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Xin 2016, Pfge&Mlst
Xin 2016, Pfge&Mlst
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Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences,
Beijing, China. 2Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing,
China. 3Charles Sturt University, Leeds Parade, Orange, New South Wales, Australia. 4Centre for Infectious Diseases
and Microbiology Laboratory Services, ICPMR Pathology West, University of Sydney, Westmead Hospital, Darcy
Road, Westmead, New South Wales, NSW 2145, Australia. Correspondence and requests for materials should be
addressed to Y.C.X. (email: [email protected])
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almost all -lactams. The two major groups of carbapenemases commonly found in P. aeruginosa, namely IMP
and VIM, are Ambler Class B metallo-lactamases (MBLs). Ambler class A carbapenemases, such as Klebsiella
pneumoniae carbapenemase (KPC), are more frequently reported in Enterobacteriaceae, although they have
started to be detected in P. aeruginosa isolates as well6. Given the increasing prevalence of carbapenemases and
the limited information available about their prevalence in mainland China, we decided to investigate the frequency of different types of carbapenemases among MDR-PA isolates in this geographic area (Fig.1).
Although several studies have shown that the P. aeruginosa population structure is typically non-clonal7,8,
several other studies have described the presence of MBL-producing clones in hospitals9,10. For a comprehensive
understanding of the MDR-PA problem in China, we have established a nationwide surveillance network of
27 tertiary hospitals from diverse geographical areas (Fig.1) to monitor this pathogen, including antimicrobial
susceptibilities, molecular epidemiology, clonal structure and prevalence rate of carbapenemase in nosocomial
MDR-PA.
Results
General information. A total of 254 non-duplicate MDR-PA isolates were collected from nosocomial
infections from 23 of the 27 participating hospitals (four hospitals did not isolate any). The mean age of these
254 patients was 54.621.6 years, and 66.1% (168 patients) were male. Among them, 49 patients had skin and
soft tissue infection, 44 had bacteraemia, and 42 had urinary tract infections. The main clinical wards in which
MDR-PA were isolated included surgical (99 patients, 39.0%) and ICU (68 patients, 26.8%), while the medical
ward accounted for 18.9% (48 patients) and all other wards combined accounted for 15.4% (39 patients).
Pus was the most common specimen, accounting for 19.3% of all MDR isolates, followed by blood cultures
(17.3%), urine (16.5%), and broncho-alveolar lavage fluid (12.6%). The isolates were also obtained from venous
catheters (7.5%), ascitic fluid (5.9%), surgery incision (5.9%), bile (5.5%), hydrothorax (4.7%) and cerebrospinal
fluid (3.2%). Tissue biopsy specimens, peritoneal dialysate fluid and bone marrow only yielded one or two isolates
each (Table1).
Among the 254 MDR-PA isolates included in this study, 40 (15.7%) isolates were collected from hospital
KM, followed by 26 (10.2%) from hospital SD, 17 (6.7%) from hospital JL. A further 14 isolates (5.5%) each were
collected from 5 hospitals (SC, ZZ, TJ, Z1, TZ), followed by 13 (5.1%) from hospital XY. The remaining hospitals
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No. of
isolates
No. of carbapenemases
producer (%)
49
4 (8.2%)
Blood
44
3 (6.8%)
Urine
42
8 (19.0%)
BALF
32
2 (6.3%)
IMP-9 (2)
Venous catheter
19
1 (5.3%)
IMP-9 (1)
Ascitic fluid
15
None
Surgery incision
15
None
Bile
14
None
Hydrothorax
12
1 (8.3%)
IMP-10 (1)
Cerebrospinal fluid
None
Tissue
None
None
Bone marrow
None
19 (7.5%)
Specimen source
Total
254
Antimicrobial agent
S%
I%
R%
MIC50
(mg/L)
Cefoperazone/Sulbactam
N/A
N/A
N/A
>32
Piperacillin/Tazobactam
6.7
20.5
72.8
128
Ceftazidime
20.9
16.1
63.0
32
Cefepime
6.7
18.1
75.2
>32
Aztreonam
10.6
13.8
75.6
>16
Imipenem
13.8
5.9
80.3
>8
Meropenem
15.0
9.0
76.0
>8
Amikacin
65.0
1.5
33.5
Ciprofloxacin
19.7
7.1
73.2
16
Colistin
72.4
19.7
7.9
contributed on average less than 2.5% of the isolates each (Fig.1). The ICU (15 isolates), neurosurgery (9 isolates),
haemodialysis centre (8 isolates), and urology surgery (5 isolates) were the main wards/departments from which
MDR-PA isolates were collected from hospital KM. However, such isolates were also collected in other wards e.g.
respiratory and gastroenterology wards.
Antimicrobial susceptibility. More than 70% of MDR-PA isolates were resistant to piperacillin/tazobac-
tam, cefepime, aztreonam, imipenem, meropenem and ciprofloxacin (Table2). Colistin was the most active agent
(7.9% resistance), followed by amikacin with a resistance rate of 33.5%.
Eighty-six antimicrobial resistance patterns were detected among the 254 MDR-PA isolates. The most common pattern was resistance to piperacillin/tazobactam, cefepime, aztreonam, imipenem, meropenem and ciprofloxacin, but susceptible to ceftazidime, amikacin and colistin (44 isolates, 17.3%). The second most common
pattern was resistance to all the agents except colistin (31 isolates, 12.2%). The other antibiotic susceptibility
patterns were highly variable, with less than 20 isolates per pattern. The resistance patterns showed no obvious
correlation with specimen sources, but with geographical areas. Among 44 isolates sharing the most common
pattern, 40 were from KM hospital, and were identified as XDR-PA. Furthermore, among 31 isolates of the second
most common pattern, 11 isolates were from the SD hospital.
Genotyping analysis. Multilocus sequence typing (MLST) analysis revealed significant genetic diversity
among MDR-PA, with 91 sequence types (STs) (17 of which were novel) identified among the 254 isolates studied. All the STs belonged to different singletons. The most common ST was ST292 (47 isolates, 18.5%), followed
by ST244 (24 isolates, 9.4%), ST277 (18 isolates, 7.1%), ST235 (13 isolates, 5.1%) and ST699 (10 isolates, 3.9%).
Seventeen novel STs identified in the present study have been deposited in the MLST database, with assigned
numbers of ST1950, ST1956, ST1960 and ST1963 to ST1976 (Supplementary Table S1).
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Pulsed-field gel electrophoresis (PFGE) was performed on all the 47 ST292 (the single most common ST) isolates, and these were discriminated into six different genotypes (AF) (Fig.2). A high degree of genetic similarity
(90%) was observed within the 40 isolates collected from KM hospital and belonged to genotype A. The other 7
isolates from 3 hospitals in different cities, belonged to five genotypes (BF) (Fig.2).
Discussion
According to previous studies, clonal dissemination of P. aeruginosa is considered to be low7,11,12. In the present
study, except for the isolates collected in KM hospital, the MDR-PA isolates exhibited a high genetic diversity
and varied resistant patterns. However, it was unusual that all the 40 MDR-PA isolates (40 patients) collected
from one hospital (KM) presented the same XDR resistant profiles, belonged to ST292 clone and PFGE genotype
A. Clinical records showed that these 40 patients were diagnosed with different infectious diseases and hospitalized in different wards. So we highly suspected that an outbreak of XDR P. aeruginosa was present in KM hospital
(Yunnan province) during the surveillance period.
There are very limited clinical reports on P. aeruginosa ST292 lineage worldwide, with some detailed information on this clone available in a study by Lee et al. from Korea13. In contrast to the P. aeruginosa ST292 isolates
in the present study (from KM hospital), the ones in the Korean study were carbapenem-susceptible but had
reduced susceptibility to colistin13. It is worth noting that the isolates from KM hospital did not produce any carbapenemases. According to previous studies, apart from production of carbapenemases, the major mechanism of
carbapenem-resistance in P. aeruginosa is the loss of outer membrane porin OprD and over-expression of efflux
pump systems MexAB-OprM16,17. Furthermore, ST292 isolates in the present study had an unusual phenotype,
being resistant to cefepime but susceptible to ceftazidime. Previous studies suggested this uncommon phenotype
may be due to hyper-expression of the multidrug efflux pump named MexXY-OprM efflux pumps14,15.
However, possible nosocomial outbreaks of carbapenemase-producing P. aeruginosa were actually noted in
some other hospitals9,1820. In this study, we detected three ST357 MDR-PA isolates, all from three urinary tract
infection patients hospitalized in the same neurosurgery ward in HN within 45 days, suggesting possible clonal
dissemination. These strains produced IMP-9 and were only susceptible to colistin. ST357 strains have been
detected in other geographical regions, but usually produce IMP-7 in Europe2123, and other IMP enzymes like in
Japan24, which is different from the present study.
Recent studies have revealed significant differences in the prevalence and distribution of
carbapenemase-producing P. aeruginosa strains from Europe and Asia. In a very recent survey in Europe, performed on 529 carbapenem-non-susceptible P. aeruginosa, 20.0% (106 isolates) were positive for MBL, with the
VIM-type enzymes12 predominating. Only two IMP-type enzymes (IMP-15 and IMP-33) were detected in two
strains. However, studies in Japan in 2004 and 2006, detected 2.3% and 2.1% MBL-producers, respectively24. A
nation-wide survey in mainland China involving 258 P. aeruginosa isolates collected from 2006 to 2007 at 28
hospitals, detected MBL in only 22 isolates (8.5%)11. Thus comparing to the European data12, the carbapenemase
prevalence in China and Japan was much lower. In this study, the carbapenemase producing isolates were frequently detected in east and south of China (Fig.1), regions with larger population densities and considered to
be relatively more developed economically than others, and hence probably used drugs more often than other
regions.
Previous studies show that the prevalence of MBL producers in Europe kept increasing during the years 2009
to 2011, rising from 12.3% to 30.6%12. Based on this background, it is important to adopt and implement continuous surveillance programs for such organisms to assess the effectiveness of current control strategies as well
as formulation of new ones. Our study is an important update on the prevalence of carbapenemase producing
P. aeruginosa in China. The MBL were detected in only 19 isolates (8.15%), and with IMP-9 the most common
carbapenemase, which appears to be an important feature of Chinese strains according to previous studies11,25.
ST235, another recognized international drug-resistant lineage26, is more often associated with worldwide outbreaks of nosocomial infections27,28. In the present study, ST235 was detected in seven hospitals and accounted for
13 isolates (5.1%), but none of them produced carbapenemase. Another potential outbreak case was observed in
SD hospital, where ten ST244 P. aeruginosa isolates collected from this hospital exhibited a similar resistant profile
(Fig.1). Interestingly, ST244 is a lineage known world-wide to cause outbreaks of nosocomial infections7,29,30.
In conclusion, this study has demonstrated that MDR-PA of nosocomial origin in mainland China are of
diverse genetic backgrounds, but suspected nosocomial outbreaks were actually noted in KM hospital, which
would suggest strengthening the existing infection control measures for containment of MDR strains.
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Carbapenemase (no.
of isolates)
ST
Gender
Age
Hospitalsa
Specimen source
Ward
Province
Resistant
profile
Collection
date
IMP-9 (10)
357
male
55
HN
Urine
Neurosurgery ward
Hainan
PCFAIMKR
2012/2/17
357
male
27
HN
Urine
Neurosurgery ward
Hainan
PCFAIMKR
2012/4/2
357
female
58
HN
Urine
Neurosurgery ward
Hainan
PCFAIMKR
2012/3/12
919
female
27
SZ
BALF
ICU
Guangdong
PCF IMKR
2012/5/14
919
male
84
SZ
BALF
ICU
Guangdong
PCF IMKR
2012/6/26
267
male
49
TJ
Urine
Hubei
PCFAIMKR
2011/10/21
IMP-1 (2)
273
female
62
ZZ
Urine
Henan
PCF IMK
2012/3/23
773
male
25
XY
Pus
ICU
Hunan
CFA MK
2012/9/25
1967
male
61
SC
Pus
Burn ward
Sichuan
PCF MKR
2011/9/8
1973
male
12
XY
Venous catheter
Pediatrics ward
Hunan
PCF IMK
2011/10/24
463
male
17
SX
Blood
ICU
Shanxi
PCFAIM R
2012/5/15
1420
male
39
ZR
Blood
Burn ward
Beijing
PCFAIMKR
2011/2/16
IMP10 (1)
1976
male
36
ZZ
Hydrothorax
Henan
CFAIM R
2012/6/14
VIM2 (5)
390
male
XY
Pus
Neonatology ward
Hunan
PC IM
2012/6/12
882
male
48
XY
Urine
ICU
Hunan
PCF IMK L
2012/7/22
1974
male
45
XY
Blood
ICU
Hunan
PCFAIM
2012/8/13
277
female
66
SC
Pus
Sichuan
P IM R
2011/9/24
234
female
81
SD
Urine
ICU
Shandong
PCF IM
2011/10/21
463
male
68
Z1
Urine
Zhejiang
PCFAIM R
2012/6/2
KPC (1)
Ethical approval. This study was approved by the Human Research Ethics Committee of Peking Union
Medical College Hospital (Beijing, China) [No. PUMCHBC-C-2-Q01-1], and was carried out strictly in accordance with the approved guidelines. Informed consent was obtained from all subjects.
Study design. This study was part of a Chinese nationwide prospective surveillance project for investigat-
ing the antimicrobial resistance and molecular epidemiology of major MDR bacterial pathogens causing nosocomial infections between August 2011 and July 2012. Twenty-seven hospitals from 26 provinces in mainland
China participated in the surveillance voluntarily (see Acknowledgments section for full hospital list) (Fig.1).
Non-duplicate MDR-PA isolates causing nosocomial infections (from either hospitalized patients (48 h after
admission) or in patients with a history of more than 48-h hospitalization within the last 30 days)31 were consecutively collected in the study (Fig.1), and isolates from sputum and from screening swabs (for example throat swab
and rectal swab) were excluded. For each MDR-PA isolate, clinical data was obtained from medical records by
the collecting hospital, and completed on a standard electronic report form. All the isolates meeting the inclusion
criteria were forwarded to our central laboratory (Department of Clinical Laboratory, Peking Union Medical
College Hospital) for confirmative identification, further antibiotic susceptibility testing, carbapenemase screening and genotyping. Isolates were stored at 80C prior to testing.
by Curran et al.33. Standard DNA amplification of the seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD,
ppsA, and trpE) was performed for all isolates. The PCR products were sequenced in both directions using the
Scientific Reports | 6:26522 | DOI: 10.1038/srep26522
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DNA analyser ABI 3730XL system (Applied Biosystems, Foster City, CA). The nucleotide sequences were compared to existing sequences in the MLST database (www.pubmlst.org/paeruginosa) for assignment of allelic numbers. The isolates were assigned a ST number according to their allelic profiles. The phylogenetic analysis was
performed by the MLST clustering software eBURST v3.0 (http://eburst.mlst.net/). Novel alleles in each novel ST
were confirmed twice by sequencing in both directions and then submitted to MLST database.
To further investigate the genetic relationship of the isolates belonging to the most common ST, and to have
some insight into intra-hospital epidemiology of isolates, PFGE analysis was performed as described by Hu
et al.34. Briefly, genomic DNA was digested with 10U restriction enzyme SpeI (New England BioLabs Inc.) and
fragments separated by using CHEF MAPPER system (Bio-Rad, Richmond, USA). Dendrogram and cluster analysis were performed by BioNumerics software. Percentage similarities were identified on a dendrogram derived
with the un-weighted pair-group method with arithmetic means (UPGMA) and Dice coefficients. The PFGE
patterns of isolates with coefficient of similarity 90% were considered to belong to the same genotype35.
Screening of carbapenemase producing isolates. The presence of acquired carbapenemases was determined by genetic approaches for the 233 carbapenem-resistant isolates. Described specific primers and conditions were used to amplify blaVIM, blaIPM, blaKPC, blaNDM, blaNMC, blaSME, blaIMI, blaGES, blaSPM, blaGIM, blaSIM,
blaOXA-24, or closely related carbapenemase genes36,37. The positive amplification products were sequenced and the
results compared with those available in the GenBank database (www.ncbi.nil.gov/BLAST). Multiple-sequence
alignments were performed with the CLC Sequence Viewer (version 7.0.2).
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Acknowledgements
The authors thank Professor Yu-Pei Zhao (Peking Union Medical College Hospital, Beijing, China) for the great
support in organising this study. The authors are also grateful to all of the 27 hospitals that participated in this
study. They are The First Peoples Hospital of Kunming, Kunming (KM); Shandong Provincial Hospital, Jinan
(SD); Jilin Province Peoples Hospital, Changchun (JL); Sichuan Provincial Peoples Hospital, Chengdu (SC);
The First Affiliated Hospital of Zhengzhou University, Zhengzhou (ZZ); Tongji Hospital, Wuhan (TJ); The First
Affiliated Hospital of Zhejiang University, Hangzhou (Z1); Tianjin Medical University General Hospital, Tianjin
(TZ); Xiangya Hospital, Changsha (XY); Guiping Peoples Hospital, Guiping (GP); Haikou Peoples Hospital,
Haikou (HN); The First Affiliated Hospital of PLA General Hospital, Beijing (ZR); Shengjing Hospital of China
Medical University, Shengyang (SJ); Shenzhen Peoples Hospital, Shenzhen (SZ); The Second Hospital of Hebei
Medical University, Shijiazhuang (HB); Neimenggu Xinganleague Peoples hospital, Xinganleague (XA); Sir Run
Run Shaw Hospital, Hangzhou (ZS); The First Affiliated Hospital of Shanxi Medical University, Taiyuan (SX); The
First Teaching Hospital of Xinjiang Medical University, Wulumuqi (XJ); San Er Ling Yi Hospital, Hanzhong (HZ);
Qinghai Provincial Peoples Hospital, Xining (QR); The First Affiliated Hospital of Harbin Medical University,
Harbin (H1); Ningxia Peoples Hospital, Yinchuan (NX); The Second Affiliated Hospital of Nanchang University,
Nanchang (NC); Gansu Provincial hospital, Lanzhou (GS); The First Affiliated Hospital of Anhui Medical
University, Hefei (AH); Qinghai Red Cross Hospital, Xining (QH). This work was supported by the Research
Special Fund for Public Welfare Industry of Health [grant no. 201402001]. The funders had no role in study
design, data collection and interpretation, or the decision to submit the work for publication.
Author Contributions
F.X., X.M. and X.Y.C. were responsible for the conception and design of the study; F.X., W.Y., X.M. and X.Z.P.
performed laboratory work. F.X., X.M., K.T., K.F. and B.A. analysed the results. F.X., X.M., K.T. and K.F. drafted
the paper. All authors reviewed the manuscript.
Additional Information