Journal of Medical Microbiology (2014), 63, 222–228 DOI 10.1099/jmm.0.

064865-0

Hospital clonal dissemination of Enterobacter

aerogenes producing carbapenemase KPC-2 in a
Chinese teaching hospital
Xiaohua Qin,1,23 Yang Yang,1,23 Fupin Hu1,2,3 and Demei Zhu1,2
Correspondence 1
Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, PR China
Fupin Hu 2
Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, PR China
hufupin@163.com
3
Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Received 28 June 2013
Accepted 22 November 2013
Carbapenems are first-line agents for the treatment of serious nosocomial infections caused by
multidrug-resistant Enterobacteriaceae. However, resistance to carbapenems has increased
dramatically among Enterobacteriaceae in our hospital. In this study, we report clonal
dissemination caused by carbapenem-resistant Enterobacter aerogenes (CREA). In 2011, CREA
was identified from 12 patients admitted to the neurosurgical ward. All 12 clinical isolates were
non-susceptible to cefotaxime, ceftazidime, cefoxitin, ertapenem, imipenem or meropenem. All
isolates carried the gene encoding Klebsiella pneumoniae carbapenemase-2 (KPC-2), except for
the isolate E4. However, a remarkably lower expression level of the porin OmpF was detected in
the non-KPC-2-producing isolate E4 on SDS-PAGE compared with the carbapenem-susceptible
isolate. Epidemiological and molecular investigations showed that a single E. aerogenes strain
(PFGE type A), including seven KPC-2-producing clinical isolates, was primarily responsible for
the first isolation and subsequent dissemination. In a case-control study, we identified risk factors
for infection/colonization with CREA. Mechanical ventilation, the changing of sickbeds and
previous use of broad-spectrum antibiotics were identified as potential risk factors. Our findings
suggest that further studies should focus on judicious use of available antibiotics, implementation
of active antibiotic resistance surveillance and strict implementation of infection-control measures
to avoid the rapid spread or clonal dissemination caused by carbapenem-resistant
Enterobacteriaceae in healthcare facilities.

INTRODUCTION moniae carbapenemase (KPC) and metallo-b-lactamase

Carbapenems are the most potent and reliable b-lactam
antibiotics for the treatment of serious infections caused
by multidrug-resistant Enterobacteriaceae (Vardakas et al.,
2012; Yang & Guglielmo, 2007). However, carbapenem
resistance has been emerging and increasing in a wide variety
of Enterobacteriaceae species worldwide (Gupta et al., 2011;
Hu et al., 2012; Nordmann et al., 2012). Although the
production of extended-spectrum b-lactamases and/or
plasmid-borne AmpC b-lactamases, coupled with porin
loss, can be responsible for carbapenem resistance, the main
mechanism underlying resistance to carbapenems is the
production of carbapenemases, especially Klebsiella pneu-
3These authors contributed equally to this work.
Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; CLSI,
Clinical and Laboratory Standards Institute; CRE, carbapenem-resistant
Enterobacteriaceae; CREA, carbapenem-resistant Enterobacter
aerogenes; CSEA, carbapenem-susceptible Enterobacter aerogenes;
KPC, Klebsiella pneumoniae carbapenemase; PBA, 3-phenylboronic
acid.
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(Hirsch & Tam, 2010). Carbapenemases have been detected
in a wide range of species in the family Enterobacteriaceae,
including K. pneumoniae, Citrobacter freundii, Escherichia
coli and Enterobacter aerogenes (Peleg & Hooper, 2010).
Because carbapenemase-producing Enterobacteriaceae iso-
lates are usually extensively drug resistant, infections
caused by carbapenem-resistant Enterobacteriaceae (CRE)
present a serious clinical challenge for physicians in
healthcare settings (Lledo et al., 2009). Treatment options
for these infections are limited and few clinical data are
available on which to base antibiotic recommendations
(Falagas et al., 2011). Moreover, the use of inappropriate
empirical antibiotic therapy or delayed appropriate anti-
biotic therapy can lead to worse outcomes. Previous
studies have found crude mortality rates ranging from
30 % to 44 % for diverse infections caused by CRE (Borer
et al., 2009). The prevalence of CRE clinical isolates has
increased significantly in our hospital since 2005 (Hu et al.,
2012). In particular, the detection rate of carbapenem-
resistant E. aerogenes (CREA) among CRE isolates rose
064865 G 2014 SGM Printed in Great Britain

from 0 % (0/7 isolates) in 2005 to 21.1 % (12/57 isolates)
in 2011 (data not shown). Therefore, we keep a record of
CREA strains isolated from the inpatient department in
our hospital.
The purpose of this study was to describe the emergence and
dissemination of CRE clinical isolates caused by CREA in a
teaching hospital in Shanghai, China. Our findings provide
valuable insight into possible strategies for treatment and
controlling the spread of CREA isolates.
METHODS
Bacterial strains. All E. aerogenes isolates (n557) obtained in 2011
at Huashan Hospital (Fudan University, Shanghai, China), a 1300-
bed tertiary-care hospital, were screened to investigate the prevalence
of carbapenem-resistant isolates. These isolates were non-duplicate
and were collected on routine workdays without any specific
exclusion criteria. Among the 57 isolates, a total of 12 carbape-
nem-resistant isolates were identified (the inhibitory diameter of
imipenem or meropenem was ¡19 mm). Of the 12 isolates, 91.7 %
(11/12) were isolated from sputum and one was isolated from
abdominal fluid. All of the isolates were identified from 12
hospitalized patients. A well-characterized strain of C. freundii
1641 (producing an IMP-9-type metallo-b-lactamase), K. pneumo-
niae 2528 (producing a KPC-2-type carbapenemase) (Chen et al.,
2011b) and E. coli strain ATCC 25922 were used as positive and
negative controls for screening carbapenemase genes and antimi-
crobial susceptibility testing, respectively. In addition, E. coli EC600
(resistant to rifampicin) was used as a recipient for conjugation. The
carbapenem-susceptible E. aerogenes (CSEA) isolate Es was randomly
chosen as the control for analysis of outer-membrane protein
profiles and real-time reverse transcriptase PCR.
Antimicrobial susceptibility testing. Antimicrobial susceptibility
testing was performed using the disc diffusion and the agar dilution
methods recommended by the Clinical and Laboratory Standards
Institute (CLSI, 2010). MICs of tigecycline, minocycline, doxycycline,
amikacin, cefoxitin, cefepime, cefotaxime, ceftazidime, ciprofloxacin,
polymyxin B and polymyxin E were determined following criteria of
the CLSI, while the MIC of fosfomycin was determined using an
Etest strip (bioMérieux) following the manufacturer’s instructions.
Breakpoint MICs of tigecycline were determined following the
guidelines of the US Food and Drug Administration (with MICs
¡2 mg ml21 denoting susceptibility and ¢8 mg ml21 denoting
resistance). The MICs of imipenem, meropenem and ertapenem were
analysed with or without the KPC inhibitor 3-phenylboronic acid
(PBA; 400 mg ml21) or the efflux pump inhibitor carbonyl cyanide m-
chlorophenylhydrazone (CCCP; 25 mg ml21), respectively.
Genotypic detection of b-lactamase genes. The presence of genes
encoding the b-lactamases TEM, SHV, PER, SFO, VEB, CTX-M and
GES and genes encoding the plasmid-borne AmpC b-lactamases and
carbapenemases KPC, NDM-1, VIM, IMP, SPM, SME, GIM, NMC,
IMI, IND and OXA was investigated in all of the 12 clinical isolates by
using previously described primers (Chen et al., 2011c; Murray et al.,
2007; Pérez-Pérez et al., 2002). PCR amplicons were sequenced and
the DNA sequences obtained were compared with those available in
the NCBI GenBank database using BLAST searches.
Transfer of carbapenem resistance. Conjugation experiments
were carried out in Luria–Bertani broth with rifampicin-resistant E.
coli EC600 as the recipient, as described previously (Zhang et al.,
2011). Transconjugants were selected on Trypticase soy agar plates
containing rifampicin (600 mg ml21) and ampicillin (50 mg ml21) to
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Hospital clonal dissemination of CREA
select for plasmid-encoded resistance. The colonies grown on the
selective medium were selected and identified using the Vitek 2
(bioMérieux) compact system and KPC-2 carbapenemase determina-
tion by PCR and DNA sequencing.
PFGE analysis. Clonal relationships were analysed using PFGE,
which was performed according to a previously described protocol
(Schlesinger et al., 2005). The DNA fingerprints generated were
analysed according to the criteria proposed by Tenover et al. (1995).
Analysis of outer-membrane protein profiles. The outer-mem-
brane proteins of all isolates were examined by SDS-PAGE, as
described previously (Gayet et al., 2003).
Real-time reverse transcriptase PCR. Real-time reverse transcrip-
tase PCR was performed to determine the expression of ompD and
ompF porin genes, and of the acrB efflux pump gene relative to the
rpoB housekeeping gene according to a previously described protocol
(Szabó et al., 2006).
Clinical epidemiology. Epidemiological data were collected via
chart review for each patient from the hospital’s uniform electronic
database. The following parameters were assessed: (1) general
demographics, such as age, sex and other background information;
(2) the ward to which the patient was assigned after admission; (3)
previous use of antibiotics, particularly carbapenems, extended-
spectrum cephalosporin and quinolones; and (4) potential risk factors
for the occurrence and spread of CREA. Infection or colonization
with a CREA isolate was defined according to the Centers for Disease
Control and Prevention’s definition of nosocomial infections (Garner
et al., 1988). The medical histories of 12 randomly chosen patients
from whom non-duplicated CSEA had been isolated were reviewed as
controls.
RESULTS
Antimicrobial susceptibility testing
All 12 clinical isolates were non-susceptible to cefotaxime,
ceftazidime, cefoxitin, ertapenem, imipenem or merope-
nem. The proportions of the isolates that were susceptible
to doxycycline, minocycline, ciprofloxacin and tigecycline
were 50.0, 58.3, 66.7 and 83.3 %, respectively. All remained
susceptible to amikacin, fosfomycin, polymyxin B and
polymyxin E (Table 1). The resistance patterns could be
divided into two major groups according to the results
obtained with carbapenem-susceptibility tests performed in
the presence of the KPC inhibitor PBA or the efflux pump
inhibitor CCCP (Table 2). For imipenem, PBA and CCCP
exerted significant effects on the MICs, which were reduced
four- or twofold for all isolates except E4. For meropenem
and ertapenem, PBA and CCCP also showed significant
effects on the MICs of most isolates, where they were
reduced by four- or eightfold. For isolates E5 and
E7, the efflux pump inhibitor CCCP reduced the MIC of
meropenem to a greater degree than the KPC inhibitor
PBA.
Characterization of b-lactamases
All of the E. aerogenes strains carried the gene encoding
KPC-2, except for E4. Isolates E3, E6, E8, E9, E11 and E12
223
X. Qin and others

Table 1. In vitro activities of antimicrobial agents against CREA isolates

Drug MIC (mg ml”1) Resistant (%) Susceptible (%)

Range MIC50 MIC90

Imipenem 4–32 16 32 100 0

Meropenem 4–32 16 32 100 0
Ertapenem 32–128 64 128 100 0
Tigecycline 0.5–8 1 4 8.3 83.3
Fosfomycin 2–12 8 12 0 100
Minocycline 2–64 4 64 25 58.3
Doxycycline 2–128 4 64 50 50
Cefepime 2–1024 16 1024 41.7 16.7
Ceftazidime 8–64 16 64 83.3 0
Cefotaxime 32–1024 64 1024 100 0
Cefoxitin 32–1024 1024 1024 100 0
Amikacin 0.25–8 0.25 4 0 100
Ciprofloxacin 0.25–8 0.25 4 25 66.7
Polymyxin B 0.5–2 1 1 0 100
Polymyxin E 0.25–0.5 0.5 0.5 0 100

carried also blaTEM, while isolates E6 and E10 were PFGE

blaSHV-positive and isolate E9 carried the gene blaCTX-M-15.
No PCR products were obtained for any of the other genes
investigated.
Transfer of carbapenem resistance
Two KPC-2-producing E. aerogenes were randomly selected
for conjugation. Carbapenem resistance was successfully
transferred from E. aerogenes to E. coli EC600. The two E.
coli transconjugants exhibited significantly reduced carba-
penem susceptibility, consistent with detection of blaKPC-2
in these transconjugants. The MICs of imipenem, mer-
openem and ertapenem ranged from 4 to 8 mg ml21 and
the antimicrobial susceptibility patterns of E. coli trans-
conjugants were similar to those of the donor. They were
resistant to cefoxitin, cefotaxime and cefepime, but were
susceptible to doxycycline, minocycline and tigecycline.
Five distinct PFGE groups (PFGE types A–E) were
observed among the 12 CREA isolates; one group (type
A) included 58 % (7/12) of the E. aerogenes isolates (E1, E2,
E3, E6, E8, E11, E12) and another group (type B) included
17 % (2/12) isolates (E7 and E9). The remaining three
groups (C–E) included a single isolate each (Fig. 1).
Analysis of outer-membrane proteins and gene
expression of ompF, ompD and acrB
SDS-PAGE analysis of outer-membrane proteins revealed a
remarkably lower level of expression of the porin OmpF in
the clinical strain E4 compared with CSEA. The other 11
CREA isolates had comparable outer-membrane profiles to
the carbapenem- susceptible strain (Fig. 2). The transcrip-
tion levels of both acrB and ompD were equivalent between
carbapenem-resistant and -susceptible isolates. However,

Table 2. MICs of carbapenems, with or without PBA or CCCP, for 12 CREA isolates

Antimicrobial agent MIC (mg ml”1)

E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12

Imipenem 16 16 16 4 32 16 32 16 16 16 16 16
Imipenem + PBA 4 4 4 2 8 4 8 4 4 4 4 4
Imipenem + CCCP 8 8 8 2 16 8 8 8 8 8 8 8
Meropenem 32 16 16 4 32 16 32 16 32 32 16 16
Meropenem + PBA 4 2 4 2 16 4 16 4 4 4 4 4
Meropenem + CCCP 4 4 4 4 4 2 4 4 4 2 4 4
Ertapenem 64 32 64 32 128 64 128 64 64 64 64 64
Ertapenem + PBA 16 8 8 16 32 8 32 16 16 16 8 8
Ertapenem + CCCP 8 8 8 32 16 8 16 8 16 4 16 8

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 Hospital clonal dissemination of CREA

100

100
70
80
90

20

40

60

80
0
E 11-2580 E. aerogenes E7
11-2680 E. aerogenes E9
D 11-2738 E. aerogenes E10
C 11-2100 E. aerogenes E4
B 11-2209 E. aerogenes E5
11-2740 E. aerogenes E11
11-2778 E. aerogenes E12
11-1244 E. aerogenes E2
A
11-1471 E. aerogenes E3
11-2625 E. aerogenes E8
11-991 E. aerogenes E1
11-2421 E. aerogenes E6 Fig. 1. DNA fingerprinting of CREA by PFGE.

the transcription levels of ompF were five- to 100-fold
lower in the E4, E10, E11 and E12 isolates compared with the
susceptible control, while transcription was 16 times higher
in isolate E9.
Epidemiology
CREA isolates were identified from 12 patients (10 men
and two women, ages 41–83 years) hospitalized on the
neurosurgery ward of Huashan Hospital. The patients’
clinical characteristics are shown in Table 3. The first
patient (patient 1), from whom the CREA isolate E1 (PFGE
type A) was identified (from sputum), had undergone
surgery in May 2011 and experienced pneumonia post-
operatively. Nearly 6 weeks after this first CREA isolate was
identified, two patients (patients 2 and 3) on the same
ward were found to have invasive infections with CREA
isolates (E2 and E3, respectively; PFGE type A). In the next
10 days, a further two patients (patients 4 and 5) tested
positive for CREA isolates (E4 and E5, respectively), which
were considered colonizers. During the subsequent 5 weeks,
seven additional patients tested positive for CREA isolates
(PFGE type A for four isolates and PFGE type E for two
isolates). Of these, six patients experienced an actual
infection caused by CREA.
Overall, 75 % of patients (9/12) in the CREA group and
58 % (7/12) in the CSEA control group had undergone
one or more surgical procedures (P50.387). Half of the
patients in the CREA group but only one patient in the
CSEA group had received mechanical ventilation (P5
0.025). Ten patients (83 %) with CREA strains isolated had
undergone one or more sickbed changes during hospit-
alization, compared with none in the control group (P,
0.00001). In addition, eight patients with CREA (67 %)
harboured other multidrug- or pandrug-resistant patho-
gens (Falagas & Karageorgopoulos et al., 2008), mostly
K. pneumoniae, Pseudomonas aeruginosa, Acinetobacter
baumannii, E. coli or meticillin-resistant Staphylococcus
aureus. However, 42 % (5/12) of CSEA patients harboured
susceptible pathogens. Patients in the CREA group were
treated with various antimicrobial agents before isolation

M E1 E2 E3 E5 E4 E6 E7 E8 E9 E10 E11 E12 Es

97.2 kDa
66.4 kDa

44.3 kDa OmpC

29.0 kDa OmpF

20.1 kDa

14.3 kDa

Fig. 2. Outer-membrane fractions of the various E. aerogenes strains. Outer-membrane proteins were stained with Coomassie
blue. CSEA (Es) presented the major porin, while strain E4 presented loss of a porin at 39 kDa (presumed OmpF analogue) and
an additional band at about 23 kDa (triangle). M, marker.
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X. Qin and others

AMK, Amikacin; BIA, biapenem; CAS, caspofungin; CAZ, ceftazidime; CIP, ciprofloxacin; CRO, ceftriaxone; CSL, cefoperazone/sulbactam; CXM, cefuroxime; CZO, cefazolin; DOX, doxycycline;
ETP, ertapenem; FLU, fluconazole; GEN, gentamicin; LZD, linezolid; MEM, meropenem; MNO, minocycline; PAN, panipenem; TEC, teicoplanin; TZP, piperacillin/tazobactam; VAN, vancomycin.
Status on hospital of CREA (Table 3). However, no antimicrobials were
discharge used before CSEA isolation in eight patients (67 %, 8/12)

Recovered

Recovered
Recovered

Recovered
in the control group (P50.0005).

Improved
Improved
Improved

Improved
Improved
Improved
Improved

Improved
DISCUSSION
Although carbapenemase-producing Enterobacteriaceae
MNO, MEM, PAN, VAN, BIA, CSL, CAS

isolates are usually extensively drug resistant, some isolates

CRO, MEM, CIP, ETP, CSL, DOX, LEV,

can still remain susceptible to other antimicrobial agents
(Petrosillo et al., 2013). In the present study, 50 % or
Antimicrobial therapy

more of the isolates were susceptible to minocycline and

GEN, CXM, MEM, CAZ, FLU

CZO, MEM, VAN, TEC, FLU

BIA, VAN, DOX, MEM, CAS

doxycycline, and 83.3 and 66.7 % were susceptible to

tigecycline and ciprofloxacin, respectively. None of the
isolates was found to be resistant to amikacin,
MEM, VAN, CSL fosfomycin, polymyxin B or polymyxin E. Therefore,
TZP, MEM, LZD

CIP, TZP, CAS

CSL, VAN, CIP

these agents, either alone or in combination, may be

MEM, CSL

further explored as options for the treatment of

CSL, AMK

CSL, TZP
TZP, CIP

infections caused by CREA. Overall, 92 % (11/12) of

CREA carried the gene encoding KPC-2, and the KPC
inhibitor PBA reduced the MICs of the three tested
carbapenems to these 11 isolates to various degrees.
Colonization
Colonization

Colonization
colonization
Infection/

Other than b-lactamases, loss of outer-membrane porins

Infection
Infection
Infection

Infection
Infection
Infection

Infection
Infection

Infection

or overexpression of genes that regulate or encode efflux

pumps are among the mechanisms that contribute to
CREA (Pfeifer et al., 2010). Apart from in the E4 isolate,
Abdominal fluid

the efflux pump inhibitor CCCP reduced the MICs

Specimen

of meropenem and ertapenem by four- or eightfold

(Table 2).
Sputum
Sputum
Sputum

Sputum
Sputum
Sputum
Sputum
Sputum
Sputum
Sputum

Sputum

KPC-producing Enterobacteriaceae have become a public

health concern worldwide, including in China (Cantón
et al., 2012; Hu et al., 2012). In our hospital, CRE isolates
Days of hospital stay
Table 3. Clinical characteristics of patients colonized or infected with CREA

before infection/

have been detected in a wide range of species, especially

total days

K. pneumoniae (Chen et al., 2011a; Hu et al., 2012). In

22/37
12/74
15/87

13/27
11/29
20/41
17/30
5/14

5/15
1/12
5/15

6/31

this study, epidemiological and molecular investigations

showed that a single E. aerogenes strain (PFGE type A),
including seven clinical isolates (E1, E2, E3, E6, E8, E11,
E12), that produced KPC-2 was primarily responsible for
the first isolation and subsequent dissemination (Fig. 1).
Ventricular haemorrhage

Intracranial haematoma
Underlying condition

Results of epidemiological and molecular investigations

Craniocerebral trauma

Craniocerebral trauma
Craniocerebral trauma
Craniocerebral trauma

Craniocerebral trauma
Epidural haematoma
Epidural haematoma

Epidural haematoma

suggest that the increased prevalence of carbapenem-

Adenoid tumour

resistant isolates in our hospital may have been caused by

Cerebral trauma

a failure to control the spread of these resistant strains.

Studies have found reduced expression of omp genes in
CREA (Fernández-Cuenca et al., 2006; Thiolas et al.,
2004). The CRE isolate E4 was resistant to carbapenems
but did not possess any b-lactamase genes implicated
Sex (age,

F (41)

F (76)
M (58)
M (47)
M (83)
M (57)
M (78)
M (68)
M (53)
M (55)
M (42)

M (56)
years)

in carbapenem resistance, and its resistance was not

compromised by CCCP. For this isolate, reduced
expression of ompF was identified with SDS-PAGE, and
Isolate no.

a decreased gene transcription level of ompF was detected

E10
E11

E12

with real-time PCR. Therefore, we propose that the

E1
E2
E3
E4
E5
E6
E7
E8
E9

reduced expression of outer-membrane protein served as

the major resistance mechanism for isolate E4. Several
Patient

KPC-2-producing isolates exhibited identical MIC values

despite different expression levels of ompF (e.g. E9
10
11

12
1
2
3
4
5
6
7
8
9

compared with E10–12). We suggest that compared with

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outer-membrane protein, KPC plays a conclusive role in
carbapenem resistance mechanisms.
In a previous study on carbapenem-resistant C. freundii
among hospitalized patients, the risk factors for acquiring
this organism included invasive procedures (especially
surgical operations), indwelling urine catheters, the chan-
ging of sickbeds and previous in-hospital cephalosporin use
(Chen et al., 2011b). We found very similar risk factors for
the acquisition of CREA among hospitalized patients,
including mechanical ventilation, the changing of sickbeds
and previous use of broad-spectrum antibiotics. Determi-
nation of risk factors is important, because it will help to
identify patients who require close monitoring and also
optimize empirical antimicrobial drug therapy.
Because carbapenemase genes located on plasmids are
readily transferable, infections caused by carbapenemase-
producing Enterobacteriaceae isolates may prove difficult to
control once they have emerged (Lledo et al., 2009). Rapid
dissemination of extensively drug-resistant isolates is a
serious concern in clinical patient care. Therefore, prompt
detection is critical for containing carbapenemase-pro-
ducing strains and preventing nosocomial transmission. As
only few novel antimicrobials are in development for the
treatment of these extensively drug-resistant infections
(Petrosillo et al., 2013), further studies should focus on the
judicious use of available antibiotics and implementation
of strict infection-control measures to avoid the rapid
spread or clonal dissemination of carbapenemase-pro-
ducing Enterobacteriaceae in healthcare facilities.
ACKNOWLEDGEMENTS
We kindly thank Hongyou Chen from the Department of
Microbiology, Shanghai municipal centres for disease control and
prevention, for guiding the PFGE analysis. We thank Yohei Doi from
the Division of Infectious Diseases, University of Pittsburgh School of
Medicine, for critical review of the manuscript. This work was
supported by the Shanghai Municipal Natural Science Foundation
(grant no. 11ZR1404700 to F. H.) and the National Natural Science
Foundation of China (grant no. 81273559 to F. H.). The funders had
no role in study design, data collection and analysis, decision to
publish or preparation of the manuscript.
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