Prevalence and mechanisms of carbapenem resistance among Klebsiella aerogenes in a tertiary hospital in China

Background: Klebsiella aerogenes has emerged as one of the most important nosocomial pathogens for patients in intensive care units (ICU) in recent years. This study aims to evaluated the prevalence and molecular characteristics of clinical carbapenem-resistant K. aerogenes (CRKA) isolates in a tertiary hospital in China. Results: Twenty CRKA were identied among all the isolates, with the rate of 5.5% (20/364). Six CRKA isolates produced KPC-2 and 1 CRKA isolate produced NDM-1. PFGE and MLST indicated that the 20 CRKA strains were clonal diversity. All the bla KPC-2 gene and bla NDM-1 gene were located on plasmids and all the plasmids with bla KPC-2 and bla NDM-1 genes could successfully transferred to EC600 or J53. Twelve of 13 CRKA strains without any carbapenemase genes were positive for eux pump inhibition test. Conclusion: Overall, the prevalence of CRKA in the tertiary hospital in Zhejiang Province of China is 5.5%. Only 35% of CRKA produce carbapenemase and eux pumps might play an important role in the carbapenem resistance of K. aerogenes. It is necessary to strengthen the surveillance of carbapenem resistance in the hospital to prevent the horizontal and clonal spread of CRKA. CRKA once caused an epidemic and outbreak in hospitals of China [9, 21]. In our study, S1-PFGE followed by Southern blot demonstrated that all the bla KPC−2 and bla NDM−1 genes were all located at plasmids. the lter mating experiments conrmed that all the bla KPC−2 and bla NDM−1− carrying plasmids could transfer to the recipient bacteria. of the shows that plasmids play important roles in the transmission of carbapenemase genes and indicate the potential threat of outbreak and prevalence of CRKA. Eux pump inhibition test In addition, to investigate the role of eux pumps in the carbapenem resistance mechanisms, the MICs of carbapenems were determined in the presence of the eux pump inhibitor Phe-Arg β-naphthylamide dihydrochloride (PAβN:25ug/ml) [27] and carbonyl cyanide m-chlorophenylhydrazone (CCCP:25ug/ml) [9], respectively. A 4-fold or greater reduction in the MIC values after addition of CCCP or PAβN was considered as signicance [28].

(92.3%, 12/13) of the CRKA strains, respectively. This indicated that the activation of e ux pumps might contribute to the carbapenem resistance of CRKA isolates (except the isolate 33127) (Table3).
aerogenes is the fth highest Enterobacteriaceae and the seventh highest Gram-negative Bacillus responsible for notorious nosocomial infections in France [17].
Carbapenamase production is one of the main resistance mechanisms of CRE. Carbapenemases consist of ambler class A or D serine β-lactamases and ambler class B metallo-β-lactamases (MBLs). K. pneumoniae carbapenemase (KPC, Class A) is the most common carbapenemase in carbapenemase producing Enterobacteriaceae (CPE) [3]. It was rst discovered in the USA in 1996 [18], and has spread worldwide since then. Recently years, KPCproducing Enterobacteriaceae have become a public health concern worldwide, including in China [19,20]. However, in this study, only six CRKA and one CRKA were identi ed as producing KPC-2 and NDM-1. Further analysis of PFGE and MLST revealed that most of the KPC-2-producing CRKA were nonclonal spread, except two CRKA (34366 and 34602), which were isolated from the same department. Several study had reported that KPC-2-producing CRKA once caused an epidemic and outbreak in hospitals of China [9,21]. In our study, S1-PFGE followed by Southern blot demonstrated that all the bla KPC−2 and bla NDM−1 genes were all located at plasmids. Furthermore, the lter mating experiments con rmed that all the bla KPC−2 and bla NDM−1− carrying plasmids could transfer to the recipient bacteria. All of the above shows that plasmids play important roles in the transmission of carbapenemase genes and indicate the potential threat of outbreak and prevalence of CRKA.
In addition to carbapenemases, other mechanisms such as the overexpression of e ux pumps contributing to the carbapenem resistance in K. pneumoniae are now well documented [22]. The e ux pump inhibitors PAβN and CCCP are active against RND pumps in Gram-negative bacteria, including E. cloacae, E. coli, K. pneumoniae, P. aeruginosa, S. enteric and so on [23]. Despite of the potential toxic effect of PAβN and CCCP on bacteria, concentrations of 25 µg/ml of PAβN and 25 µg/ml of CCCP could not kill bacteria [23,24]. Our results showed that the MICs of CRKA isolates could decreased signi cantly in the presence of PAβN and CCCP, indicating that e ux pumps might play an important role in the carbapenem resistance of K.
aerogenes. Moreover, there were still 1 isolates of CRKA without carbapenase and had no e ux pump inhibitor inhibition phenotype in the present study. Further research is needed to con rm the mechanisms of carbapenem resistance.

Conclusion
Overall, the prevalence of CRKA in the tertiary hospital in Zhejiang Province of China is 5.5% during the years from 2011 to 2017. Only 35% of CRKA produce carbapenemase and e ux pumps might play an important role in the carbapenem resistance of K. aerogenes. bla KPC−2 and bla NDM−1 are major carbapenemase genes and always transfer through plasmids. It is necessary to strengthen the surveillance of carbapenem resistance in the hospital to prevent the horizontal and clonal spread of CRKA.

Methods
Bacterial isolates All the K. aerogenes isolates (n=364) were collected from 2011-2017 in a tertiary hospital in Zhejiang province, China (isolates of 2014 were lost). These isolates were non-duplicate and were collected on routine workdays without any speci c exclusion criteria. Escherichia coli strain ATCC 25922 was used as control for antimicrobial susceptibility testing. In addition, E. coli EC600 (resistant to rifampicin) and E. coli J53 (resistant to Sodium azide) were used as recipients for conjugation test.
Detection of carbapenemase genes The presence of genes encoding the carbapenemases KPC, NDM, VIM, IMP, and OXA-48 were investigated in all of the CRKA isolates using the primers described in Supplemental table 1. The ampli ed products were observed and con rmed by agarose gel electrophoresis and the positive ones were sequenced with Sanger sequencing. The sequences were further con rmed using BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Pulsed eld gel electrophoresis (PFGE) Clonal relationships of CRKA were analysed using PFGE, which was performed according to a previously described protocol. Brie y, the DNA fragments were separated with a CHEF-Mapper XA PFGE system (Bio-Rad, USA). Electrophoresis was performed for generated were analysed according to the criteria proposed by TENOVER et al [19]. S1-PFGE and Southern blot hybridization The location of the carbapenemase genes were determined by S1-nuclease digestion and pulsed-eld gel electrophoresis (S1-PFGE) combined with Southern blotting hybridizations. Gegomic DNA of all carbapenemases-producing isolates were extracted and embedded in gold agarose gel plugs (SeaKem® Gold Agarose, Lonza, Atlanta, GA USA). The plugs were digested with S1 nuclease (TaKaRa, Dalian, China), and the DNA fragments were separated by PFGE. The plasmid was characterized by S1-PFGE, and the location of carbapenemase genes was identi ed by Southern hybridization with digoxigenin-labelled probe using the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics).
Transferability of carbapenemase genes Conjugation experiments were carried out in Luria-Bertani broth with E. coli EC600 (resistant to rifampicin) or E. coli J53 (resistant to Sodium azide) as the recipient as described previously [26]. E. coli transconjugants were selected on Mueller-Hinton agar containing rifampicin (600ug/ml) or Sodium azide (200ug/ml) and ertapenem (2ug/ml). The colonies that grew on the selecting medium were picked up and identi ed using the MALDI-TOF and PCR analysis.
Whole genome sequencing The whole genomes of all the CRKA strains were extracted and sequenced by Illumina HiSeq2000 platform. Sequencing data was assembled by CLC Genomics Workbench software (Version 9.0). The multi-locus sequence type (MLST) and other resistant genes were analyzed on the website of Center of Genomics Epidemiology (http://www.genomicepidemiology.org/). E ux pump inhibition test In addition, to investigate the role of e ux pumps in the carbapenem resistance mechanisms, the MICs of carbapenems were determined in the presence of the e ux pump inhibitor Phe-Arg β-naphthylamide dihydrochloride (PAβN:25ug/ml) [27] and carbonyl cyanide mchlorophenylhydrazone (CCCP:25ug/ml) [9], respectively.

Declarations
Ethics approval and consent to participate The clinical isolates were part of the routine hospital laboratory procedure. The present study mainly focused on bacteria, but not the patients. So the ethical approval was not required.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information les.

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