Molecular Characteristics of First IMP-4-Producing Enterobacter cloacae Sequence Type 74 and 194 in Korea

The worldwide dissemination of carbapenemase-producing Enterobacteriaceae (CPE) has become a major therapeutic concern in clinical settings. Enterobacter cloacae is a major pathogen that causes serious hospital-acquired infections. We investigated the clinical characteristics and molecular mechanisms of the first IMP-4-producing E. cloacae clinical isolates in Korea. Five carbapenemase-producing E. cloacae strains out of 792 E. cloacae clinical isolates, which have been identified at a university hospital in Korea between March 2014 and February 2016, were included in this study. Antimicrobial susceptibilities to imipenem, meropenem, and ertapenem were tested using E-test. Carbapenemase determinant screening, genetic environment, and multilocus sequence typing were conducted using PCR and sequencing analysis. All isolates were not susceptible to at least one of the tested carbapenems and presented highly similar pulsed-field gel electrophoresis (PFGE) patterns, evidencing hospital-wide clonal dissemination. Among all isolates harboring the blaIMP-4 carbapenemase gene, four isolates identified as predominant ST74, also contained blaCMY−2. One strain, designated as rare ST194, carried blaCMY-1. The E. cloacae strain, harboring both blaIMP-4 and blaCMY-1, was resistant to all three tested carbapenems. The blaIMP-4 gene was located on a highly mobile class 1 integron, showing a new form of the blaIMP-4-qacG-aacA4 array. This is the first description of IMP-4-producing E. cloacae strains in Korea. This observation implicates the widespread of blaIMP-4 in Enterobacteriaceae clinical isolates and provides insights into the epidemic potential and clinical therapeutic importance of IMP-4-producing E. cloacae for healthcare-associated infections.

Until now, carbapenem-resistant E. cloacae has rarely been reported in Korea since the initial VIM-2-producing isolate in 2003 (Jeong et al., 2003). Here, we described the clinical characteristics and molecular mechanisms of the first IMP-4producing E. cloacae clinical isolates in Korea.

Bacterial Strains
A total of 792 E. cloacae clinical isolates have been identified at a university hospital in Korea between March 2014 and February 2016. Among the isolates, five carbapenemaseproducing E. cloacae strains (0.6%), YUMC1, YUMC2, YUMC3, YUMC4, and YUMC5 were included in this study. The isolates were identified as E. cloacae using the Vitek GNI card (bioMérieux, Marcy l'Étoile, France) and 16S rRNA sequencing (Lane et al., 1985;Mao et al., 2012;Mezzatesta et al., 2012;Jeong et al., 2015). This study was carried out in accordance with the recommendations of Institutional Review Board of Kosin University Gospel Hospital, Busan, Korea; with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. We primarily focused on the analysis of the isolated strains and made our effort to anonymize private information of infected patients.

Pulsed-Field Gel Electrophoresis
Pulsed-field gel electrophoresis (PFGE) was performed to confirm the clonality of the IMP-4-producing E. cloacae isolates. XbaI (Roche, Mannheim, Germany)-digested genomic DNA was prepared at 37 • C for 12-14 h. DNA fragments were separated using a CHEF-DRII System (Bio-Rad, Hercules, CA, USA). Banding patterns were analyzed with InforQuestFP software version 4.5 (Bio-Rad) to generate a dendrogram.

Polymerase Chain Reaction and Sequencing
The genomic DNA of five isolates were extracted via the boiling lysis method (L. Chen et al., 2011). The genes for 16S rRNA, carbapenemase, integron components, fluoroquinolones, ESBLs, and plasmid-mediated AmpCs were amplified by polymerase chain reaction (PCR) and sequenced using the primers (Lane et al., 1985;Jeong et al., 2003;Bae et al., 2007Bae et al., , 2011Mao et al., 2012;Hong et al., 2015) described in Table 1. Briefly, the PCR program was as follows: 94 • C denaturation for 5 min, followed by 30 cycles of 94 • C denaturation for 30 s, then 55-60 • C annealing for 30 s, and subsequently 72 • C extension for 30 s, followed by 72 • C final extension for 7 min. The amplified products were sequenced and the nucleotide sequences were compared by the Basic Local Alignment Search Tool (BLAST) (https://www.ncbi. nlm.nih.gov/BLAST) . Genetic organization of class 1 integron carrying the bla IMP-4 gene cassette of a plasmid was investigated by PCR mapping and sequencing of the regions surrounding the gene using the primers described in

Description of the Patients
The clinical characteristics of the patients infected with five isolates are summarized in  overall patterns are similar to those of five IMP-4-producing isolates, except for the carbapenems.

Resistance to Carbapenems
All five isolates were positive as carbapenem producers in the modified Hodge test and KPC+MBL Confirm ID Kit (Rosco Diagnostica). The MICs were determined using E-test strips (AB Biodisk) and the results for imipenem, meropenem, and ertapenem are presented in Table 3. All isolates were not susceptible to at least one of the carbapenems using CLSI breakpoints. Notably, YUMC2 was resistant to all tested carbapenems and had higher MICs than other isolates.

Clonality of the Isolates
YUMC4 and YUMC5 strains presented identical PFGE patterns and the other isolates also showed highly similar patterns based on the criteria of 85% similarity ( Table 3). The strains, isolated same years, presented close relationship.

Sequence Type
The MLST assay assigned the isolates to two STs (Table 3). YUMC2 was assigned to predominant ST74. Four out of the five IMP-4-producing E. cloacae strains were rare ST194, showing significant clonal similarity.

DISCUSSION
E. cloacae is frequently implicated in serious nosocomial infections with high mortality. Majority of patients were reported to be immunocompromised, similar to our patients (Qureshi et al., 2011). Clinical outbreaks of E. cloacae in the hematology ward, burns unit, and intensive care unit have persisted, despite of concerted infection control to prevent ongoing transmission (Leung et al., 2013;Chapuis et al., 2016;Pang et al., 2016). VIM-2, NDM-1, and IMP-1, frequently found in Asia, have been previously reported mechanisms of carbapenem-resistant E. cloacae in Korea (Jeong et al., 2003;Kim et al., 2015;Lee et al., 2017). Meanwhile, IMP-4-producing E. cloacae isolates have been mainly found in Australia (Peleg et al., 2005;Leung et al., FIGURE 2 | Schematic representation of the class 1 integron gene cassettes bearing the bla IMP-4 genes in E. cloacae isolates. Genes and their directions of transcription are described as broad arrows. The gray box indicates recombination site. The primers, detailed in Table 1, for PCR mapping are depicted as narrow arrows with numbers. The red arrow of bla IMP-4 is related to carbapenemase. The yellow arrow of qacG and aacA4 are associated with resistance to quaternary ammonium compounds and fluoroquinolone, respectively. The 5 ′ conserved segment (CS) of IntI1 and 3 ′ CS of sul1 are presented with green arrow. (A) E. cloacae YUMC2 in this study with Genbank accession no. KY884003. (B) E. cloacae EI1573 from Sydney, Australia with Genbank accession no. JX101693.1. (C) E. cloacae from Queensland, Australia reported by Sidjabat et al. (2015). 2013). The first detection of IMP-4 in this study implicates that the plasmid-mediated bla IMP-4 eventually spread in E. cloacae clinical isolates in Korea.
IMP-4 was reported to be strongly active against imipenem and meropenem, with 0.25-16 MIC range (Chu et al., 2001). The MICs of our isolates showed that all five strains were not susceptible to at least one of the carbapenems, including imipenem, meropenem, and ertapenem. Antibiotic resistance profiles of bla IMP -positive Enterobacteriaceae isolates showed 25% resistance, 57% intermediate resistance, and 18% susceptibility to meropenem and 6% resistance, 33% intermediate resistance, and 61% susceptibility to imipenem in a previous study (Dolejska et al., 2016). Natural antibiotic susceptibility of E. cloacae complex to carbapenems were reported to be susceptible (Stock et al., 2001), however, the presence of IMP-4 would influence on the antibiotic profiles.
The antimicrobial susceptibility profiles of E. cloacae isolates in this study were similar to the intrinsic patterns of antibiotics (Mezzatesta et al., 2012). However, 5 strains containing bla  were not susceptible to carbapenems and YUMC2 was resistant to ciprofloxacin. The detected genes, aac(6 ′ )-Ib-cr and qnrS1 relevant to fluoroquinolones might be associated with this results. Nevertheless, the cr variant of aac(6 ′ )-Ib confers reduced susceptibility to ciprofloxacin by N-acetylation of its piperazinyl amine , ciprofloxacin resistance was not related to aac(6 ′ )-Ib-cr prevalence . Interestingly, the isolates co-carrying aac(6 ′ )-Ib-cr and qnrS1 were also reported to be sensitive to quinolones (Huang et al., 2012). Therefore, these genes seems to supplement other quinolone resistance mechanisms rather than confer directly to resistance. Although, the aac(6 ′ )-Ib-cr and qnrS1 genes were frequently found to be co-carried with various ESBLs, becoming therapeutic threats (Huang et al., 2012;Mezzatesta et al., 2012), our isolates harbored bla IMP-4 without ESBLs.
The homogeneity of five strains was analyzed using PFGE. Although the strains were isolated from various clinical departments, the high similarity of PFGE patterns of isolates, especially in the same years, might be the evidence of hospitalwide clonal dissemination.
According to MLST results, YUMC2 was designated to ST74, the most predominant clonal lineage with increased epidemic potential based on previous E. cloacae clonality studies (Fernández et al., 2015;Guillard et al., 2015;Izdebski et al., 2015). E. cloacae ST74 had higher carbapenems MICs than other isolates, similar to the results of previous studies, and was assumed to confer with the spread of the resistance to carbapenems (Guillard et al., 2015;Izdebski et al., 2015). The other four IMP-4-producing E. cloacae strains were ST194, presenting significant genetic similarity. To the best of our knowledge, available studies for E. cloacae ST194 were rare, indicating that this is the first report of clinical E. cloacae ST194.
PCR results showed the presence of CMY-1 in YUMC2 and CMY-2 in the other strains as well as IMP-4. Prior studies demonstrated that the most frequently reported AmpC βlactamase was CMY, consisting of 92.7% of CMY-2 among Enterobacteriaceae isolates in the Asia-Pacific region (Sheng et al., 2013). The combination of bla IMP-4 and bla CMY−2−like was found from one clinical E. cloacae isolate among the CPE in Australia (Sidjabat et al., 2015). In addition, the coexistence of bla IMP-4 and bla CMY-1 in E. cloacae strain was not reported previously and this is the first description of E. cloacae, coproducing IMP-4 and CMY-1 with resistance to all three carbapenems.
When comparing the product of sequencing of our study to E. cloacae pEI1573 (GenBank accession no. JX101693.1) (Partridge et al., 2012), both of the bla IMP-4 genes of our study and pEI1573 were located on class 1 integrons. However, the gene cassettes compositions were slightly different between YUMC2 and pEI1573, containing a reference sequence of typical Australian class 1 integron array (Figure 2). The bla IMP-4 -qacG-aacA4-catB3 cassette array of pEI1573 from Sydney, Australia is almost identical to those of pJIBE401 from Sydney index isolate K. pneumoniae (GenBank accession no. AJ609296) (Espedido et al., 2005), pCTX-M3 from Citrobacter freundii in Poland (GenBank accession no. AF550415) (Gołebiewski et al., 2007), and pCTX-M360 from K. pneumoniae in China (GenBank accession no. EU938349) (Zhu et al., 2009). Meanwhile, the class 1 integron of our study consisted of bla IMP-4 -qacG-aacA4 and a different array form composed of bla IMP-4 -aacA4, which was reported previously from Queensland, Australia (Sidjabat et al., 2015). These cassette arrays, found in diverse isolates with slightly different genetic contexts, suggest movement of the array by homologous recombination and the worldwide dissemination potential of bla IMP-4 gene.
In the respect of epidemiological relationship, the class 1 integrons of Australia and Korea, containing bla IMP-4 genes of E. cloacae isolates, revealed similar gene cassettes, except for catB3 or qacG. Geographically, Australia and Korea are located at the rim of Asian-pacific region. Further, a largescale transmission of bla IMP-4 of E. cloacae isolates, predominant from of CPE in Australia (Sidjabat et al., 2015), through silver gulls of Australia was previously reported (Dolejska et al., 2016).
In conclusion, we report the first IMP-4-producing E. cloacae strains identified as predominant ST74 and rare ST194 in Korea. Furthermore, it is the first description of bla IMP-4 and bla CMY-1 coexistence and a new class 1 integron cassette array form in Enterobacteriaceae. This finding implicates the emergence of plasmid-mediated bla IMP-4 on the highly mobile class 1 integron in Enterobacteriaceae clinical isolates in Korea with great concern for widespread and therapeutic threats. In addition, it provides insights into the epidemic potential and clinical importance of IMP-4-producing E. cloacae for hospitalacquired infections.

AUTHOR CONTRIBUTIONS
SJ: analyzed the data, and wrote the manuscript; IKB: designed and performed the experiments, and revised the manuscript; JHL and CHL: helped the experiments and the writing of the manuscript.