Dissemination of Carbapenemases (KPC, NDM, OXA-48, IMP, and VIM) Among Carbapenem-Resistant Enterobacteriaceae Isolated From Adult and Children Patients in China

This study aimed to investigate the dissemination and characteristics of blaKPC, blaNDM, blaOXA-48-like, blaIMP, and blaVIM among the carbapenem-resistant Enterobacteriaceae (CRE) strains isolated from adult and children patients. A total of 935 non-duplicate CRE strains were collected from 36 hospitals in 24 provinces or cities across China from 2016 to 2018. Antimicrobial susceptibility testing was performed by broth microdilution method and carbapenemase genes blaKPC, blaNDM, blaOXA-48-like, blaIMP, and blaVIM were screened by PCR and confirmed by DNA sequencing. Overall, carbapenemases were produced in 97.4% (911/935) of CRE strains, including KPC-2 (51.6%, 482/935), NDM (35.7%, 334/935), and OXA-48-like carbapenemases (7.3%, 68/935). Overall, the most prevalent carbapenemase gene was blaKPC-2 among Klebsiella pneumoniae (64.6%, 457/709) and the CRE strains isolated from adult patients (70.3%, 307/437), and blaNDM among Escherichia coli (96.0%, 143/149) and the CRE strains from children (49.0%, 247/498). The blaOXA-232-positive carbapenem-resistant K. pneumoniae (9.3%, 66/709) were all isolated from children. Sixteen strains were positive for blaIMP and 9 strains produced multiple carbapenemases. No strain was positive for blaVIM. Most of the CRE strains (>90%) were resistant to cephalosporins and carbapenems, more than half (>50%) were resistant to aminoglycosides and fluoroquinolones, but the majority (95.8 and 98.4%) were susceptible to polymyxin B and tigecycline. Ceftazidime-avibactam showed excellent in vitro activity against blaKPC-2 and blaOXA-48-like positive strains (100% susceptible). In China, KPC-2, NDM, and OXA-48-like carbapenemases were predominant among CRE clinical isolates. The most prevalent carbapenemase gene was blaKPC-2 among K. pneumoniae isolates from adult patients, and blaNDM among E. coli isolates from children.


INTRODUCTION
Enterobacteriaceae are opportunistic pathogens causing severe hospital-acquired infections (Feil, 2016). The spread of carbapenemase-producing Enterobacteriaceae (CPE) has been a global threat to public health. Carbapenems have conventionally been used for treating infections caused by extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae, and are still considered as last resort antibiotics to date (van Duin and Doi, 2016). According to the data from China Antimicrobial Surveillance Network (CHINET, www.chinets.com), the resistance rate of K. pneumoniae to meropenem and imipenem rapidly increased from 2.9 and 3.0% in 2005 to 26.3 and 25% in 2018, respectively. In Europe, carbapenem-resistant K. pneumoniae are most widespread in the Mediterranean and Balkan countries with a prevalence of 60% in Greece and 40% in Italy, respectively (Perez and Villegas, 2015;Feil, 2016). The production of carbapenemases including KPC, NDM, and OXA-48-like is the most common resistance mechanism among carbapenem-resistant Enterobacteriaceae clinical isolates (Nordmann et al., 2012;Goodman et al., 2016). The bla KPC -positive Enterobacteriaceae were widespread in the United States, Latin America, Italy, Greece, the Middle East, and China (Albiger et al., 2015;Feil, 2016;Villegas et al., 2016;Iovleva and Doi, 2017). The bla NDM -positive Enterobacteriaceae were widespread in India, Pakistan, Bangladesh, Italy, Poland, Denmark, Latin America, and African countries (Yong et al., 2009;Albiger et al., 2015;van Duin and Doi, 2016). The bla OXA−48−like -positive strains remained rare in the US, in contrast to the prevalence in Turkey, Spain, France, Belgium, Romania, Middle East, Africa, Asia, and South America as well (Albiger et al., 2015). These infections are usually associated with very poor prognosis and high mortality, especially in neonates or high-risk immunocompromised patients (Falagas et al., 2014;Feil, 2016). In China, the presence of bla KPC and bla NDM is responsible for phenotypic resistance in most of the CRE strains Wang et al., 2018). Most researches currently focus on the dissemination of carbapenemases among CRE strains isolated from adult patients, while only a few are available to investigate the distribution of carbapenemases among CRE strains isolated from children. To obtain the comprehensive characteristic of carbapenemases among CRE isolated from both adults and children patients in China, we conducted this study to characterize the dissemination and characteristics of carbapenemases (including KPC, NDM OXA-48, IMP, and VIM) among CRE clinical isolates and the susceptibility to antimicrobial agents.

Detection of Carbapenemase and mcr-1 Genes
All the CRE strains were tested for the presence of the most common carbapenemase genes (bla KPC , bla NDM , bla OXA-48-like , bla IMP , and bla VIM ) by polymerase chain reaction (PCR) with specific primers and conditions as described previously (Poirel et al., 2011;Liu et al., 2016). The colistin resistance gene mcr-1 was also detected by PCR, as previously described (Liu et al., 2016). The positive PCR amplicons were sequenced and compared with the reported sequences from GenBank by Blast (www.ncbi.nlm.nih.gov/blast/).

Statistical Analysis
Descriptive statistics were used to summarize the epidemiologic characteristics of CRE strains. For categorical variables, the percentage of CRE strains in each category was calculated. All analyses were performed using WHONET (version 5.6) and the IBM SPSS Statistics (version 21).

DISCUSSION
Previous studies have proved that the presence of carbapenemase genes, including bla KPC-2 and bla NDM , was the major mechanism of carbapenem resistance among CRE strains in China, which   were the most prevalent in K. pneumoniae and E. coli, respectively Wang et al., 2018). However, the researches on CRE strains isolated from children patients are limited in China. This study provided a comprehensive and updated carbapenemase profile of 935 CRE strains isolated from both adult and children patients. We found that bla KPC-2 (51.6%) and bla NDM (35.7%) were the most common carbapenemase genes among CRE strains, while the emergence of bla OXA-232 , bla IMP , and other multi-carbapenemase genes have been increasing in recent years. KPC-2 was the most frequently detected carbapenemase gene in K. pneumoniae, while NDM was the most prevalent one in E. coli. This pattern in China is significantly different from that in Europe. In Europe, the prevalence of OXA-48-like producing Enterobacteriaceae was 38% (333/927), next to KPC-(42%, 393/927), but higher than NDM-producing Enterobacteriaceae (12%, 113/927) (Grundmann et al., 2017). The distribution of carbapenemase-producers also varied with bacterial species. In K. pneumoniae, KPC-producers were the most prevalent, followed by OXA-48-like (37%, 310/850) and NDM-producers (11%, 93/850). In E. coli, OXA-48-like consistently bactericidal against NDM-5-bearing mcr-1-positive E. coli, which might be an alternative therapeutic option for the treatment of lethal infections .

CONCLUSIONS
In conclusion, KPC-2, NDM, and OXA-48-like enzymes were the most prevalent carbapenemases among CRE clinical isolates in China. The most prevalent carbapenemase gene was bla KPC-2 among K. pneumoniae isolated from adult patients, and bla NDM among E. coli isolates from both children and adult patients. The bla OXA-232 was only detected among K. pneumoniae isolates from children.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
The study protocol was approved by the Institutional Review Board of Huashan Hospital, Fudan University (Number: 2018-408).

AUTHOR CONTRIBUTIONS
FH and RZ designed the study. RH, QS, SW, and MP performed the experimental work. RH and DY collected the data. FH analyzed the data. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by the National Natural Science Foundation of China (grant nos. 81871690, 81902101, and 81861138051), the National Mega-project for Innovative Drugs (2019ZX09721001-006-004) and China Antimicrobial Surveillance Network (WI207259).