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, Ray S, et al. Invasive methicillin-resistant Staphylococcus aureus infections in the United States. JAMA 2007;298:1763–71. Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin susceptible clones of Staphylococcus aureus. J Clin Microbiol 2000;38:1008–15. Milheirico C, Oliveira DC, de Lencastre H. Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus. Antimicrob Agents Chemother 2007;51:3374–7. Koreen L, Ramaswamy SV, Graviss EA, Naidich S, Musser JM, Kreiswirth BN. Spa typing method for discriminating among Staphylococcus aureus isolates: implications for use of a single marker to detect genetic micro- and macrovariation. J Clin Microbiol 2004;42:792–9. Li J, Wang L, Ip M, Sun M, Sun J, Huang G, et al. Molecular and clinical characteristics of clonal complex 59 methicillin-resistant Staphylococcus aureus infections in mainland China. PLoS One 2013;8:e70602. Li M, Diep BA, Villaruz AE, Braughton KR, Jiang X, DeLeo FR, et al. Evolution of virulence in epidemic communityassociated methicillin-resistant Staphylococcus aureus. Proc Natl Acad Sci U S A 2009;106:5883–8. Jarraud S, Mougel C, Thioulouse J, Lina G, Meugnier H, Forey F, et al. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect Immun 2002;70:631–41. Zhang S, Iandolo JJ, Stewart GC. The enterotoxin D plasmid of Staphylococcus aureus encodes a second enterotoxin determinant (sej). FEMS Microbiol Lett 1998;168: 227–33. Holtfreter S, Bauer K, Thomas D, Feig C, Lorenz V, Roschack K, et al. egc-Encoded superantigens from Staphylococcus aureus are neutralized by human sera much less efficiently than are classical staphylococcal enterotoxins or toxic shock syndrome toxin. Infect Immun 2004;72:4061–71. Lina G, Boutite F, Tristan A, Bes M, Etienne J, Vandenesch F. Bacterial competition for human nasal cavity colonization: role of Staphylococcal agr alleles. Appl Environ Microbiol 2003;69:18–23. Shukla SK, Karow ME, Brady JM, Stemper ME, Kislow J, Moore N, et al. Virulence genes and genotypic associations in nasal carriage, community-associated methicillin-susceptible and methicillin-resistant USA400 Staphylococcus aureus isolates. J Clin Microbiol 2010;48:3582–92.
Supplementary material available online Supplementary Tables I and II
[22] Campbell SJ, Deshmukh HS, Nelson CL, Bae IG, Stryjewski ME, Federspiel JJ, et al. Genotypic characteristics of Staphylococcus aureus isolates from a multinational trial of complicated skin and skin structure infections. J Clin Microbiol 2008;46:678–84. [23] Chen CJ, Huang YC, Su LH, Wu TL, Huang SH, Chien CC, et al. Molecular epidemiology and antimicrobial resistance of methicillin-resistant Staphylococcus aureus bloodstream isolates in Taiwan, 2010. PLoS One 2014;26;9:e101184. [24] Liu Y, Wang H, Du N, Shen E, Chen H, Niu J, et al. Molecular evidence for spread of two major methicillin-resistant Staphylococcus aureus clones with a unique geographic distribution in Chinese hospitals. Antimicrob Agents Chemother 2009;53:512–8. [25] Boyce JM. Community-associated methicillin-resistant Staphylococcus aureus as a cause of health care-associated infection. Clin Infect Dis 2008;46:795–8. [26] Huang YC, Su LH, Wu TL, Lin TY. Changing molecular epidemiology of methicillin-resistant Staphylococcus aureus bloodstream isolates from a teaching hospital in northern Taiwan. J Clin Microbiol 2006;44:2268–70. [27] DeLeo FR, Otto M, Kreiswirth BN, Chambers HF. Community-associated meticillin-resistant Staphylococcus aureus. Lancet 2010;375:1557–68. [28] Chi CY, Lin CC, Liao IC, Yao YC, Shen FC, Liu CC, et al. Panton-Valentine leukocidin facilitates the escape of Staphylococcus aureus from human keratinocyte endosomes and induces apoptosis. J Infect Dis 2014;209:224–35. [29] Chini V, Petinaki E, Foka A, Paratiras S, Dimitracopoulos G, Spiliopoulou I. Spread of Staphylococcus aureus clinical isolates carrying Pantone Valentine leukocidin genes during a 3-year period in Greece. Clin Microbiol Infect 2006;12:29–34. [30] Wannet WJ, Heck ME, Pluister GN, Spalburg E, van Santen MG, Huijsdans XW, et al. Panton-Valentine leukocidin positive MRSA in 2003: the Dutch situation. Euro Surveill 2004;9:28–9. [31] Raab U, Kahlau D, Wagenlehner F, Reischl U, Ehrenstein V, Lehn N, et al. Prevalence of and risk factors for carriage of Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus among residents and staff of a German nursing home. Infect Control Hosp Epidemiol 2006;27:208–11. [32] Köck R, Brakensiek L, Mellmann A, Kipp F, Henderikx M, Harmsen D, et al. Cross-border comparison of the admission prevalence and clonal structure of meticillin-resistant Staphylococcus aureus. J Hosp Infect 2009;71:320–6. [33] Deurenberg RH, Rijnders MI, Sebastian S, Welling MA, Beisser PS, Stobberingh EE. The Staphylococcus aureus lineage-specific markers collagen adhesin and toxic shock syndrome toxin 1 distinguish multilocus sequence typing clonal complexes within spa clonal complexes. Diagn Microbiol Infect Dis 2009;65:116–122.
