Molecular Characterization of Carbapenem-Resistant Enterobacter cloacae in 11 Chinese Cities

Carbapenem-resistant Enterobacteriaceae (CRE) are usually resistant to most of antibiotics. Infections caused by such bacteria have a high mortality and pose a serious threat to clinical management and public health. Enterobacter cloacae ranks third among Enterobacteriaceae that cause nosocomial infections. In this study, the molecular characteristics of carbapenem-resistant E. cloacae in China were investigated. From November 2012 to August 2016, 55 non-repetitive strains of carbapenem-resistant E. cloacae were collected from 12 hospitals in 11 Chinese cities. The bacteria were identified with matrix-assisted laser desorption/ionization time of flight mass spectrometry. Antimicrobial susceptibility tests were determined by agar dilution method. Carbapenemase and other β-lactamase genes were detected with PCR and sequencing. Multilocus sequence typing and plasmid conjugation tests were performed. Among the 55 E. cloacae strains, 50 strains were detected to produce 8 types of carbapenemase including NDM-1, NDM-5, IMP-4, IMP-26, IMP-1, KPC-2, and VIM-1. NDM-1 accounted for 68.0% (34/50) among the carbapenemase-producing E. cloacae. A total of 24 sequence types were identified and ST418 was the most common, accounting for 20% (11/55). For further investigation, a pulsed-field gel electrophoresis (PFGE) assay was conducted to identify the PFGE patterns of the strains. These 23 isolates yielded 13 PFGE patterns, which were designated as type A–M. Eight isolates obtained from Shenzhen had the same PFGE pattern (type A) and the remaining 15 isolates belonged to the other 12 PFGE patterns (type B–M). The observation that 8 of the 15 blaNDM−1-positive E. cloacae isolates obtained from Shenzhen with the same PFGE pattern (type A) suggested a transmission outbreak of a common strain. S1-nuclease PFGE and Southern blotting were also conducted to estimate the size of plasmids harbored by blaNDM−1-positive strains. The results showed that the plasmids harboring the blaNDM−1 gene ranged in size from approximately 52–58 kilobases. Our study indicates that carbapenem-resistant E. cloacae strains that produce NDM carbapenemase have strong resistance. Early detection and monitoring of the prevalence of these strains are urgent.


INTRODUCTION
In recent years, the emergence of carbapenem-resistant Enterobacteriaceae (CRE) has become a serious issue both on community-acquired infections and healthcare-associated infections (van Duin and Doi, 2017). As well as other Enterobacteriaceae, Enterobacter cloacae (E. cloacae) is a conditional pathogen found in the intestine. Healthcareassociated infections caused by E. cloacae ranked third among all the Enterobacteriaceae (Dai et al., 2013). Enterobacter cloacae can produce chromosome mediated AmpC β-lactamase and has resistance to ampicillin, amoxicillin/clavulanic, cephamycin and first and second generation cephalosporin. A wide spectrum of antibacterial drugs such as carbapenems may be used in treatment more often. Thus, multidrug resistance has emerged rapidly under antibiotic selection pressure. Carbapenem-resistant E. cloacae infections have been reported in many countries such as Spain, Australia, the United States, India, and China (Kiedrowski et al., 2014;Fernández et al., 2015;Liu et al., 2015;Sidjabat et al., 2015). The emergence of carbapenem-resistant E. cloacae is an enormous challenge to clinical treatment. It is well known that the main mechanism for reduced susceptibility to carbapenems in E. cloacae is the deregulation of ACT (the natural cephalosporinase of E. cloacae), which is associated with a decrease in membrane permeability. In addition to this, producing carbapenemases is another important mechanism of Enterobacteriaceae in carbapenem resistance (Walsh et al.,  2005; Nordmann et al., 2009;Tzouvelekis et al., 2012). Also, the mechanism of combinations of either ESBL or AmpC and mutation of porins may hold a certain proportion (Yang et al., 2010).
Up until now, there was a lack of multicenter research on carbapenem-resistant E. cloacae in China. So, we conducted this molecular epidemiological study on carbapenem-resistant E. cloacae to further understand the prevalence of the bacteria in China.

Plasmid Conjugation Test
The plasmid conjugation test was used to test carbapenemresistant gene transfer. Ten strains were selected for the test. Escherichia coli EC600 (rifampicin resistant) was used as the recipient. Conjugants were screened using China blue lactose agar plates containing rifampicin (300 µg/ml) and imipenem (1 µg/ml). The donor and the recipient were mixed at a ratio of 1:1 for 24 h. Transconjugants were selected on China blue lactose agar plates (OXOID, Basingstoke Hampshire, UK), supplemented with rifampicin (100 µg/ml) and imipenem (1 µg/ml). PCR was used to screen for bla NDM−1 , bla VIM−1 , bla KPC−2 , and bla IMP−1 as previously described (Wang et al., 2014).

Pulsed-Field Gel Electrophoresis (PFGE)
Enterobacter cloacae isolates were characterized by PFGE according to the previously published protocol by Ribot et al., with modifications (Ribot et al., 2002). We selected 23 bla NDM−1positive isolates (including the 15 isolates from Shenzhen, 6 isolates involved in the conjugation experiments, and another 2 representative isolates). Electrophoresis conditions were altered to have an initial switch time of 2.16 s and a final switch time of 54.17 s, and gels were run for 18 h. The resulting PFGE patterns were analyzed in BioNumerics software (Applied Maths, Austin, TX, USA) with dendrograms based on the Dice coefficient with a band position tolerance of 1%. Patterns with no discernible differences were considered indistinguishable and given the same PFGE pattern designation. S1-Nuclease PFGE and Southern Blotting S1-nuclease PFGE and Southern blotting were performed to estimate the size of plasmids harbored by bla NDM−1 -positive Frontiers in Microbiology | www.frontiersin.org strains as described previously. We selected 23 bla NDM−1positive isolates as mentioned above. The bla NDM−1 gene was detected by digoxigenin-labeled specific probes (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche Diagnostics, Mannheim, Germany). Salmonella enterica H9812 was used as a size marker.

Ethical Approval
This study was approved by the research ethics board at Peking University People's Hospital. Informed consent was not needed as this study was retrospective and participants were anonymized. Medical records and patient's information were retrospectively reviewed and collected.

PFGE, S1-Nuclease PFGE and Southern Blotting
When typed by PFGE to determine if they were related, the 23 isolates yielded 13 PFGE patterns, which were designated as type A-M. Eight isolates (ecl768, ecl771, ecl774, ecl776, ecl779, ecl782, ecl784, and ecl786) obtained from Shenzhen had the same PFGE pattern (type A) and the remaining 15 isolates belonged to the other 12 PFGE patterns (type B-M) ( Table 2).
The observation that 8 of the 15 bla NDM−1 -positive E. cloacae isolates with the same PFGE pattern (type A) and the same sequence type (ST418) suggested a transmission outbreak of a common strain.
The results of S1-nuclease PFGE and Southern blotting showed that the plasmids harboring the bla NDM−1 gene ranged in size from approximately 52-58 kilobases, respectively ( Table 2). The plasmids harboring the bla NDM−1 gene of the 8 isolates obtained from Shenzhen were the same size (approximately 52 kilobases).
The plasmid conjugation test was completed with 6 bla NDM−1 -positive strains. Conjugants were all detected to have the bla NDM−1 . Susceptibility results showed that compared with the receptor bacteria EC600, the conjugants have a higher MIC value on cephalosporins and carbapenems. There were no MIC promotions on polymyxin B and tigecycline between conjugants and EC600. Many studies have demonstrated that the plasmid owned bla NDM also have other resistant genes, such as bla TEM−1 , bla CMY , qnrA6, and qnrB1 for quinolone resistance, armA, rmtA, and rmtC for aminoglycoside resistance (Poirel et al., 2011a,b;Kocsis et al., 2016). But all strains in this study have no bla TEM−1 and bla CMY . Five of the conjugants have no MIC difference on quinolone with the EC600. Maybe the relative plasmid did not harbor the quinolone resistant gene.
MLST showed subtype diversity. A total of 24 sequence types were detected in 55 E. cloacae strains. ST418 was detected the most frequently (11/55, 20%), and the second was ST93 (14.5%, 8/55). Three new sequence types were found, namely ST916, ST919, and ST920. Our study reveals the diversity of carbapenem-resistant E. cloacae and the difference in genetic affinity, which is consistent with the study of Gomez-Simmonds et al. (2016). Our study showed that ST418 is the main epidemic strain in Shenzhen in China; while in America, Central de Asturias of Spain, and the Henan province of China, the main epidemic strains were ST171, ST74, and ST120, respectively (Fernández et al., 2015;Liu et al., 2015;Gomez-Simmonds et al., 2016). We found that ST418 was genetically closer to ST127 and ST755 with START2 analysis. Studies have found that all ST418 strains produced NDM-1 carbapenemase, indicating that there might be a small outbreak of NDM-1-ST418 carbapenem-resistant E. cloacae in Shenzhen and Dongguan City of Guangdong province of China. In this study, ST78-NDM-1type carbapenem-resistant E. cloacae was also found in Xuzhou, Xi'an, Wuhan, and Xiamen, which should be taken seriously concern.
In conclusion, our study indicates that ST418, which produces NDM-1 carbapenemase, is the main epidemic strain of carbapenem-resistant E. cloacae in Shenzhen and Dongguan City of China. Early detection and monitoring are necessary to prevent the further spread of the bacteria.

AUTHOR CONTRIBUTIONS
HW conceived and designed the study. CJ and JZ wrote this paper. CJ, QW, and JZ performed the experiments. QW and JZ analyzed the data. HC, XW, and YZ assisted CJ and JZ to finish the experiments. All authors approved the final version.

FUNDING
This study was supported by National Natural Science Foundation of China (grant No.81625014).