Molecular epidemiology of blaOXA-23-producing carbapenem-resistant Acinetobacter baumannii in a single institution over a 65-month period in north China
verfasst von:
Nian-zhi Ning, Xiong Liu, Chun-mei Bao, Su-ming Chen, En-bo Cui, Ju-ling zhang, Jie Huang, Fang-hong Chen, Tao Li, Fen Qu, Hui Wang
Carbapenem-resistant Acinetobacter baumannii poses a significant threat to hospitalized patients, as few therapeutic options remain. Thus, we investigated the molecular epidemiology and mechanism of resistance of carbapenem-resistant A.baumannii isolates in Beijing, China.
Methods
Carbapenem-resistant A.baumannii isolates (n = 101) obtained between June 2009 and November 2014 were used. Multilocus sequence typing (MLST) and PCR assays for class C and D β-lactamase were performed on all isolates. S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization were performed to identify the resistance gene location.
Results
All 101 A.baumannii isolates were highly resistant to frequently used antimicrobials, and were considered multidrug resistant. A total of 12 sequence types (STs) were identified, including 10 reported STs and 2 novel STs. Eighty-seven isolates were classified to clonal complex 92 (CC92), among which ST191 and ST195 were the most common STs. The blaOXA-23 gene was positive in most (n = 95) of the A.baumannii isolates. Using S1-nuclease digestion PFGE and Southern blot hybridization, 3 patterns of plasmids carrying blaOXA-23 were confirmed. ST191 and ST195 (both harboring blaOXA-23) caused outbreaks during the study period, and this is the first report of outbreaks caused by ST191 and ST195 in north China.
Conclusion
blaOXA-23-producing A.baumannii ST191 and ST 195 isolates can disseminate in a hospital and are potential nosocomial outbreak strains. Surveillance of imipenem-resistant A.baumannii and antimicrobial stewardship should be strengthened.
Abkürzungen
CC
Clonal complex 92
CRAB
Carbapenem-resistant A. baumannii
DLVs
Double-locus variants
MLST
Multilocus sequence typing
PFGE
Pulsed-field gel electrophoresis
SLVs
Single-locus variants
ST
Sequence type
Background
Acinetobacter baumannii is an opportunistic pathogen involved in outbreaks occurring in burn units, surgical wards or intensive care units (ICUs), as well as important cause of nosocomial septicemia, pneumonia and urinary tract infections [1]. A.baumannii is of interest due to increasing the increase in antimicrobial resistance [2]. This organism is generally intrinsically resistant to many frequently-used antibiotics, including aminopenicillin, first- and second-generation cephalosporins and chloramphenicol [3, 4]. Carbapenems are important antibiotics to treat A.baumannii because they are highly efficacious and have low toxicity [5]. However, the emergent and rapid spread of carbapenem-resistant A.baumannii isolates pose a severe threat to public health and are a global concern [6]. Carbapenem resistance, such as to imipenem, increased in China from 31.0% in 2005 to 62.4% in 2014 [7]. Recent studies also suggest high resistant of A.baumannii against carbapenems across the world [8‐11].
Carbapenem resistance in A.baumannii is mainly mediated by the production of carbapenem-hydrolyzing enzymes [6]. Class D OXA-type enzymes are the most prevalent carbapenemases in A.baumannii [12]. In addition to the intrinsic OXA-51-like enzymes, 3 unrelated groups of these carbapenem-hydrolysing enzymes have been identified OXA-23-like, -40-like and -58-like [13]. Outbreaks of blaOXA-23-producing A.baumannii have been reported across the world [14‐16] and a previously study has pointed out that blaOXA-23 was the predominant group of carbapenem-hydrolysing enzymes in China [17].
Anzeige
Multilocus sequence typing (MLST) is used for global and long-term epidemiological studies [18], and data from MLST show that CC92 was the most widely distributed A.baumannii clone globally [19‐21]. Studies from China indicate that blaOXA-23-producing CC92clones are prevalent in most provinces of China [17, 22]. Although the molecular epidemiology of carbapenem-resistant A.baumannii has been investigated, the epidemiology of carbapenem-resistant A.baumannii over long time periods in single institution may allow new insights into the behavior of this pathogen.
Thus, we sought to investigate carbapenem-resistance mechanisms and the molecular epidemiology of carbapenem-resistant A. baumannii in a single hospital over a 65-month period.
Methods
Bacterial isolates
Between June 2009 and November 2014, a total of 101 nonduplicate carbapenem-resistant (Zone Diameter of imipenem ≤18 mm; Clinical Laboratory Standards Institute [CLSI] breakpoint) A. baumannii (CRAB) isolates were collected from a single hospital in Beijing, China. A single isolate per patient was included. All isolates were identified by conventional biochemical techniques using VITEK 2 system (BioMérieux France). PCR confirmation of the blaOXA-51-like carbapenemase gene was performed to help identify A. baumannii simultaneously, because this gene is intrinsic to A. baumannii [23, 24].
Antimicrobial susceptibility testing
The disk diffusion method was used to evaluate susceptibility to the following antimicrobial agents: imipenem (IPM: 10 μg), ceftazidime (CAZ: 30 μg), amikacin (AMK: 30 μg), piperacillin/tazobactam (TZP: 100/10 μg), levofloxacin (LVX: 5 μg), ticarcillin/Clavulanic acid (TCC: 75/10 μg), minocycline (MNO: 30 μg) (Oxoid, UK). Results were interpreted in accordance with CLSI guidelines from 2011. Isolates with intermediate susceptibility were classified as non-susceptible.
Anzeige
Molecular typing methods
Multilocus sequence typing (MLST) was performed on all A.baumannii isolates as described previously [18]. Analysis of allele sequences and sequence type (ST) assignment made use of the Oxford Acinetobacter baumannii MLST website (http://pubmlst.org/abaumannii/). The eBURST algorithm (version 3; http://eburst.mlst.net/) was used to assign clonal complexes (CCs).
Screening of ambler classes C and D β-lactamase genes
PCR experiments were carried out using primers specific for the genes encoding Ambler C and D β-lactamase (AmpC, MOX-1, MOX-2, CMY-1 to CMY-11, BIL-1, DHA-1, DHA-2, ACC, ACT-1, MIR-1 T, FOX-1 to FOX-5b, blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, and blaOXA-58-like and blaOXA-143)as described previously [25‐28]. Primers are depicted in Table 1. For each gene detected, some PCR products were randomly selected, and then sequenced to confirm genes.
To detect plasmids of A.baumannii isolates, an agarose gel plug containing total cellular DNA was prepared and digested with S1 nuclease (Takara, Japan) as described previously [29]. Digested plugs were subjected to PFGE using a CHEF-Mapper system (pulse times, 5 to 30 s; running time, 15 h; 6 V/cm). Gels were blotted onto nylon membranes (Millipore, USA) using standard techniques. The membrane was hybridized with a digoxigenin-labeled probe consisting of a blaOXA-23 fragment which was amplified by primers.
Results
A total of 101 A.baumannii isolates were resistant to imipenem and considered carbapenem-resistant and enrolled in our study. CRAB isolates were obtained from various sources, including sputum (n = 72 isolates), blood (n = 14 isolates), abdominal fluid (n = 9 isolates), secretion (n = 2 isolates), catheter (n = 1 isolates), eyes (n = 1 isolates), pus (n = 1 isolates) and throat swabs (n = 1 isolates). Of 101 CRAB isolates, 87% (n = 88) were collected from the ICU. The temporal distribution of CRAB isolates is showed as follows. 2, 3, 45, 4, 18 and 29 isolates were obtained in 2009, 2010, 2011, 2012, 2013 and 2014, respectively. CRAB isolates resistance data appear in Table 2. All CRAB isolates were resistant to at least 3 classes of antibiotic and were considered multidrug resistant. CRAB isolate data for AmpC and blaOXA-51-like genes appear in Table 2.
Table 2
Details of A. baumannii isolates, by sequence type
To investigate the molecular epidemiology of isolates, MLST was performed to characterize CRABs and data are summarized in Table 2. The eBURST analysis data appear in Fig. 1.
×
ST191 was the largest sequence type (32 of 101 isolates) and was found in our institution from August 2009 to May 2012. Only 1 isolate was obtained in August 2009 from the Liver Failure ward, but most (96.9%) of ST191 isolates were collected between February 2011 and May 2012. Importantly, 28 of the 32 ST191 strains were isolated from ICU ward. All ST191 isolates were resistant to piperacillin/tazobactam, ticarcillin/clavulanic acid, ceftazidime and levofloxacin but had variable susceptibilities to amikacin and minocycline (Table 2). All of ST191 isolates were blaOXA-23-positive. One strain was selected randomly to be subjected to PFGE digested with S1 nuclease, and results show that this ST strain contains a plasmid of approximately 78 Kb. Southern blot hybrid hybridization assays confirmed that blaOXA-23 gene was located on this plasmid (Fig. 2).
×
Thirty-one (30.7%) isolates were defined as ST195, and this ST was first detected in the infectious liver diseases ward in March 2013. Twenty-eight ST195 isolates were collected from ICU ward from then on. It is notable that all ST195 isolates were resistant to almost all antibiotics tested in this study except 1 isolate was susceptible to amikacin. All of ST195 isolates were also blaOXA-23 positive. This ST strain does not contain any plasmid, and the blaOXA-23 gene is located on the chromosome (Fig. 2).
Another 4 sequence types, ST208, ST368, ST218 and ST369, were found in 15 (14.8%), 6 (5.9%), 2 (2.0%) and 1 (1.0%) isolates, respectively, and carried the blaOXA−23 gene but had different resistance profiles. The blaOXA−23 gene is located on the plasmid in the A.baumannii ST218 and ST368 strain, but is found on the chromosome in ST208 and ST369.
Anzeige
Six isolates were ST469. This is a double-locus variant (DLV) of multiple STs within CC92, implying a close relationship. However, it does not agree with the conservative definition of sharing alleles at 6/7 of the loci, and thus ST469 cannot be considered a CC92 member. All ST469 isolates were collected during December 2012 and November 2013. ST469 isolates were blaOXA-23 positive and were resistant to allantibiotics. PFGE and hybridization results show that this ST isolate harbored 2 plasmids. The blaOXA-23 gene was located on a ca. 120 kb plasmid and on a plasmid of approximately 245 kb, with another copy of blaOXA-23 on a chromosome (Fig. 2).
Two unreported singleton STs were identified. ST1309 presented in this hospital in August 2011 only and was resistant to all the 7 classes of antimicrobials. The carbapenem-resistant determinant of this isolate remains unclear. The ST1302 isolate was unique among imipenem-resistant strains; it was ceftazidime, amikacin, levofloxacin and minocycline susceptible and was the only isolate that carried the blaOXA-40 gene.
Discussion
This study offers insight into the longitudinal evaluation of the molecular epidemiology of carbapenem-resistant A.baumannii in a single institution over a 65-month period.
The blaOXA-23 gene was positive in most (n = 95) of the A.baumannii isolates in this institution. The first report of this enzyme in A.baumannii was ARI-1, which was identified in an isolate from Scotland collected in 1985 [30]. In 2000, enzyme sequence analysis (re-named OXA-23) indicated that it was a member of the ambler class D group of β-lactamases [31]. Since then, outbreaks of OXA-23 carbapenemase-producing A.baumannii have been reported all over the world [32‐36]. Our finding was consistent with other reports from China. Zhou’s group investigated resistance determinants of 342 imipenem-resistant A.baumannii isolates which were collected from 16 Chinese cities in 2005, and found that most CRAB isolates contained the blaOXA-23 gene [37]. Recent studies confirm a high prevalence of the blaOXA-23 gene in carbapenem-resistant A.baumannii in different Chinese cities (80.6-100%) [38‐40]. Southern blotting revealed that the blaoxa-23 gene is plasmid-mediated in some STs (ST191, ST218 and ST368), but chromosome borne in others (ST195, ST208, ST369 and ST429). Chromosomal locations of blaoxa-23 make it less likely for A.baumannii to lose caarbapenem resistance. Investigation of OXA-23 producing A.baumannii isolates collected from 28 hospitals in 18 provinces of China showed that OXA-23 was mainly located on a ca.78-kb plasmid or on a chromosome [41].
Anzeige
We were concerned that A.baumanii isolates harboring the blaOXA-23 gene were multidrug resistant in our study and had few antibiotic therapeutic options for treating CRAB infection. Thus, controlling the spread of blaOXA-23 producing A.baumannii is important.
We identified ST191, ST195, ST208, ST218, ST368 and ST369 as classified into CC92 which was the largest and most widely distributed A.baumannii clone in China [17]. CC92 represented the most epidemic CRAB STs in this hospital, accounting for 86.1% of isolates in this study. For blaOXA-23-producing CRAB of CC92, the ability to disseminate in a single institution for a long time suggests that adaptation to the hospital environment may be important for the success of A.baumannii.
It has been suggested that any clinical A.baumannii isolates with resistance to multiple antibiotics can cause a nosocomial outbreak [42]. We found that imipenem-resistant A.baumannii of CC92, compared with other clonal complexes, may be more prone to cause severe outbreaks during long-term dissemination. Two outbreaks of CRAB CC92 were observed in our institution. Most ST191 isolates (31/32) were identified between February 2011 and May 2012 in the ICU ward, suggesting an outbreak of blaOXA-23-producing ST191. Deng’s group has reported the prevalence of an A.baumannii ST191 clone in a southern Chinese hospital [43]. To our knowledge, this is the first identification of an outbreak of blaOXA-23 harboring A.baumannii ST191 isolate in north China. A second outbreak of blaOXA-23-producing CRAB occurred in this ICU later. Thirty-one blaOXA-23-producing ST195 isolates were also found in the ICU between March 2013 and November 2014and this sequence type was more resistant to frequently-used antimicrobial agents compared with ST191. ST195 has frequently been identified in Asian countries, including Japan, Vietnam, and Malaysia [44‐46]. To our knowledge, Li’s group was first to identify ST195 in a teaching hospital in Guangzhou, in southern China [47]. Since then, ST195 clones has been identified in western and eastern China [38, 48]. Here, we offer the first report of an outbreak of blaOXA-23-producing ST195 in north China, suggesting that ST195 has been successfully disseminated in this country.
We collected no environmental strains from the work place, so we lack surveillance for source identification, which is a significant limitation of our study.
Anzeige
Conclusions
In summary, blaOXA-23-producing CC92 isolates were prevalent in this hospital over a 65-month period. Successive outbreaks of ST191 and ST195 demonstrated that persisting clinical carbapenem-resistant A.baumannii isolate can cause a nosocomial outbreak. Periodic investigation of molecular epidemiology and resistance determinant of A.baumannii is necessary.
Acknowledgements
This publication made use of the Acinetobacter baumannii MLST website (http://pubmlst.org/abaumannii/) sited at the University of Oxford (Jolley & Maiden 2010, BMC Bioinformatics, 11:595). The development of this site has been funded by the Wellcome Trust.
Funding
This work was supported by National Key Basic Research Program (973) of China (2015CB554202), the State Key Research Development Program Of China (2016YFC1200301), the National Science and Technology Major Project for Creation of Major New Drugs of China (2013ZX09304101),National Natural Science Foundation of China (81401643), the State Key Laboratory of Pathogen and BioSecurity Program (SKLPBS1530 and SKLPBS1424).
Availability of data and materials
The datasets generated during and/or analysed during the current study are available in the PubMLST repository, [http://pubmlst.org/abaumannii/].
Authors’ contributions
HW, EBC, FQ and TL designed and supervised the experiments. NZN, JH, CMB, SMC, EBC, JLZ and FHC performed experiments. NZN, XL and FQ interpreted and analyzed the data. NZN and XL wrote the paper. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Microbiology isolation and identification were routine work in our hospital. Only strains that have been routinely collected for diagnosis were studied. No extra sampling from the patients was performed. No personal information about patients was requested. Therefore, a written personal informed consent and ethics committee approval were not required and Chinese law was strictly complied.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Molecular epidemiology of bla OXA-23 -producing carbapenem-resistant Acinetobacter baumannii in a single institution over a 65-month period in north China
verfasst von
Nian-zhi Ning Xiong Liu Chun-mei Bao Su-ming Chen En-bo Cui Ju-ling zhang Jie Huang Fang-hong Chen Tao Li Fen Qu Hui Wang
Ob bei einer Notfalloperation nach Schenkelhalsfraktur eine Hemiarthroplastik oder eine totale Endoprothese (TEP) eingebaut wird, sollte nicht allein vom Alter der Patientinnen und Patienten abhängen. Auch über 90-Jährige können von der TEP profitieren.
Wenn unter einer medikamentösen Hochdrucktherapie der diastolische Blutdruck in den Keller geht, steigt das Risiko für schwere kardiovaskuläre Ereignisse: Darauf deutet eine Sekundäranalyse der SPRINT-Studie hin.
Insektenstiche sind bei Erwachsenen die häufigsten Auslöser einer Anaphylaxie. Einen wirksamen Schutz vor schweren anaphylaktischen Reaktionen bietet die allergenspezifische Immuntherapie. Jedoch kommt sie noch viel zu selten zum Einsatz.
Beginnen ältere Männer im Pflegeheim eine Antihypertensiva-Therapie, dann ist die Frakturrate in den folgenden 30 Tagen mehr als verdoppelt. Besonders häufig stürzen Demenzkranke und Männer, die erstmals Blutdrucksenker nehmen. Dafür spricht eine Analyse unter US-Veteranen.
Update Innere Medizin
Bestellen Sie unseren Fach-Newsletter und bleiben Sie gut informiert.