Characteristics of non-carbapenemase producing carbapenem-resistant Klebsiella pneumoniae from a tertiary hospital in China

Introduction: The spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a substantial severe global public health burden. Non-carbapenemase-producing CRKP (non-CP-CRKP) is increasingly recognized as the source of severe infections. Methodology: We analyzed the genotypic, and phenotypic profiles of non-CP-CRKP strains with the whole-genome sequences isolated between 2017 and 2019 and the clinical characterization of non-CP-CRKP infection. Results: A total of 91 CRKP strains were collected, of which 5 (5.49%) strains were non-CP-CRKP. Four strains were from male patients; three strains were isolated from the bile of patients who underwent biliary interventional surgery and four had a history of antibiotic exposure. Three strains were sequence type (ST)11, one was ST1, and one was ST5523. The non-CP-CRKP strains were insusceptible to ertapenem. Three strains were susceptible to amikacin. All the strains were susceptible to imipenem, meropenem, tigecycline, ceftazidime/avibatam and polymyxin B. The β -lactamases of non-CP-CRKP predominantly included blaCTX-M , blaSHV , and blaTEM subtypes. Two site mutations in ompK36 (p.A217S and p.N218H) and four in ompK37 (p.I70M, p.I128M, p.N230G, and m233_None234insQ) were detected accounting for carbapenem resistance. Plasmids IncFI and IncFII were found in most strains. Genes encoding aerobactin, yersiniabactin and allantoin utilization were not detected in several isolates, and all non-CP-CRKP strains did not carry rmpA gene. Conclusions: Non-CP-CRKP infected patients had a history of previous antibiotic exposure or invasive procedures. Non-CP-CRKP strains were insusceptible to ertapenem. The mechanism of resistance includes β -lactamases production and the site mutations in ompK36 and ompK37 . Several virulence genes were not detected in non-CP-CRKP.


Introduction
Carbapenem-resistant Enterobacteriaceae (CRE) infections are a serious healthcare issue and have been on the global priority list of the World Health Organization (WHO) for research and development of effective drugs [1].CRE infection is highly endemic in China, with an annual incidence rate of 4.0 per 10,000 patients in 2015 calculated through a multicenter study that covered 25 tertiary hospitals in 14 provinces [2].Carbapenem-resistant Klebsiella pneumoniae (CRKP) causes more than 70% of these CRE infections [2].According to the 2020 national surveillance in China, the resistance rate of Klebsiella pneumoniae to imipenem and meropenem was 21.5% and 22.4% [3].The production of carbapenemase enzymes, including Klebsiella pneumoniae carbapenemase (KPC), metalloβ-lactamases (MBLs), Guiana extended-spectrum (GES) β-lactamase and OXA-like enzymes, is the primary mechanism underlying carbapenem resistance [4].Other mechanisms of carbapenem resistance include overexpression of efflux pumps, mutation or downregulation of porins, and target modification and overproduction of extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases (AmpC) [4].
Non-carbapenemase-producing Enterobacteriaceae (NCPE) are the predominant CRE strains in some area [5].In the CRACKLE-2 study, NCPE accounted for 19% of CRE infections and 30-day outcomes were similar between patients with CRE and NCPE infections [6].Ertapenem is the most likely carbapenem antibiotic to be ineffective because of carbapenem non-susceptibility mechanisms.Ertapenem-resistant NCPE, which are susceptible to imipenem and meropenem, have a wider susceptibility profile than other CRE [7].Higher mortality rates are associated with ertapenem-resistant Enterobacteriaceae infection than with ertapenemsensitive Enterobacteriaceae infection [8].
Hypervirulent strains of K. pneumoniae (hvKp) have been prevalent for the past 30 years and some hypervirulence associated genes have been identified including rmpA, rmpA2, iroBCDN, iutA, iucABCD and ybt [9,10].ST11 hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) strains were reported in China which may cause severe infections in healthy individuals and were highly resistant to antibiotics [11].Subsequently, an increasing number of ST11 hv-CRKP strains have been discovered in different Chinese provinces [12].The hv-CRKP evolved when drugresistant strains acquired virulence genes or highvirulence strains acquired drug-resistant genes [13].The analysis of the genomic sequence of K. pneumoniae, especially drug resistance and virulence related genes, have progressed due to the development of sequencing technology [14].However, to our knowledge, there is lack of analysis of non-CP-CRKP genome structure, especially the virulence gene.
The aim of the present study was to obtain the comprehensive characteristic of non-CP-CRKP, by both collecting clinical data of patients and performing genome sequencing of non-CP-CRKP strains isolated from a tertiary care hospital in China from 2017 to 2019 to investigate their clinical history, antibiotics resistance profile, and molecular characteristics of resistant genes and virulence genes.

Data collection
A retrospective epidemiologic surveillance study of carbapenem non-susceptible K. pneumoniae infection was conducted in our hospital from July 2017 to December 2019.A total of 91 CRKP were collected, and among them the 5 cases (5.49%) that were without carbapenemase production were included in this study.The clinical and epidemiologic data were collected by reviewing the medical records of 5 patients, including patient demographics, underlying medical conditions, location in the hospital, healthcare and antimicrobial therapy exposures during the prior year, clinical manifestations, specimen source, sample date, indwelling devices, treatment, and outcomes.The study was approved by the Institutional Review Board (No.002).

Bacterial isolates and microbiological methods
Isolates identification and antibiotic susceptibility testing were carried out using an automated VITEK-2 compact system (Merieux, Lyon, France).The broth microdilution method was used to further confirm the susceptibility of CRKP strains to cefuroxime, piperacillin/tazobactam, ceftazidime/avibatam, aztreonam, amikacin, levofloxacin, meropenem, polymyxin B, and tigecycline.The susceptibility to ertapenem and imipenem were determined by the epsilometer test (E-test).The susceptibility interpretations are based on Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints [15].Carbapenem non-susceptible K. pneumoniae was defined according to the recommendation of CLSI as K. pneumoniae strains are non-susceptible to at least one carbapenem agent (ertapenem, imipenem, or meropenem) [16].We defined carbapenem insusceptibility as minimum inhibitory concentration (MIC) > 1 µg/mL for meropenem or imipenem or MIC > 0.5 µg/mL for ertapenem.The E. coli American Type Culture Collection (ATCC) 25922 (negative control) and K. pneumoniae ATCC 700603 [positive extended spectrum ß-lactamase (ESBL) control] were used as quality control strains.Data were only included when the quality control test results were in acceptable ranges.

DNA preparation, genome sequencing, and annotation
The genomic DNA of the strain of carbapenem nonsusceptible K. pneumoniae without carbapenemase production was extracted using the bacterial genomic DNA extraction kit (Tiangen Biotech, Beijing, China).The sequencing of the strain was performed by Hisep2000 (llumina, San Diego, USA) and assembled with Unicycler v 0.5.0 [17].Gene prediction for strains were conducted using the RAST server [18].Multilocus sequence typing (MLST) was typed by MLST 2.0 provided with the Center for Genomic Epidemiology (CGE) [19].The acquired antimicrobial resistance genes and chromosomal mutations mediating antimicrobial resistance were investigated by using  [22].The whole-genome single nucleotide polymorphism (SNP) tree was constructed using CSI Phylogeny [23] and labeled using iTOL software [24].

Genomes from sequence database
Genomic sequences available for K. pneumoniae were downloaded from the National Center for Biotechnology Information (NCBI) genome sequence repository [25].The downloaded sequences comprised

General clinical characteristics
Ninety-one CRKP strains were collected from July 2017 to December 2019, of which five strains (5.49%) were non-CP-CRKP.We identified three strains as sequence type (ST) 11, one as ST1, and one as ST5523.Four strains were isolated from male patients.Three strains were isolated from the bile of patients who underwent endoscopic retrograde cholangiopancreatography and stone removal procedures.The other two strains were isolated from the urine of patients who had a history of indwelling urethral catheterization.Four of the five patients had a history of pre-exposure to antibiotics (Table 1).

Distribution of resistance genes
The main β-lactamases subtype genes expressed by the five strains included blaCTX-M, blaSHV, and blaTEM (Table 2).Four strains carried the tigecycline resistance gene tet (A).The 18_674 strain carried the acrR gene which contained two tigecycline-resistant mutation sites, namely, p.M123* and p.E122K (Table 2).All strains carried the fosA gene.The network was constructed by using the CSI Phylogeny 1.4 [23].The reference K. pneumoniae sequence is HS11286 (NC_016845.1).

Carbapenem-resistant antibiotic mutations
Collectively, six mutations associated with carbapenem resistance were detected.The p.A217S and p.N218H mutations were identified in the ompK36 gene, whereas the p.I70M, p.I128M, p.N230G, and m233_None234insQ mutations were identified in the ompK37 gene (Table 2).P.I70M and p.I128M mutations were detected in all five strains, and p.N230G and m233_None234insQ mutations were detected in four strains (Table 2).No mutations were observed in the ompK35 gene.The ompK36 deletion mutation was detected in the strains 19_469, 19_832, and 19_839 (Table 2).

Virulence genes of non-CP-CRKP
All strains contained genes encoding type I and III pili, and all strains, except 18_674, expressed the IV flagellum gene.The efflux pump gene (acrAB), iron uptake genes (Ent siderophore, and secretion system genes (T6SS-I, T6SS-II, and T6SS-III) were detected in all strains (Table 3).The strains 18_674 and 19_340 did not carry aerobactin-related genes, and 19_340 did not carry yersiniabactin-related genes.The allantoin utilization-related gene allABCERS was only detected in strain 18_674.All strains expressed the regulatory gene rcsAB, whereas none expressed the rmpA genes (Table 3).

Phylogenetic network by SNPs
Phylogenetic network of the 16 K.pneumoniae genomes as determined on the basis of the concatenated alignment of the high quality SNPs is presented in Figure 1.A total of 11 sequences including the reference K. pneumoniae sequence (NC_016845.1)were taken from GenBank.

Discussion
Carbapenems possess the broadest spectrum of antibacterial activity and are the "last resort" antibiotics used to treat multidrug-resistant bacterial infection [26].The prevalence of carbapenem-resistant organisms (CROs) is increasing and has become a public health concern because of limitations to antibiotic therapy [27].The high prevalence of non-CP-CRKP strains in the Asia-Pacific region needs to be addressed [28].Researchers isolated 41 CRKP strains in Texas between 2011 and 2019, of which 39% were non-CP-CRKP [5].Three major mechanisms of carbapenem resistance in non-CP-CRKP are the overexpression of ESBLs or AmpC β-lactamase, resistance-nodulation-division efflux pumps, and decreased membrane permeability due to porin loss [4].In our study, we isolated five non-CP-CRKP strains that were not susceptible to ertapenem, but were susceptible to imipenem and meropenem.Ertapenem is most likely hydrolyzed by βlactamases in CRO isolates [7].Insusceptibility to ertapenem is a sensitive initial screening tool for potential CRO isolates [29].
A retrospective study showed that intensive care unit stay, exposure to any antibiotic over 30 days, and prior central venous catheterization or mechanical ventilation were risk factors for ertapenem-resistant Enterobacteriaceae infection [30].It was found that previous hospitalization and quinolone exposure were also risk factors for ertapenem resistance [8].All five patients in this study had experienced the risk factors described in the previous studies [8,30].Increased ertapenem exposure can lead to increased ertapenem resistance [31].An in vitro study suggested that ertapenem exposure can lead to ertapenem resistance in ESBL-producing E. coli strains and that the combination of ESBL production and porin loss may cause ertapenem resistance [32].
Researchers analyzed 404 cases of ertapeneminsensitive non-carbapenemase-producing strains from the study for monitoring antimicrobial resistance trends (SMART) surveillance, including E. coli (n = 83), K. pneumoniae (n = 91), and Enterobacter species (n = 210).This study showed that the majority (> 84%) of these strains were sensitive to imipenem and amikacin and that the strains isolated from the hepatobiliary system displayed lower cefepime MICs than those isolated from the peritoneal space [33].In our study, three of the five strains were sensitive to amikacin, and all strains were sensitive to imipenem, meropenem, tigecycline, ceftazidime/avibactam, and polymyxin, and resistant to cefepime.However, cefepime has not been approved for the treatment of non-CP-CRKP infections in our district.A multicenter, large-scale prospective study is needed to better understand the non-CP-CRKP burden and the mechanisms involved in drug resistance in these strains in mainland China.
The blaCTX-M and blaSHV genes were shown to be crucial for ESBL production in ertapenem-insensitive non-CP-CRKP in previous studies [7,34].In contrast to previous studies, no ampC was detected in our study [7].The spread of the β-lactamase gene is associated with the presence of the antibiotic resistance-associated plasmids IncFI, IncFII, and IncR in epidemic clones [35].Similar to a previous study, our study demonstrated that the outer membrane proteins ompK36 and ompK37 had multiple carbapenemresistant mutation sites, which was also one of the important drug resistance mechanisms [5].
The aac(6')-Ib-cr gene is one of the major determinants for plasmid-mediated quinolone resistance and was found in 89% of K. pneumoniae strains in a previous study [36].Therefore, Muggeo et al. suggested that fluoroquinolones should not be used as alternative antibiotics for the treatment of ertapenem non-susceptible K. pneumoniae [36].It has been suggested that the qnrS and aac(6')-Ib-cr genes are responsible for the underlying quinolone resistance in carbapenem-insensitive K. pneumoniae and E. coli [37].The presence of the qnrS1 and aac(6')-Ib-cr in our strains indicated that quinolone was not the appropriate choice for therapy.
The susceptibility rate of aminoglycosides, especially amikacin, to CRKP was previously high and considered in the combination therapy of CRKP [38].According to the CHINET surveillance conducted in 2019, the susceptibility rate of K. pneumoniae to amikacin was 82% [39].The presence of the mutant genes aac(6')-Ib, aac(3)-II, and aph(3')-IIIa is a major mechanism of aminoglycoside resistance in K. pneumoniae [40].In our study, two strains expressed the aac(6')-Ib, aac(3)-II and aph(3')-IIIa genes and were still susceptible to amikacin.Two strains with high MIC to amikacin had the aadA2b mutant gene.This suggests that this gene may play a major role in drug resistance.
Tigecycline is recommended by guidelines for the treatment of CRKP [41].Infection caused by tigecycline-insensitive K. pneumoniae leads to a high rate of mortality at 14 and 28 days post infection [41].The 2019 CHINET surveillance report showed that tigecycline sensitivity of K. pneumoniae was 86.5% [39].We detected the expression of the tet(A) gene in four strains and two acrR gene mutation sites in the 18_674 strain.These genetic factors are associated with tigecycline resistance.Despite this result, all five strains were sensitive to tigecycline.The reason for tigecycline sensitivity in these strains is unclear, as tigecycline exposure can increase the resistance rate of susceptible strains [42].Overexpression of acrAB and/or oqxAB genes, together with the upregulation of the regulators ramA and/or rarA, can lead to tigecycline resistance [41].Inhibition of ramR translation can also result in tigecycline resistance [42].
Recently, hvKP infections, especially carbapenemresistant infections, have attracted considerable attention [11,43,44].hvKP is a hypermucoviscous strain that lacks a genetic profile description [45].Factors that are associated with virulence in K. pneumoniae include capsular lipopolysaccharides, siderophores, and pili.Genes involved in allantoin utilization, iron transport systems, efflux pumps, and a type VI secretion system have been identified as new virulence factors in K. pneumoniae [46].It has been shown that the hypermucoviscosity of K. pneumoniae is associated with the presence of K1, K2, and rmpA genes and that hypervirulent K. pneumoniae strains cause invasive infections, including liver abscess, bloodstream infection, and sepsis [47].It was demonstrated that peg-344, iroB, iucA, plasmid-borne rmpA, and rmpA2 genes were associated with high virulence in K. pneumoniae [48].IucA is a gene associated with hypervirulence and is highly prevalent in virulent CRKP [49].IucA of the aerobactin pathway is a siderophore synthetase that acquires iron in irondepleted environments such as in a human host [50].
We identified aerobactin gene deletions in strains 18_674, 19_340, and 19_832, but it is not clear whether these mutations would cause reduced virulence.We detected only the allantoin utilization gene (allS) in the 18_674 strain.The allS gene enhances the allantoinutilizing capability of bacteria to compete for nitrogen sources [51].An animal study in BALB/c mice demonstrated that K. pneumoniae liver isolate, which had an allS deletion mutation, showed a significant decrease in virulence in intragastric infection [51].The prevalence of virulence factors varied in the isolates found in China and the United States with aerobactin present in 62% of CRKP in China and 1% in the United States.The percentage of colibactin in CRKP strains is 21% and 1% in the United States and China, respectively [52].We did not detect colibactin in any of the five strains tested in this study.These differences may be because of the differences in the prevalence of strains in China and the United States, with ST11 being the dominant strain in China and ST258 being the dominant strain in the United States.

Conclusions
We isolated five non-CP-CRKP K. pneumoniae strains insusceptible to ertapenem.The genomic profile analysis revealed these strains carried β-lactamases and mutations in ompK36 and ompK37 genes accounting for ertapenem resistance.The patients infected with non-CP-CRKP had a history of antibiotic exposure and interventional operations.Some strains lacked the aerobactin, yersiniabactin and allantoin utilizationrelated genes and all non-CP-CRKP strains did not carry rmpA gene.Although non-CP-CRKP is not the main epidemic strain in our district, clinical staff should also raise awareness of non-CP-CRKP infection for better control of CRKP infection.Prospective, multicenter, large-scale studies should be conducted to better understand the prevalence of non-CP-CRKP.

Table 1 .
Demographic and clinical characteristics of the patients.

Table 2 .
The K-types, ST types, resistant genotypes, plasmid types and resistant phenotypes of the 5 Klebsiella pneumoniae isolates.