Transmission and characterization of blaNDM-1 in Enterobacter cloacae at a teaching hospital in Yunnan, China

In recent years, New Delhi metallo-beta-lactamases 1 (blaNDM-1) has been reported with increasing frequency and become prevalent. The present study was undertaken to investigate the epidemiological dissemination of the blaNDM-1 gene in Enterobacter cloacae isolates at a teaching hospital in Yunnan, China. Antimicrobial susceptibility testing was performed using VITEK 2 system and E test gradient strips. The presence of integrons and insertion sequence common region 1 were examined by PCR and sequencing. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Conjugation experiments and Southern blot hybridization were performed to determine the transferability of plasmids. Ten E. cloacae isolates and their Escherichia coli transconjugants were exhibited similar resistant patterns to carbapenems, cephalosporins and penicillins. 8 (80%) of E. cloacae isolates carried class 1 integron and 1 (12.5%) carried class 2 integron. Integron variable regions harbored the genes which encoded resistance to aminoglycosides (aadA1, aadA2, aadA5, aadB, aac(6′)-Ib-cr), sulfamethoxazole/trimethoprim (dfrA17, dfrA12, dfrA15) and Streptozotocin (sat2). Six E. cloacae isolates belonged to ST74 and exhibited highly similar PFGE patterns. Each isolate shared an identical plasmid with ~33.3 kb size that carried the blaNDM-1 gene, except T3 strain, of which the blaNDM-1 gene was located on a ~50 kb plasmid. Our findings suggested that plasmid was able to contribute to the dissemination of blaNDM-1. Hence, more attention should be devoted to monitor the dissemination of the blaNDM-1 gene due to its horizontal transfer via plasmid. In addition, nosocomial surveillance system should actively monitor the potential endemic clone of ST74 to prevent their further spread.


Background
Enterobacter cloacae is an important nosocomial pathogen, which can cause various infections including urinary tract, skin and soft tissue, respiratory tract, surgical site, biliary tract, sepsis, intravenous catheters, central nervous system and outbreaks at neonatal units [1,2]. New Delhi metallo-beta-lactamases 1 (bla NDM-1 ) are Ambler class B Metallo-β-lactamases(MBLs)with carbapenemase activity that confers resistance to all β-lactams except aztreonam, was first identified in a carbapenem-resistant Klebsiella pneumoniae strain recovered from a Swedish patient who was hospitalized in India in 2008 [3], and mainly detected in carbapenem-resistant Acinetobacter spp. in mainland China. However, NDM-1-mediated carbapenem resistance in E. cloacae has been rarely reported in China.
The widespread dissemination of bla NDM-1 is mainly due to plasmids, integrons, insertion sequence common region (ISCR) and clonal outbreaks [4]. Plasmids are extrachromosomal DNA molecules capable of autonomous replication, and can confer resistance

Annals of Clinical Microbiology and Antimicrobials
to the major antimicrobials [5]. Integrons are bacterial genetic elements able to capture and express genes contained within mobile gene cassettes. Typically, integrons are composed of two conserved regions, a 3′ conserved segment (3′CS) and a 5′ conserved segment (5′CS), as well as an internal variable region containing gene cassettes that encode antimicrobial resistance determinants [6]. ISCR elements can transpose adjacent DNA sequences by a process called rolling-circle replication, now it is recognized as powerful antibiotic resistance gene capture systems and playing a major role in spread of antibiotic resistance genes. However, whether these mobile elements mediate the dissemination of bla NDM-1 gene is still an unsolved mystery in the region. Therefore, the aim of this study is to explore the epidemiological dissemination of the bla NDM-1 gene in E. cloacae isolates at a teaching hospital in Yunnan, China.

Bacterial strains
In total, ten NDM-1-producing E. cloacae isolates were collected from hospitalized patients in the First Affiliated Hospital of Kunming Medical University between June 2012 and January 2016. All isolates were identified by VITEK2 automated identification system (bioMerieux, France). Carbapenemase activity was assessed by the modified Hodge test, MBL production was examined by imipenem-ethylenediaminetetraacetic acid (EDTA) disk method and the bla NDM-1 gene was determined by PCR in the early stage of the study. All patients were of Yunnan descent and none had a recent history of travelling epidemic area (Table 1).

Antimicrobial susceptibility testing
Antimicrobial susceptibilities for the NDM-1-producing isolates and transconjugants were initially tested using the VITEK2 system. MICs of imipenem, meropenem and ertapenem were re-evaluated using E test gradient strips (bioMerieux, France) on Mueller-Hinton agar plates and the results interpreted according to the CLSI guidelines [7]. Escherichia coli ATCC 25922 was used as quality control strain.

Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)
Bacterial genomic DNA was prepared in agarose plugs and digested with the restriction enzymes XbaI (Promega, USA). The DNA fragments were separated by use of a CHEF Mappar XA PFGE system (Bio-Rad, USA), with running time of 22 h and pulse times ranging from 5 to 40 s. The running buffer was 0.5 × Tris-boric acid-EDTA (TBE). Salmonella ser. Braenderup H9812 was used as a standard for comparison. PFGE patterns were compared visually following previously described criteria [12]. Multilocus sequence typing (MLST) was performed on representative isolates. Internal fragments of the seven housekeeping genes were amplified using the primers given at the Institute Pasteur MLST Databases web site (http://pubmlst.org/ecloacae/). The PCR products were sequenced. Sequence types (STs) were assigned using online database tools.

Conjugation experiments and Plasmid analysis
The transfer of carbapenem resistance was tested using a conjugation test, E. coli 600 (rifampicin-resistant) was used as the recipient strain, NDM-1-producing E. cloacae clinical isolates were used as the donor strains. Donor and recipient cells from M-H broth cultures were mixed in a ratio of 2:1 and transconjugant clones were screened on M-H agar plates containing rifampicin (256 mg/L) and Imipenem (1 mg/L). Conjugation events occurred at 37 °C. The presence of the bla NDM-1 gene in transconjugants was determined by PCR and sequencing. Genomic DNA was digested with S1 nuclease (Promega, USA). The linearized plasmids and partially digested genomic DNA were separated through the CHEF-Mapper XA PFGE system with a switch time from 2.16 to 63.8 s for 18 h at 6 V/cm at 14 °C. Linear plasmids generated by S1-PFGE were transferred to nvlon membrane (Millipore, USA) and hybridized with a digoxigenin-labeled probe specific to bla NMD-1 . Probe labeling and signal detection were carried out with DIG high primer DNA labeling and detection starter kit according to the manufacturer's instructions (Roche Applied Sciences, Germany).

Antimicrobial susceptibility testing
The antibiotic susceptibility results showed that all the NDM-1-producing E. cloacae isolates exhibited resistance to carbapenems, cephalosporins and penicillins. Only one isolates remained susceptible to aztreonam, which was not hydrolysed by metallo-carbapenemases, thus suggesting the presence of additional β-lactamases in the remaining isolates. Ten isolates exhibited different level resistance to tetracycline, amikacin, ciprofloxacin and tigecycline, seven isolates were resistance to tetracycline, one to amikacin, nine to ciprofloxacin and one to tigecycline. These results are summarized in Table 3.

PFGE and MLST typing
According to Tenover's criteria [12], Six E. cloacae isolates, of which four obtained from transplantation center, one from general practice and one from emergency intensive care unit, belonged to ST74 and shared the same PFGE fingerprint pattern (Fig. 1), suggesting they were clonally related, the remaining strains were

Plasmid analysis
Conjugation experiments revealed that plasmids harboring bla NDM-1 were transformed into E. coli 600. All transconjugants conferred resistance to carbapenems, cephalosporins and penicillins while all of them remained susceptible to ciprofloxacin and amikacin (Table 1).
Isolates harbored three to five plasmids according to S1-PFGE electrophoresis. Southern hybridization analysis with a bla NDM-1 -specific probe revealed that bla NDM-1 was located on a ~33.3 kb plasmid in nine isolates and on a ~50 kb plasmid in one isolates (Fig. 2).

Discussion
The bla NDM-1 was first identified in a clinical isolate of K. pneumoniae in New Delhi, India, and suddenly got disseminated around the world. The bla NDM-1 -carrying  bacteria conferred resistance to all most β-lactam antibiotics. Thus, treatment of infections with NDM-producing bacteria was a major challenge, leaving few options for clinical treatment beside tigecycline or colistin. The bla NDM-1 gene have been identified in a variety of gramnegative bacilli, including Acinetobacter spp. [13], Enterobacteriaceae [14] and Pseudomonas aeruginosa [15]. Lack of uncontaminated potable water and abuse of over-thecounter antibiotic administration offered the ideal setting for the development of a latent endemic situation, and international "medical tourism" played an important role in the spread of bla NDM-1 gene [16]. Yunnan Kunming is a famous tourist city in the world, and large numbers of domestic and foreign tourists come here every year, thus "medical tourism" may play an important role in the spread of bla NDM-1 among carbapenem-resistant bacteria in this region. In China, plasmid-carrying bla NDM-1 have been identified in Enterobacteriaceae isolates in several regions including Beijing, Shanghai, Hong Kong, Shandong Henan and Yunnan province, the size of plasmids harboring bla NDM-1 were vary from ~50 to 360 kb [17,18]. Our research shows that bla NDM-1 gene was mainly located on a plasmid with ~33.3 kb size, were different from previously reported in China before. The bla NDM-1 gene was not detected in integrons and ISCR1 mobile elements. Integrons only carried these genes encoding resistance to aminoglycosides [aadA1, aadA2, aadA5, aadB, aac(6′)-Ib-cr], sulfamethoxazole/trimethoprim (dfrA17, dfrA12, dfrA15) and Streptozotocin (sat2). Those results suggested that plasmid was the most important mobile element that mediated the dissemination of bla NDM-1 and integrons were the basis for the formation of multiple drug-resistant bacteria in the region. In addition, it was worth noting that there had been some evidences which demonstrated that integrons and ISCR mobile elements were located on plasmids [4], thus, the resistance gene through the plasmids spreading was not a single element, but by 1 + 1 or 1 + 2 mode. This mode of dissemination should arouse high concern of the relevant departments.
Clonal spread was an important factor involved in the prevalence of NDM-1-producing Enterobacteriaceae. Outbreak of NDM-1-producing K. pneumoniae ST105 and ST147 have been reported in Yunnan [17] and Xi'an [19], and NDM-1-producing E. cloacae ST120 have been reported in Henan [18], China, respectively. Our study showed that 5 clusters for 10 strains, 1 cluster from 6 closely related isolates was found to exhibit similarities which is more than 90%. The result suggested that 6 NDM-1-producing E. cloacae isolates were clonally related. However, there was no significant epidemiological relatedness among them. Hence, we could not trace their origin. MLST analysis revealed four ST types including ST74, ST754, ST175 and ST182, among them, ST74 was the major type in NDM-1-producing E. cloacae isolates, which was different from previous types reported before such as ST66, ST78, ST108, ST114 and ST120 [18]. ST74 has been identified among non-susceptible to ertapenem of E. cloacae isolates in North-Eastern France, and was associated with OXA-48-producing E. cloacae isolates in Spain [20,21]. The present study is the first to report on an outbreak of NDM-1-producing E. cloacae ST74.
Infection control measures were strengthened to prevent the further transmission of bla NDM-1 . They included hand hygiene, contact isolation, active screening, environmental surface disinfection, standard aseptic manipulation techniques, among them, hand hygiene was the most effective and economical strategy for reducing cross infection [16].