Molecular Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae in a Tertiary Hospital in Northern China

Background In recent years, carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged rapidly in China with the abuse and overuse of antibiotics, and infections caused by CRKP pose a serious threat to global public health safety. The present study aimed to explore the epidemiological characteristics of CRKP isolates in Northern China and to elucidate their drug resistance mechanisms. Methods 45 CRKP strains were consecutively collected at a teaching hospital from March 1st, 2018 to June 30th, 2018. Antimicrobial susceptibility was determined by the VITEK2 compact system and microbroth dilution method. Polymerase chain reaction (PCR) and sequencing were used to analyze multilocus sequence typing (MLST), drug resistance determinants, and plasmid types. The transfer of resistance genes was determined by conjugation. All statistical analysis was performed using SPSS 22.0 software. Results All 45 isolates showed multidrug resistance (MDR). MLST analysis showed ST11 (48.9%, 22/45) was the most frequent type. All of the 45 CRKP isolates contained carbapenemase genes, extended-spectrum β-lactamase (ESBL) genes, and plasmid-mediated quinolone resistance (PMQR) genes. For carbapenemase genes, KPC-2 (93.3%, 42/45) was the main genotype, and followed by GES (37.8%, 17/45) and NDM-1 (11.1%, 5/45). Plasmid typing analysis showed that IncFII and IncFIB were the most prevalent plasmids. The carbapenem resistance rate of K.pneumoniae was 11.4% and ICU was the main CRKP infection source. Conclusions ST11 is the most frequent sequence type and KPC-2 is the predominant carbapenemase of CRKP strains in Northern China. KPC-2-ST11 are representative clonal lineages.


Introduction
Klebsiella pneumoniae, a common opportunistic pathogen, can cause severe pneumonia, bloodstream infection, urinary tract infection, wound or surgical site infection, and meningitis, especially, in immunocompromised patients [1], which accounts for approximately 10% of hospital-acquired infections [2]. In recent years, with the abuse and overuse of antibiotics, particularly the third-generation cephalosporins and carbapenems, carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged rapidly in China, with the carbapenem resistant rate increasing from 3% in 2005 to 25.5% in 2021 based on the data of CHINET [3,4]. Te majority of CRKP isolates was resistant to more than three kinds of antibiotics and was defned as multidrug resistance (MDR) [5]. Terefore, infections caused by CRKP pose serious threats to global public health security and an enormous challenge to antimicrobial therapy.
Te spread of CRKP is rapidly occurring in hospitalacquired infections, as the emergence of mobile gene elements carrying the carbapenem-resistance gene can shuttle easily through horizontal or vertical gene transfer in the same or diferent species, especially, result in accelerated growth of resistant strains across many species of enterobacterial species [11]. Terefore, studying the molecular epidemiology of CRKP was necessary to limit the spread of antimicrobial resistance and can provide an empirical treatment strategy for the clinicians.
In this study, we investigated the molecular characteristics, plasmid typing, and antimicrobial resistance gene of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from a tertiary hospital in Northern China, so as to provide a basis for the control and prevention of nosocomial infections.

Study Design and Setting.
We performed a retrospective study from March 1st, 2018 to June 30th, 2018 in the First Afliated Hospital of Harbin Medical University, a 6498-bed tertiary hospital in Northern China. We reviewed the microbiology laboratory database of any patients with CRKP infection during this period. Only the frst positive culture of CRKP for each patient was included in this study, and patients with insufcient medical records were excluded from this study. Tis study was approved by the ethics committee of the First Afliated Hospital of Harbin Medical University.

Bacterial Strains.
In this study, a total of 395 strains of non-repetitive K. pneumoniae were collected consecutively from March 1st, 2018 to June 30th, 2018 in the First Afliated Hospital of Harbin Medical University. Tese strains come from diferent clinical samples, including sputum, blood, pus, ascites, cerebrospinal fuid, bile, and secretions, among which 45 strains were identifed as carbapenem-resistant K. pneumoniae. Te CRKP strains were defned as resistant to at least one of carbapenem antibiotic (imipenem, meropenem, or ertapenem). Te clinical information was obtained from the medical record. Identifcation of the K. pneumoniae isolates was performed by the VITEK2 compact system (BioMérieux, Marcy l'Etoile, France) and the matrixassisted laser desorption ionization-time of fight-mass spectrometry (MALDI-TOF-MS) (BioMérieux, Marcy l'Etoile, France). Te Escherichia coli (ATCC25922, ATCC8739), Pseudomonas aeruginosa (ATCC27853) were used as reference strains. Te rifampin-resistant strain E. coli EC600 was used as the recipient strain for conjugation experiments.

Multilocus Sequence Typing (MLST).
Te MLST analysis was conducted by amplifying seven housekeeping genes including gapA, infB, mdh, pgi, phoE, rpoB, and tonB for the genetic relatedness of 45 CRKP isolates. Te sequence types (STs) of each strain were sequenced and blasted using the online database at https://bigsdb.pasteur.fr/klebsiella/ klebsiella.html. Te phylogenetic tree was conducted by BioNumerics software with the maximum likelihood method, and a minimum spanning tree was also generated by BioNumerics software.

Conjugation Experiment.
Te conjugation experiments were carried out with the method described previously with slight modifcations [19]. In brief, the rifampin-resistant E. coli EC600 was used as the recipient, and the 45 CRKP isolates were used as donors. Te isolates were grown on blood agar plates at 37°C overnight. Each of the donor and recipient bacteria were grown in 3 ml of LB broth at 37°C for 4 h. Te mixture of the donor and recipient isolates at a volume ratio of 3 : 1 was spotted on a 1 cm 2 hydrophilic nylon membrane flter with a 0.22 μm pore size that was placed on an blood agar plate and then incubated for mating at 37°C for 24 h. Transformants were selected on LB agar containing imipenem (0.5 μg/ml) and rifampin (100 μg/ml).

Plasmid Replicons
Analysis. PCR-based replicon typing was performed for the determination of the following plasmid types: IncFII, IncFIIK, IncFIA, IncFIB, IncHII, IncII, IncX, IncA/C, IncL/M, IncB/O, and CoIE, and the primers were as described by Johnson et al. and Potron et al. [20,21]. Te DNA of each isolate was extracted by the boiling method. All PCR amplifcations, except the IncFII, were performed with the following amplifcation scheme: initial denaturation of 5 min at 94°C, followed by 30 cycles of denaturation (94°C, 60 s), annealing (60°C, 30 s), extension (72°C, 60 s), and with a fnal extension of 5 min at 72°C. Te IncFII amplifcation was performed with an annealing temperature of 52°C. Te PCR products were sequenced and blasted in PubMed (https://blast.ncbi.nlm.nih.gov/) to confrm the plasmid types.

Antimicrobial Susceptibility Testing.
Te antimicrobial resistance profles of 45 CRKP isolates against 20 common antibiotics were shown in Table 2. Te drug resistance rates of ICU patients infected with CRKP to aztreonam, ciprofoxacin, and levofoxacin were higher than those of non-ICU patients, while the drug resistance rates of ICU patients infected with CRKP to cefoxitin and colistin were lower, and the diferences were statistically signifcant (P < 0.05). In addition, MICs of imipenem were tested by broth microdilution and all of the 45 isolates were resistant to imipenem with the MIC 50 (8 μg/ml) and MIC 90 (64 μg/ ml), respectively. Te MIC distribution of imipenem was shown in Figure 1. Notably, all of the CRKP isolates were MDR. Te resistance rates to carbapenems, cephalosporins, and fuoroquinolones were relatively high, with the resistance rates more than 70%, and the resistance rates to tigecycline and colistin were low, with the resistance rates about 10%.

Resistance Genes Transfer and Plasmid Incompatibilities.
Te carbapenemase genes of eighteen CRKP strains were successfully transferred in the conjugation experiment. Te antimicrobial MICs of the 18 CRKP donors and E. coli EC600 transconjugants were presented in Table 5. In general, the resistance profles to carbapenems and cephalosporins of the eighteen transconjugants were similar to those of the CRKP donor strains, demonstrating the transfer of resistance genes.

Discussion
With the use of antibiotics, CRKP has gained extensive attention globally [22]. While previous studies have seldom focused on the molecular epidemiological data of CRKP infections in Northern China. Terefore, we aim to explore the epidemiological characteristics of CRKP isolates in Northern China and to elucidate their drug resistance mechanisms, so as to guide the clinicians to empirically use of antibiotics.
In this study, we investigated the antimicrobial resistance profles, molecular typing, antibiotic resistance genes and plasmid replicon types of 45 consecutively collected CRKP isolates in Northern China. From the clinical department distribution, most of the CRKP isolates (48.9%, 22/45) originated from the ICU department and showed multidrug resistance, which was consistent with previous studies [23][24][25]. As K. pneumoniae is an opportunistic pathogen, ICU patients with impaired immunity are at an increased risk of infection. In particular, some ICU patients also required mechanical ventilation and other invasive operation, so they prone to sufer from a nosocomial infection.
Te antimicrobial resistance test showed that they had higher resistance rates to carbapenems, cephalosporins, and fuoroquinolone and lower resistance rates to amikacin, tigecycline, and colistin, which was consistent with CHINET data in 2018 [26]. Because carbapenemase genes and ESBL genes often coexisted in CRKP isolates, the resistance rate of CRKP isolates to cephalosporins was also high, while amikacin, tigecycline and colistin were rarely used in clinical practice, so the resistance rates to these antibiotics were low. It was reported that combination therapy was related to a lower mortality rate than monotherapy in CRKP, and imipenem combined with polymyxin and tigecycline showed high synergistic antibacterial efects in in vitro drug susceptibility tests [27].
All CRKP isolates were found to produce carbapenemases, one of the major mechanisms for carbapenem resistance in CRE. Our results showed that 88.9% isolates produced serine carbapenemase, and 11.1% isolates produced both serine carbapenemase and metactamallo-β-lases. Among serine carbapenemases, KPC-2 was the predominant type, which accounted for 93.3% of the isolates, followed by GES (37.8%, 17/45). Tese results were consistent with the previous studies that found KPC was the most common carbapenemase in China, while GES was higher and NDM was lower compared with other regions in China [28]. Te above data indicated that the distribution of carbapenemase genes in CRKP may be varied in diferent regions. Te clinician may use ceftazidime-avibactam as the frst line drugs for CRKP infections in Northern China. Most of the CRKP isolates (60%, 27/45) harbored only one carbapenemases gene, 16 isolates carried two carbapenemases genes; with KPC-2 + GES the most frequent type and 2 isolates harbored 3 carbapenemases genes. KPC-2 + GES producing CRKP had a higher MIC 50 and MIC 90 compared to KPC-2 producing CRKP.
MLST is a nucleotide sequence-based method that is adequate for characterizing the genetic relationships among bacterial isolates [29,30]. Te most common clinical CRKP strains are those of the clonal group 258 (CG258), which was     Canadian Journal of Infectious Diseases and Medical Microbiology known to spread throughout the world [31]. Among which, ST258 and ST11 were the most prevalent STs in diferent parts of the world [32]. ST258 has contributed signifcantly to the dissemination of carbapenem resistance and has become particularly prevalent in the United States, Latin America, and several European countries [33,34], while in Asia, the dominant clone is ST11 CRKP, which accounts for up to 60% of CRKP in China [35]. Combined with precise epidemiological information and the characterization of antibiotic resistance mechanisms, MLST analysis of larger sample sets should provide a much improved understanding of the evolutionary origin and dissemination of K. pneumoniae MDR strains. In our research, ST11 is still the most common sequence type, accounting for 47.8% (22/45) CRKP isolates, which is consistent with the fact that ST11 is the primary sequence type in Asia for CRKP [36]. While no ST258 CRKP was isolated in our study. In addition, KPC-2 and GES co-producing (14/22) was the main mechanism for carbapenems resistance in ST11 isolates, and national surveillance study on carbapenem nonsusceptible K. pneumoniae found that the clonal spread of ST11 KPC-2-producing K. pneumoniae was occurring at an alarming speed [37,38]. No NDM-1 or IMP producing ST11 strains were detected. Only one ST2059 isolate was found to produce IMP-type carbapenemase and co-produced NDM-1-type carbapenemase, moreover, NDM-1-type carbapenemase was also detected in ST307, ST625, and ST1 isolates. Among 45 CRKP isolates, the second most common sequence types were ST307 (8.9%, 4/45) and ST323 (8.9%, 4/45). Te clonal spread of ST307 was consistent with previous reports, whereas the clonal spread of ST323 increased in our research [39,40]. Meanwhile, ESBL-producing isolates tend to be multidrug-resistant, and they have an increased risk of treatment failure [41,42]. Hence, it is apparent to monitor the molecular epidemiology profles of ESBL-producing isolates in our country. Based on the report by Ocampo et al. all ESBL-producing isolates produced the SHV ESBL enzyme, which was in line with our results [43]. For the CTX-M group, CTX-M-1 was the most common type, and CTX-M-15 was closely related to ST1 [44]. However, CTX-M-2 was the primary CTX-M-type (44/45, 97.8%) and CTX-M-15 was associated with ST307, ST15, and ST2059 in our study.
Plasmids have the ability to carry multiple antibiotic resistance genes (ARGs) and can transfer between the same or diferent species by conjugation, which makes them play an important role in the dissemination of antimicrobial resistance [45]. In our research, a total of 11 types of plasmid replicons were detected by PCR-based replicon typing method, but only group F (IncF) plasmids were identifed among the 45 CRKP isolates. Eight isolates (8/45, 17.8%) all belonged to the ST11 clone co-harbored IncFII, IncFIB, and IncFIIK plasmid replicons. IncF-group plasmids were the most common plasmid types, and most of them were associated with ESBLs and carbapenemases, so they were related to the spread of determinants of antimicrobial resistance in Enterobacteriaceae, and the surveillance of antimicrobial resistance should be strengthened [46]. It has also been reported that the IncFIB plasmid replicons were closely related to the majority of the antimicrobial resistance genes [47]. As we know, the IncX plasmid was an important vehicle with high mobility in the worldwide dissemination of NDM-1-type carbapenemase [48], while the IncX plasmid was not found and bla NDM-1 was less common in our study.
Tere are also some limitations to our study. We only collected CRKP isolates in one large tertiary hospital in Northern China. Multi-center isolates should be collected to provide more evidence on the clinical signifcance of these resistance strains.

Conclusions
In conclusion, ICU is the main ward for rapid and widespread transmission of CRKP in a tertiary hospital in Northern China. ST11 is the most frequently cloned and KPC-2 is the main genotype. KPC-2-ST11 is representative clonal lineages. Terefore, knowledge of the molecular epidemiology characteristics and drug-resistant mechanisms  of CRKP is crucial in preventing the occurrence and rapid spread of nosocomial infections in tertiary hospitals in Northern China.

Data Availability
Te data are obtainable from the corresponding authors upon reasonable request.

Ethical Approval
Tis study was approved by the ethics committee of the First Afliated Hospital of Harbin Medical University in accordance with Helsinki Declaration.

Consent
Written consent forms have been obtained from persons involved in this study.

Conflicts of Interest
Te authors declare that they have no conficts of interest.