Carbapenem-Resistant Klebsiella Pneumoniae  in Southwest China: Molecular Characterizations and Risk Factors Caused by NDM and KPC Producers

doi:10.21203/rs.3.rs-356729/v1

Download PDF

Research

Carbapenem-Resistant Klebsiella Pneumoniae in Southwest China: Molecular Characterizations and Risk Factors Caused by NDM and KPC Producers

https://doi.org/10.21203/rs.3.rs-356729/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background: Klebsiella pneumoniae is one of the most common Enterobacteriaceae. In recent years, carbapenem-resistant Klebsiella pneumoniae (CRKP) has become one of the most important carbapenem-resistant Enterobacteriaceae. CRKP are usually resistant to antibiotics. Up to this day, the emergence of carbapenemase-producing K. pneumoniae has been a challenge for treatment of clinical infection.

Methods: (i) 66 non-repetitive clinical CRKP isolates were identified by matrix-assisted laser analytical ionization time-of-flight mass spectrometer (MALDI-TOF-MS) and drug sensitivity analysis was performed by Vitek2 Compact. EDTA-synergy test and mCIM / eCIM test were used to detect drug-resistant phenotypes. (ii) Carbapenemase genes, extended-spectrum β-lactamase genes (ESBLs), cephalosporinase gene (AmpC), virulence genes, integron and resistance gene cassettes were amplified by PCR. (iii) Plasmid typing was performed by plasmid conjugation assay and PCR-based replicon typing (PBRT) method. (iv) The genetic environments of KPC-2 and NDM-1 were analyzed by using overlapping PCR. (v) MLST was used to analyze the molecular epidemiological characteristics of CRKP. (vi) Risk factors of CRKP infection by logistic regression model.

Results: Our study revealed that 42 of the 66 CRKP isolates obtained from patients were identified as bla_KPC-2, 24 bla_NDM-1-positive strains were identified (20 bla_NDM-1 and 4 bla_NDM-5), of which 18 were from the neonatal departments. And CRKP strains were ESBL (extended-spectrum β-lactamases) and AmpC enzymes producer, Notably, we found two CR-hvKp (carbapenem-resistant hypervirulent klebsiella pneumoniae) strains, which contains bla_KPC-2 gene and other resistant genes. Two of the 42 KPC-2-producing CRKP strains were positive for transconjugants, and the plasmid typing was the IncFII type. And two NDM-producing CRKP strains tested positive for transconjugants, which belonged to the lncX3 plasmid. Analysis of the genetic environment of these two genes has revealed that the highly conserved regions (tnpA-tnpR-ISkpn8-bla_KPC-2) and conserved regions (bla_NDM−1-ble_MBL-trpF-tat) are associated with the dissemination of KPC-2 and NDM-1. Intl1 carrying drug resistance gene cassettes were widely distributed in CRKP. According to the MLST results, a total of 13 ST types were measured in 66 CRKP strains, ST11 and ST4495 were the main ST types, and the latter was the newly discovered ST type. Hematological disease, tracheal cannula and prior use of β-lactams and β-lactamase inhibitor combination were identified as independent risk factors for CRKP infections.

Conclusion: These findings manifested the need for intensive surveillance and precautions to monitor the further spread of KPC and NDM in southwest China.

Infectious Diseases

Carbapenem-resistant Klebsiella pneumoniae

Molecular typing

Genetic characteristics

Risk factors

Klebsiella pneumoniae (K. pneumoniae) is a common cause of opportunistic infections in hospitalized patients with antimicrobial resistance (1). it can often lead to aseptic site infections such as blood and other respiratory tract infections (2). Over the past decades, due to increasing prevalence of healthcare-associated infections caused by multidrug-resistant strains producing extended-spectrum β-lactamases and / or carbapenemases, K. pneumoniae has emerged as a major clinical and public health threat. A parallel phenomenon of severe community-acquired infections caused by ‘hypervirulent’ K. pneumoniae has also emerged owing to phenomenon of strains acquired virulence factors (1). Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a particularly important problem worldwide due to the rapid increase in drug resistance and subsequent high mortality (3) (4). In China, the prevalence of CRKP infection has rapidly increased from 0.7% in 2004 to 13.4% in 2014 (5), and the mortality rate of CRKP infection was approximately 40–50% since poor prognosis.

The resistance mechanism of Enterobacteriaceae to carbapenem antibiotics is very complex. However, phenotypic drug resistance of carbapenems is usually caused by two major mechanisms: (i) β-lactamase activity-binding structural mutation and (ii) carbapenemases production (6). The former mechanism is combination of extended-spectrum β-lactamases (ESBLs) and AmpC enzyme with porin loss or reduced expression, resulting the decrease of outer membrane permeability and the change of penicillin binding protein in carbapenems (7, 8). As for later mechanism, carbapenemases are classified by molecular structure and belong to Ambler classification system A, B, D three β-lactamases, class A and D carbapenemases require serine hydrolysis of β-lactam, while class B metallo-β-lactamases (MBLs) require zinc at their hydrolysis active sites (6). In addition, KPCs is the most common class A gene in Enterobacteriaceae worldwide (9). KPC2 carbapenemases are widely disseminated, because its encoding gene bla_KPC−2 is usually located on the plasmid of bacteria, and the plasmid where it is located can often be widely transferred in different bacteria, especially in Enterobacteriaceae. Most KPC−2-producing strains belonged to clone group (CG)258, ST258 and ST11 were the dominant ST types. ST258 has spread worldwide since its emergence in the early 21st century, particularly in North America, Latin America and several European countries (10). However, in Asia, the dominant CRKP is ST11, which accounts for 60 % in China (11). NDM-1 carbapenemases in class B have been reported in China for the first time in 2013, and the NDM-1-producing clinical strains have spread rapidly in China, even causing explosive epidemic in some regions (12, 13).

Recently, not only the outbreaks of CRKP have become increasingly common in China (14), but also the prevalence of hvKp has created new challenges for the clinician. Genetic factors that confer high virulence to hvKp existing in a large virulence plasmid, perhaps an integrated binding element, therefore, we focus on predictive biomarkers located on virulence plasmid (e.g. rmpA and iucA) (15). Importantly, MDR-hvKp (multiple drug resistance hypervirulent k. pneumoniae) strains are gradually reported worldwide due to horizontal transfer of mobile genetic elements (16).

Therefore, the objectives of this study were the following: (i) To describe the prevalence of clinical CRKP isolates collected for approximately 4 years. (ii) To identify the antimicrobial resistance profiles, resistance genes, integrons, drug resistance gene cassettes, and virulence genes among these CRKP strains. (iii) To report plasmid analysis (iv) To detect genetic environment of KPC-2 and NDM-1. (v) To examine the clonal relatedness of these CRKP strains. (vi) To evaluate the risk factors CRKP infections in hospitalized patients.

Study Design and Data Collect.

During the 4 years study period (September 2016 to August 2019), this retrospective study was performed in the Affiliated Hospital of Southwest Medical University (Luzhou, China), where is a 3200-bed large teaching hospital with 43 wards and approximately 120,000 annual admissions. 565 clinical K. pneumoniae isolates were collected from this hospital (WHONET.2020). We selected 66 CRKP isolates which were from the patients for the first time and excluded duplicate isolates from the same patient. All CRKP isolates were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, Bruker Daltonics, Bremen, Germany).

We used a stepwise matching technique to identify appropriate control cases from patients infected with carbapenem-susceptible Klebsiella pneumoniae (CSKP) infection, in order to study the risk factors of CRKP infections. For each patient with CRKP infection, we selected a matched control patient from the pool of patients with CSKP infections. Patient matched to a case for the site of infection, gender, age ± 2 years and year of hospital admission.

Antimicrobial Susceptibility Testing (AST) of CRKP Isolates.

The susceptibility of CRKP isolates to 16 antibiotics, including Cefepime, Cefotaxime, Cefazolin, Cefuroxime, Ceftazidime, Cefoxitin, Piperacillin/tazobactam, Ampicillin/sulbactam, Amikacin, Tobramycin, Gentamicin, Levofloxacin, Ciprofloxacin, Sulperazone, Compound sulfamethoxazole, Aztreonam, was tested by VITEK 2 Compact system (bioMérieux, Marcy l’Etoile, Lyon, France). The MICs of Meropenem, Imipenem, Ertapenem, Tigecycline and Polymyxin B were determined by broth dilution method, and the results were interpreted according to the standards of the Clinical and Laboratory Standards Institute (CLSI) 2020-M100 (17). Pseudomonas aeruginosa ATCC27853 was used as quality control strains (purchased from China National Health Inspection Center).

Screening of CRKP and phenotypic detection of carbapenemase.

After overnight cultured, 0.5 McFarland bacterial suspension used to swab inoculate Mueller-Hinton ager plate (Haibo, Qingdao,China). When dry, 10-µg imipenem disk (OXOID, Thermo Fisher Scientific, Massachusetts, USA) and blank filter paper disk were placed 10 mm from each other, then a blank disk applied to 10 µL of 0.5 mol / L EDTA solution (Thermo Fisher Scientific, Massachusetts, USA), which concentration was about 1.5mg / mL. After 18-24h incubation, the EDTA-synergy positive presented that enlarged zone of inhibition (17) (18).

The CIM-test utilizes antibiotic susceptibility-testing disks as previously described (17) (19), 2-ml aliquots of Trypticase soy broth (TSB) (Haibo, Qingdao, China) were directly inoculated with a 1-µl loopful of K. pneumoniae colonies, a 2-mL tube of TSB supplemented with EDTA at a final concentration of 5mM. A 10-µg meropenem disk (OXOID, Thermo Fisher Scientific, Massachusetts, USA) was placed in each inoculated tube, which were incubated for 4 h (± 15 min), then meropenem disks placed on a Mueller-Hinton agar plate after lawn inoculation of 0.5 McFarland bacterial suspension of meropenem-susceptible Escherichia coli ATCC 25922, no carbapenemase activity will imply there will be a zone, whereas enzymatic inactivation will produce no zone. The mCIM is considered positive if the zone size is 6 to 15 mm or pinpoint colonies are present within a 16- to 18-mm zone (20). Only when the isolate is positive for mCIM, eCIM results are recorded. When the zone size increases by ≥ 5 mm compared to the zone size observed by mCIM, the test isolation of MBL production is positive, and if the area size increases by ≤ 4 mm, the test isolation of MBL is negative.

The Detection of Resistance Gene, Virulence Gene and Integrase-associated Gene.

After the bacterial genomic DNA was extracted by bacterial DNA Kit (Tiangen, Beijing,China). This DNA was used as a template in polymerase chain reactions (PCR) to detect related genes. The PCR was performed to amplify carbapenemase genes (bla_KPC, bla_NDM, bla_VIM, bla_IMP−4 and bla_OXA−48), extended spectrumβ-lactamase genes (bla_TEM, bla_SHV, bla_CTX−M). plasmid-mediated AmpC genes (bla_MOX, bla_DHA, bla_ACC, bla_EBC, and bla_FOX) virulence genes (rmpA, rmpA2, HI1B, iroN, iutA, magA, fimH, mrkD, entB) (Table S1) and integrase-associated genes (Intl1, Intl2, Intl3, ISCR1VR) (Table S2) were also determined by using primers as described previously. The primers were shown in Table S1 and Table S2. PCR amplification system: ddH2O 10 µL, 5’ and 3’ primer 2 µL, 2×Taq MasterMix 11 µL (Innovagene, Changsha, China), DNA 2µL, totally 25 µL reaction volume. Reaction conditions: 95 ℃ denaturation for 5min, 95 ℃ 30s, annealing temperature 30s, 72 ℃ 30s with 30 cycles, and extension at 72 ℃ for 5min and the DNA samples collected were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA); The reaction products were separated gel electrophoresis on a 1.5% agarose gel (Biorad, California, USA). Positive amplifications were subjected to Shanghai Jieli Biotechnology. The variable region of the class 1 Integron and the ISCR1 analyzed by BLAST (www.ncbi.nlm.nih.gov).

Conjugation and plasmid analysis.

To evaluate whether the carbapenemase genes are located on the plasmid and assess whether these genes can be horizontal transferred. The receptor strain was sodium azide-resistant E. coli strain J53. Implant donor and recipient strains in Mueller-Hinton plate and incubated overnight at 37°C. Then, an appropriate amount of strains was inoculated in a glass tube containing 5 mL LB medium and cultured at 37°C for 16-18h. Subsequently, 400 µL donor strain and 200 µL recipient strain were added to the glass tube containing 800 µL LB broth medium and cultured at 37°C for 16–18 h. Transconjugants were selected using LB plates containing sodium azide (180 µg/mL) and imipenem (0.5 µg/mL), meanwhile, the LB plate was used as blank control (21). Confirmation that conjugation had taken place in E. coli J53 was carried out by MALDI-TOF MS system for the presence of resistance gene by PCR analysis. In addition, plasmid analysis was performed for strains with successful conjugation which was determined by PCR-based replicon typing as described previously (22).

Genetic Environments of NDM-1-Carrying Plasmids and KPC-2-Carrying Plasmids.

To investigate the genetic environments of the NDM-1 and KPC-2, we use overlapping PCR and sequencing were applied to analyze. (23) (24). The PCR amplicon was sequenced, the DNA sequences obtained was spliced and compared to those in the NCBI gene bank database (25) (Table S3, S4).

Molecular Epidemiological Study.

Multi-locus sequence typing (MLST) was performed using amplification of internal fragments of the seven housekeeping genes: gapA, infB, mdh, pgi, phoE, rpoB, tonB. Primer pairs designed according to MLST website (26)(www.pasteur.fr/mlst). Reaction conditions: 95 ℃ denaturation for 5min, 95 ℃ 30s, 55 ℃ 45s, 72 ℃ 30s with 30 cycles, and extension at 72 ℃ for 5min. The PCR products were analyzed by agarose gel electrophoresis (Biorad, California, USA) and the positive products were sequenced by Shanghai Jieli Biotechnology, the sequencing results were then submitted to klebsiella pneumoniae MLST data (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html) for comparison.

Statistical analysis.

All analyses and graphs were performed using SPSS v.24.0 software (SPSS Inc., Chicago, USA) The chi-square test or Fisher’s exact test was used to analyze categorical variables. Continuous variables were presented as means ± standard deviation (SD), and were evaluated by a more appropriate student’s t-tests or Mann–Whitney U-test. Multivariable logistic regression analysis was performed to identify independent risk factors of CRKP infection. All biologically plausible variables with a value of P < 0.1 within univariate analysis were included in the following multiple logistic regression model. P < 0.05 was considered as statistically significant, and all probability values were two-tailed distribution.

Bacterial Isolates.

In this study, a total of 66 consecutive non-duplicate CRKP isolates were identified from September 2016 to August 2019 in southwest China. The majority of patients were in neonatology department (27.3%, n = 18) and rehabilitation department (24.3%, n = 16), followed by department of hepatobiliary surgery (10.6%, n = 7), department of hematology (7.6%, n = 5), department of respiratory medicine (6.1%, n = 4), department of pediatrics (6.1%, n = 4),department of urology (4.5%, n = 3), department of neurosurgery (4.5%, n = 3), department of gastrointestinal surgery (3.0%, n = 2), cardiothoracic surgery department (3.0%, n = 2), intensive care unit (1.5%, n = 1) and digestive medicine (1.5%, n = 1)(Fig. 1A). These isolates were mainly cultured from sputum (34.8%, n = 23), urine (25.8%, n = 17) and blood (21.2%, n = 14), followed by secretion (6.1 %, n = 4), pleuroperitoneal fluids (6.1 %, n = 4), catheter tip (3 % n = 2), pus (3 % n = 2) (Fig. 1B). We conducted a matched case–control study and at a ratio of 1:1 as the control group, therefore, 132 patients were included in the final study cohort.

Antimicrobial Susceptibility Testing (AST) and Carbapenem Resistance Phenotype.

As shown in Table 1, VITEK2-Compact results showed that 66 CRKP strains were multidrug resistance, the resistance rates to Cefepime, Imipenem, Piperacillin/tazobactam, Ceftriaxone, Ampicillin/sulbactam, Cefotaxime, Cefazolin, Ertapenem, Ceftazidime, Meropenem and Cefoxitin were all 100%. The resistance rates to Ciprofloxacin, Levofloxacin, Gentamicin, Tobramycin, Aztreonam, and Sulperazon were 80.3 %, 81.9 %, 83.3 %, 83.3 %, 87.9 %, and 95.5 %, respectively. In addition, the resistance rates of Amikacin and Cotrimoxazole were 54.5 % and 65.2 %, respectively. However, polymyxin B were effective for these strains.

The results of EDTA-synergy test, mCIM / eCIM test and drug susceptibility test were consistent. All 66 CRKPs showed positive mCIM test, namely carbapenemase. Among them, 24 strains showed eCIM positive, that is, Metallo Beta Lactamases (MBL) positive.

Molecular Testing

Among the 66 carbapenemase producers: 42 (63.6%) isolates contained bla_KPC-2, 24 (36.4%) isolates possessed bla_NDM, in which, 20 strains were bla_NDM-1,4 strains were bla_NDM-5. The results were consistent with EDTA-synergy test and mCIM/eCIM test. Notably, there were three isolates co-harbor bla_NDM and bla_IMP simultaneously. In addition to the production of carbapenemase, 100% (66/66) and 95.5% (63/66) of the CRKP isolates were positive for ESBL and AmpC genes, respectively, with 95.5% (63/66) of the strains positive for both. Otherwise, virulence genes were detected in all isolates, 54 (81.8 %) detected three different virulence genes. it was noteworthy that Kpn497 harbor 12 virulence genes and Kpn131 harbor 11 virulence genes (Table 2).

Plasmid and Integrase-associated Analysis.

Transconjugants containing bla_KPC-2-carrying plasmids were successfully obtained from kpn406 and kpn490, whose plasmids belong to IncFII. Likewise, kpn473 and kpn475 containing bla_NDM-containing plasmids were positive for transconjugants and these plasmids belonged to lncX3.

Almost all isolates class 1 integrin-positive (84.8 %, n = 56), of which 46 strains were variable region positive, and no class 2 integrin and class 3 integrin strains were detected. ISCR1 positive was detected in 25 strains, whereas whose related drug-resistance genes were not. A total of 6 different types of variable region gene cassette arrays. The most common antibiotic resistance genes conferred resistance to aminoglycoside antibiotics [addA2 (40/46), aadA1 (1/46)]. Five trimethoprim-resistance genes [dfrA12 (6/46), dfrA27 (1/46)] conferred resistance to trimethoprim antibiotics; Furthermore, we also found OXA-10 gene (1/46) in the integron variable region, a gentamycin-resistance gene arr3 (1/46) and a rifampicin-resistance gene aacA4 (1/46). The different gene cassette arrays were ddfrA12、addA2、dfrA12-addA2、dfrA27-arr3、gcuF-dfrA12 and OXA-10-addA1-aacA4 (Table 2).

Characterization of Genetic Environment of KPC-2 and NDM-1.

For KPC-2, the genetic structure compared with pKP048 (GenBank Accession No.FJ628167). 42 KPC-2-producing strains could be divided into three different types (A-C) based on the analysis of genetic structures, among which type C was the most principal (n = 32), followed by type A (n = 8), type B (n = 3). Type A harbored same structure as pKP048. In type B, Tn1721-tnpR and Tn1721-tnpA deletions downstream of ISKpn6-like. Compared with type B, type C lacks ISkpn6-ike, and its gene structure is tnpA-tnpR-ISkpn8-bla_KPC-2(Fig.2A).

For NDM-1, compared with pNDM-BJ01 (GenBank accession no. JQ001791), genetic structures surrounding the bla_NDM-1gene demonstrated there are three types A, B and C. As for type A(n=12), a remnant of insertion sequence ISAba125 was present upstream of the bla_NDM-1gene, and loss of downstream genes of tat. Compared with type A, the retention sequence ISAba125 was lacked in type B. In type C, the upstream of bla_NDM-1 was IS30, and the downstream of bla_NDM-1was ble_MBL-trpF-tat-cutA-groES-groEL. (Fig.2B)

Detection of ST types.

A total of 13 MLST was identified, which revealed that ST11 (56%, n=37) and ST4495 (19.7%, n=13) are the predominant ST types (Table 3).

Risk Factors and Multivariate Analysis of CRKP Infection.

As shown in Table 4, we present the results of the univariable analysis of matched data regarding risk factors associated with CRKP infections. Compared with the CRKP group, statistically significant differences were observed for history of respiratory disease (P = 0.034), renal disease (P = 0.039) and hematological disease (P=0.02), need for tracheal cannula (P<0.001), prior use of β-lactams and β-lactamase inhibitor combination (P=0.009). Multivariate analysis for matched data revealed that history of hematological disease [odds ratio (OR) 2.568, 95% OR 1.106–5.964, P =0.028], tracheal cannula [odds ratio (OR) 4.883, 95% OR 1.797–13.265, P =0.002] and prior use of β-lactams and β-lactamase inhibitor combination [odds ratio (OR) 4.271, 95% OR 1.760–10.365, P =0.001] were independent risk factors for CRKP infection (Table 4, 5).

In light of carbapenem-resistant Klebsiella pneumoniae being widely distributed in China, the increasing prevalence and global spread of these clinically resistant strains posed a serious threat to public health. At the same time, due to the acquisition of virulence plasmids, ‘hypervirulent’ Klebsiella pneumoniae was also emerged (1, 27). In this work, we have found some particularly noteworthy findings, first, our results revealed that the main mechanism of CRKP collected in this study could be attributed to carbapenemase producer. And, CRKP strains were ESBL and AmpC enzymes producer. bla_KPC-2 and bla_NDM-1 are two major carbapenemase genes in our study. Second, the result indicated almost CRKP strains carry virulence-associated genes (81.8%, entB+fimH+mrkD), otherwise, the genes peg-344, iroB, iucA, plasmid-borne rmpA gene (_prmpA), and_prmpA2 gene are regarded as regulator of hypervirulent phenotype. At present, in the study of CRKP it is found that it can be produced by hvKp through obtaining the plasmid encoding carbapenemases (15). Notably, we found two CR-hvKp strains, which contains bla_KPC-2 gene and other resistant genes. This conclusion proved that CR-hvKp began to appear and gradually became popular worldwide, with rapid spread (28). Third. The bla_NDM-1 gene has been rapidly prevalent worldwide and it often co-exists with other resistant genes in bacteria, leading to multidrug resistance, which further challenges the selection of clinical antibiotics (29). In this study, bla_NDM-1 and bla_IMP-4 genes co-exist in two strains, especially, we found a strain carry bla_NDM-5 and bla_IMP-4 genes. The first reported drug-resistant bacteria carrying bla_IMP-4 and bla_NDM-1 simultaneously were K. pneumoniae isolated from 2012 (30). In recent years, strains harboring bla_NDM gene and another type of carbapenemase genes have emerged worldwide (31) (32, 33). The location of these carbapenemase genes is variable, which may be located on chromosome genes and plasmids. Simultaneously, they harbor more resistant genes, making the possibility of horizontal transmission of resistant genes between strains greater, and even can produce higher resistance, which poses great challenges to clinical treatment (34).

By studying the molecular detection and genetic characteristics of CRKP. In China, K. pneumoniae ST11 were always associated with bla_KPC-2 (35). In the forty-two KPC-2-producing CRKP strains, there were 7 ST types, of which 35 belonged to ST11. The rest were ST318, ST467, ST147, ST405, ST23, ST17. In recent years, more and more studies have pointed out that mobile genetic elements may promote the transmission of bla_KPC-2gene. Almost all bla_KPC-2 genes are located on plasmids, and resistance genes spread rapidly through plasmid horizontal transfer (36). Previous studies have reported KPC-2 located on diversity of plasmids IncN, IncL / M and IncR except IncFII. In our study, KPC-2 often binds to IncF plasmid, consistent with the results of this study (37). bla_KPC-2 is often located on Tn4401 in European and American countries. The tnpA sequences of Tn4401 and Tn3 were about 22 % identical and 39 % similar, while the tnpR nucleotide sequences were completely different. In China, the immediate environment surrounding bla_KPC-2in pKP048 was considered to result from the integration of a Tn3-based transposon and a partial Tn4401 structure, with the ORFs ordered as follows: Tn3-tnpA, Tn3-tnpR, ISKpn8, the bla_KPC-2gene, the ISKpn6-like element, Tn1721-tnpR, Tn1721-tnpA (38). Our results demonstrated that the genetic environment of KPC-2 was diversity, which can be divided into three types. Twenty-four NDM-producing strains were divided into 7 ST types. Interestingly, so it is considered to be the outbreak that 18 of the 24 NDM-producing strains were from the neonatal department. In recent years, the outbreak of NDM-producing CRKP has been mostly in the neonatal department, but ST types were different. First reported outbreak of NDM-1 K. pneumoniae ST76 and ST37 in Shanghai in 2016 (39). Subsequently, NDM-1-producing K. pneumoniae broke out in Yunnan, China, and all of them belonged to ST105, meanwhile, its bla_NDM-1 gene was located on IncFI plasmid. NDM-1 producing strains present in the hospital environment also posed a potential risk and the incubator water acted as a diffusion reservoir of NDM-1-producing bacteria (28). The outbreak of ST1419, ST234 and ST1412 followed (40, 41). In 2019, the outbreak of NDM-5-Producing K. pneumoniae in China began, and all strains belonged to ST337, carrying ESBLs resistant genes, all bla_NDM-5 genes were located on IncX3 plasmid (42). In our study, the 14 NDM-1-producing strains, 13 belonged to ST4495 (4-1-99-1-9-5-5), ST4495 is the newly discovered ST sequence type in this study. Among the 4 NDM-5-producing strains, Kpn494, Kpn499 and Kpn502 belonged to ST2407 (2-1-99-1-9-5-5), while Kpn504 belonged to ST11. It is worth noting that the main molecular sequence type of NDM-1 is ST4495(4-1-99-1-9-5-5) and the main molecular sequence type of NDM-5 is ST2407 (2-1-99-1-9-5-5), the difference between them was only gapA gene. The reasons for this phenomenon are speculated to following possibilities:(i) NDM-producing CRKP may undergo gene evolution and transformation in a short time, including NDM-1 evolution into its variant NDM-5, while housekeeping gene gapA changes. (ii) The NDM-5 is not evolved from NDM-1, but a new carbapenemase gene carried by the patient. It is necessary to further study its epidemiology to clarify the causes and mechanisms. Among CRKP in neonatal department, bla_NDM gene is more common than bla_KPC, and its drug resistance is also significantly stronger than the latter. Due to the particularity of newborns, it is more restricted in the use of antibiotics compared with adults (43). The outbreak of NDM-producing CRKP poses greater challenges and threats treatment. Therefore, the emergence of NDM-producing CRKP in neonatal department should be paid enough attention. As one of the most significant metallo-β-lactamases, bla_NDM-1 has been rapidly spread worldwide recently. It was found that the rapid horizontal transmission of bla_NDM-1 was closely related to mobile genetic elements. bla_NDM-1 gene mostly located in transposon Tn125, the structure as following: ISAba125-bla_NDM-1-ble_MBL-trpF-dsbC-cutA-groES-groEL-ISCR27-ISAba125. In this study, we detected the surrounding environment of bla_NDM-1 gene and found that the surrounding environment of bla_NDM-1 gene was mainly composed of A, B and C three types. Our results demonstrated that the genetic environment of these types is partly similar to transposon Tn125.

Integrons are genetic elements that allow efficient capture and expression of exogenous genes. It plays a critical role in the dissemination of antibiotic resistance, particularly among gram-negative bacterial pathogens. In this study, almost all isolates carried IntI1 gene, indicating that integron has become an important element of drug resistance in K. pneumoniae, and to some extent, it also makes the mechanism of drug resistance in K. pneumoniae more changeable and complex. There were six genes encoding drug resistance cassette structures in 46 strains with positive variable region, including dfrA12, addA2, dfrA12-addA2, dfrA27-arr3, gcuF-dfrA12 and OXA-10-addA1-aacA4.addA was the most common resistance gene, followed by dfrA. The high detection rate of these two drug resistance genes may be related to the widespread use of the two antibiotics in clinic and environment, thus causing the prevalence of related drug resistance gene cassettes. In this study, the drug resistance gene cassette bla_OXA-10-addA1-aacA4 was identified from a CRKP strain. OXA-10, as a class D carbapenemase, has weaker hydrolysis ability to carbapenem antibiotics. In recent years, many OXA-10-like enzymes have been found to be involved in bacterial resistance (44).

The main finding of our analysis is that hematological disease, tracheal cannula and exposure to β-lactams and β-lactamase inhibitor combination were independent risk factors associated with the development of CRKP infections. Most preview studies have demonstrated that the history of tracheal cannula was independent risk factor for CRKP infection (45-48). Meanwhile, exposure to antibiotic was also associated with CRKP infection, such as exposure to quinolones (49), β-lactams and β-lactamase inhibitor combination (50) and carbapenems (51), which was partly consistent with our study. Inappropriate treatment had the potential to increase the selection and transmission of CRKP. Notably, in the present study, hematological disease was rarely found to be a risk factor for CRKP infection. Possible explanation may be that part of the isolates in this study were from neonatology department, newborns could have poor functional status and they were susceptible to be infected. There are some limitations in our study. First, we should acknowledge that the number of patients included in this study is relatively small, although this is a common problem in studies assessing risk factors for multidrug-resistant microbial infections (49). Secondly, the study was performed in a single-center setting, so that some important risk factors may be missed.

this present study revealed the high prevalence of CRKP in a large teaching hospital in Southwest China. (i) 66 clinical isolates were multidrug resistant, and polymyxin B was effective. (ii) bla_KPC-2 and bla_NDM-1 are two major types of carbapenemase genes and coexist with other types of drug resistance genes, which are important reasons for CRKP resistance to carbapenem antibiotics. The outbreak of NDM-producing CRKP was also found in neonatal department of our hospital. (iii) Plasmids of CRKP and other types of removable genetic elements may be crucial in the generation and wide spread of drug resistance and virulence. (iv) The surrounding environment of KPC-2 and NDM-1 genes showed polymorphism. Analysis of the genetic environment of these two genes has revealed that the highly conserved regions (tnpA-tnpR-ISkpn8-bla_KPC-2) and conserved regions (bla_NDM₋₁-ble_MBL-trpF-tat) are associated with the dissemination of KPC-2 and NDM-1. Intl1 carrying drug resistance gene cassettes are widely distributed in CRKP. (v) The molecular cloning types of 66 CRKP strains were diverse, with 13 ST types. ST11 and ST4495 were the main ST types, and the latter was the newly discovered ST type. (vi) logistic regression model for matched data revealed that history of hematological disease [odds ratio (OR) 2.568, 95% CI 1.106-5.964, P =0.028], tracheal cannula [OR 4.883, 95% CI 1.797-13.265, P =0.002] and prior use of β-lactams and β-lactamase inhibitor combination [OR 4.271, 95% CI 1.760-10.365, P =0.001] were independent risk factors for CRKP infection. In addition, our findings highlight an urgent need to develop effective measures to prevent and control the further spread of KPC-2 and NDM-1 in China.

ESBL: extended-spectrum β-lactamase;

MBL: metallo-β-lactamase;

hvKp: hypervirulent klebsiella pneumoniae;

CRKP: carbapenem-resistant klebsiella pneumoniae;

CSKP: carbapenem-susceptible Klebsiella pneumoniae;

CR-hvKp: carbapenem-resistant hypervirulent klebsiella pneumoniae;

MDR-hvKp: multiple drug resistance hypervirulent klebsiella pneumoniae;

ATCC: American Type Cell Cultures;

bla: Beta lactamase;

KPC: Klebsiella pneumoniae Carbapenemase;

NDM: New Delhi Metallo-β-lactamase;

OR: odds ratio;

CI: confidence interval

Int: Integron;

ISCR: Insertion sequence common region;

ORF:Open Reading Frame;

Ethics approval and consent to participate

This study was approved by the Institutional Review Board of affiliated hospital of southwest medical university（KY2020043). Written informed consent were obtained from all participants.

Consent for publication

Not applicable

Availability of data and materials

The data used and/or analyzed in this study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no conflict of interest.

Author contributions

ZL, ZD and JY designed the work; YL collected data; ZL, ZD and XJ performed experiment; JX participated in the data analysis; ZL, ZD and TL drafted the initial manuscript; YD and ZZ participated in its design and coordination. All authors read and approved the final manuscript.

Acknowledgments

This work was supported by the grants from Sichuan Science and Technology Program (2021YFH001, 21ZDYF2148, 2019YFS0038, 2019YFH0021), and the Affiliated Hospital of Southwest Medical College Science Foundation (2020ZRQNB025).

Wyres KL, Lam MMC, Holt KE. Population genomics of Klebsiella pneumoniae. Nature reviews Microbiology. 2020;18(6):344-59.
Tumbarello M, Viale P, Viscoli C, Trecarichi EM, Tumietto F, Marchese A, et al. Predictors of mortality in bloodstream infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae: importance of combination therapy. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2012;55(7):943-50.
Li M, Wang X, Wang J, Tan R, Sun J, Li L, et al. Infection-prevention and control interventions to reduce colonisation and infection of intensive care unit-acquired carbapenem-resistant Klebsiella pneumoniae: a 4-year quasi-experimental before-and-after study. Antimicrobial resistance and infection control. 2019; 8:8.
Hoxha A, Kärki T, Giambi C, Montano C, Sisto A, Bella A, et al. Attributable mortality of carbapenem-resistant Klebsiella pneumoniae infections in a prospective matched cohort study in Italy, 2012-2013. The Journal of hospital infection. 2016;92(1):61-6.
Hu FP, Guo Y, Zhu DM, Wang F, Jiang XF, Xu YC, et al. Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005-2014. Clinical microbiology and infection: the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 2016;22 Suppl1:S9-14.
Bush K, Jacoby GA. Updated functional classification of beta-lactamases. Antimicrobial agents and chemotherapy. 2010;54(3):969-76.
Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical update. Clinical microbiology reviews. 2005;18(4):657-86.
Jacoby GA. AmpC beta-lactamases. Clinical microbiology reviews. 2009;22(1):161-82, Table of Contents.
Patel G, Bonomo RA. "Stormy waters ahead": global emergence of carbapenemases. Frontiers in microbiology. 2013; 4: 48.
Chen L, Mathema B, Chavda KD, DeLeo FR, Bonomo RA, Kreiswirth BN. Carbapenemase-producing Klebsiella pneumoniae: molecular and genetic decoding. Trends in microbiology. 2014;22(12):686-96.
Zhang R, Liu L, Zhou H, Chan EW, Li J, Fang Y, et al. Nationwide Surveillance of Clinical Carbapenem-resistant Enterobacteriaceae (CRE) Strains in China. EBioMedicine. 2017; 19:98-106.
Cambray G, Guerout AM, Mazel D. Integrons. Annual review of genetics. 2010; 44:141-66.
Jové T, Da Re S, Denis F, Mazel D, Ploy MC. Inverse correlation between promoter strength and excision activity in class 1 integrons. PLoS genetics. 2010;6(1): e1000793.
Zhou J, Li G, Ma X, Yang Q, Yi J. Outbreak of colonization by carbapenemase-producing Klebsiella pneumoniae in a neonatal intensive care unit: Investigation, control measures and assessment. American journal of infection control. 2015;43(10):1122-4.
Russo TA, Marr CM. Hypervirulent Klebsiella pneumoniae. Clinical microbiology reviews. 2019;32(3).
Zhang Y, Zhao C, Wang Q, Wang X, Chen H, Li H, et al. High Prevalence of Hypervirulent Klebsiella pneumoniae Infection in China: Geographic Distribution, Clinical Characteristics, and Antimicrobial Resistance. Antimicrobial agents and chemotherapy. 2016;60(10):6115-20.
Wayne, PA: CLSI. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing, 30th edition. CLSI M100. 2020 (in press and on-line).
Lee K, Chong Y, Shin HB, Kim YA, Yong D, Yum JH. Modified Hodge and EDTA-disk synergy tests to screen metallo-beta-lactamase-producing strains of Pseudomonas and Acinetobacter species. Clinical microbiology and infection: the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 2001;7(2):88-91.
Sfeir MM, Hayden JA, Fauntleroy KA, Mazur C, Johnson JK, Simner PJ, et al. EDTA-Modified Carbapenem Inactivation Method: a Phenotypic Method for Detecting Metallo-β-Lactamase-Producing Enterobacteriaceae. Journal of clinical microbiology. 2019;57(5).
Pierce VM, Simner PJ, Lonsway DR, Roe-Carpenter DE, Johnson JK, Brasso WB, et al. Modified Carbapenem Inactivation Method for Phenotypic Detection of Carbapenemase Production among Enterobacteriaceae. Journal of clinical microbiology. 2017;55(8):2321-33.
Xiang T, Chen C, Wen J, Liu Y, Zhang Q, Cheng N, et al. Resistance of Klebsiella pneumoniae Strains Carrying bla (NDM-1) Gene and the Genetic Environment of bla (NDM-1). Frontiers in microbiology. 2020; 11:700.
Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. Journal of microbiological methods. 2005;63(3):219-28.
Poirel L, Dortet L, Bernabeu S, Nordmann P. Genetic features of blaNDM-1-positive Enterobacteriaceae. Antimicrobial agents and chemotherapy. 2011;55(11):5403-7.
Mishra S, Upadhyay S, Sen MR, Maurya AP, Choudhury D, Bhattacharjee A. Genetic acquisition of NDM gene offers sustainability among clinical isolates of Pseudomonas aeruginosa in clinical settings. PloS one. 2015;10(1): e0116611.
Jia X, Dai W, Ma W, Yan J, He J, Li S, et al. Carbapenem-Resistant E. cloacae in Southwest China: Molecular Analysis of Resistance and Risk Factors for Infections Caused by NDM-1-Producers. Frontiers in microbiology. 2018; 9:658.
Diancourt L, Passet V, Verhoef J, Grimont PA, Brisse S. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. Journal of clinical microbiology. 2005;43(8):4178-82.
Zhou K, Xiao T, David S, Wang Q, Zhou Y, Guo L, et al. Novel Subclone of Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 11 with Enhanced Virulence and Transmissibility, China. Emerging infectious diseases. 2020;26(2):289-97.
Zheng R, Zhang Q, Guo Y, Feng Y, Liu L, Zhang A, et al. Outbreak of plasmid-mediated NDM-1-producing Klebsiella pneumoniae ST105 among neonatal patients in Yunnan, China. Annals of clinical microbiology and antimicrobials. 2016; 15:10.
Paul D, Dhar D, Maurya AP, Mishra S, Sharma GD, Chakravarty A, et al. Occurrence of co-existing bla VIM-2 and bla NDM-1 in clinical isolates of Pseudomonas aeruginosa from India. Annals of clinical microbiology and antimicrobials. 2016; 15:31.
Chen Z, Wang Y, Tian L, Zhu X, Li L, Zhang B, et al. First report in China of Enterobacteriaceae clinical isolates coharboring blaNDM-1 and blaIMP-4 drug resistance genes. Microbial drug resistance (Larchmont, NY). 2015;21(2):167-70.
Xie L, Dou Y, Zhou K, Chen Y, Han L, Guo X, et al. Coexistence of bla(OXA-48) and Truncated bla(NDM-1) on Different Plasmids in a Klebsiella pneumoniae Isolate in China. Frontiers in microbiology. 2017; 8:133.
Liu X, Zhang J, Li Y, Shen Q, Jiang W, Zhao K, et al. Diversity and frequency of resistance and virulence genes in bla (KPC) and bla (NDM) co-producing Klebsiella pneumoniae strains from China. Infection and drug resistance. 2019; 12:2819-26.
Politi L, Gartzonika K, Spanakis N, Zarkotou O, Poulou A, Skoura L, et al. Emergence of NDM-1-producing Klebsiella pneumoniae in Greece: evidence of a widespread clonal outbreak. The Journal of antimicrobial chemotherapy. 2019;74(8):2197-202.
Khan AU, Maryam L, Zarrilli R. Structure, Genetics and Worldwide Spread of New Delhi Metallo-β-lactamase (NDM): a threat to public health. BMC microbiology. 2017;17(1):101.
Liu J, Yu J, Chen F, Yu J, Simner P, Tamma P, et al. Emergence and establishment of KPC-2-producing ST11 Klebsiella pneumoniae in a general hospital in Shanghai, China. European journal of clinical microbiology & infectious diseases: official publication of the European Society of Clinical Microbiology. 2018;37(2):293-9.
Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL. Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clinical microbiology reviews. 2012;25(4):682-707.
Peirano G, Bradford PA, Kazmierczak KM, Chen L, Kreiswirth BN, Pitout JD. Importance of Clonal Complex 258 and IncF(K2-like) Plasmids among a Global Collection of Klebsiella pneumoniae with bla(KPC). Antimicrobial agents and chemotherapy. 2017;61(4).
Shen P, Wei Z, Jiang Y, Du X, Ji S, Yu Y, et al. Novel genetic environment of the carbapenem-hydrolyzing beta-lactamase KPC-2 among Enterobacteriaceae in China. Antimicrobial agents and chemotherapy. 2009;53(10):4333-8.
Zhu J, Sun L, Ding B, Yang Y, Xu X, Liu W, et al. Outbreak of NDM-1-producing Klebsiella pneumoniae ST76 and ST37 isolates in neonates. European journal of clinical microbiology & infectious diseases: official publication of the European Society of Clinical Microbiology. 2016;35(4):611-8.
Huang X, Cheng X, Sun P, Tang C, Ni F, Liu G. Characteristics of NDM-1-producing Klebsiella pneumoniae ST234 and ST1412 isolates spread in a neonatal unit. BMC microbiology. 2018;18(1):186.
Yu J, Wang Y, Chen Z, Zhu X, Tian L, Li L, et al. Outbreak of nosocomial NDM-1-producing Klebsiella pneumoniae ST1419 in a neonatal unit. Journal of global antimicrobial resistance. 2017; 8:135-9.
Kong Z, Cai R, Cheng C, Zhang C, Kang H, Ma P, et al. First Reported Nosocomial Outbreak Of NDM-5-Producing Klebsiella pneumoniae In A Neonatal Unit in China. Infection and drug resistance. 2019; 12:3557-66.
Logan LK. Carbapenem-resistant enterobacteriaceae: an emerging problem in children. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2012;55(6):852-9.
Porto A, Ayala J, Gutkind G, Di Conza J. A novel OXA-10-like beta-lactamase is present in different Enterobacteriaceae. Diagnostic microbiology and infectious disease. 2010;66(2):228-9.
Akgul F, Bozkurt I, Sunbul M, Esen S, Leblebicioglu H. Risk factors and mortality in the Carbapenem-resistant Klebsiella pneumoniae infection: case control study. Pathogens and global health. 2016;110(7-8):321-5.
Jiao Y, Qin Y, Liu J, Li Q, Dong Y, Shang Y, et al. Risk factors for carbapenem-resistant Klebsiella pneumoniae infection/colonization and predictors of mortality: a retrospective study. Pathogens and global health. 2015;109(2):68-74.
Zhang Y, Guo LY, Song WQ, Wang Y, Dong F, Liu G. Risk factors for carbapenem-resistant K. pneumoniae bloodstream infection and predictors of mortality in Chinese paediatric patients. BMC infectious diseases. 2018;18(1):248.
Zheng X, Wang JF, Xu WL, Xu J, Hu J. Clinical and molecular characteristics, risk factors and outcomes of Carbapenem-resistant Klebsiella pneumoniae bloodstream infections in the intensive care unit. Antimicrobial resistance and infection control. 2017; 6:102.
Falagas ME, Rafailidis PI, Kofteridis D, Virtzili S, Chelvatzoglou FC, Papaioannou V, et al. Risk factors of carbapenem-resistant Klebsiella pneumoniae infections: a matched case control study. The Journal of antimicrobial chemotherapy. 2007;60(5):1124-30.
Hu Y, Ping Y, Li L, Xu H, Yan X, Dai H. A retrospective study of risk factors for carbapenem-resistant Klebsiella pneumoniae acquisition among ICU patients. Journal of infection in developing countries. 2016;10(3):208-13.
Li Y, Li J, Hu T, Hu J, Song N, Zhang Y, et al. Five-year change of prevalence and risk factors for infection and mortality of carbapenem-resistant Klebsiella pneumoniae bloodstream infection in a tertiary hospital in North China. Antimicrobial resistance and infection control. 2020;9(1):79.

Table 1 Susceptibility of 66 CKRP strains to antimicrobial agents.

Antimicrobial agent	No.	%R	No.	%I	No.	%S
Amikacin	36	54.5	0	0	30	45.5
Cefepime	66	100	0	0	0	0
Imipenem	66	100	0	0	0	0
Piperacillin/tazobactam	66	100	0	0	0	0
Ceftriaxone	66	100	0	0	0	0
Ampicillin/sulbactam	66	100	0	0	0	0
Cefotaxime	66	100	0	0	0	0
Cefazolin	66	100	0	0	0	0
Cefuroxime	66	100	0	0	0	0
Ertapenem	66	100	0	0	0	0
Tobramycin	55	83.3	4	6.1	7	10.6
Sulperazon	63	95.5	2	3.0	1	1.5
Ceftazidime	66	100	0	0	0	0
Meropenem	66	100	0	0	0	0
Levofloxacin	54	81.9	0	0	12	18.1
Cefoxitin	66	100	0	0	0	0
Aztreonam	58	87.9	0	0	8	12.1
Ciprofloxacin	53	80.3	0	0	13	19.7
Gentamicin	55	83.3	0	0	11	16.7
Tigecycline	1	1.5	0	0	65	98.5
Compound sulfamethoxazole	43	65.2	0	0	23	34.8

Table 2 Genotypes of the 66 CRKP strains.

N	Carbapenemase	ESBLs	AmpC	Int	Virulence gene	Gene Cassette	ISCR	MLST





16	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2	ISCR1	ST-11
26	NDM-1	SHV+CTX-M	DHA		entB+mrkD		ISCR1	ST4495
27	NDM-1	SHV+CTX-M	DHA		entB+fimH+mrkD		ISCR1	ST4495
30	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2	ISCR1	ST-11
32	NDM-1	SHV+CTX-M	DHA		entB+fimH+mrkD		ISCR1	ST4495
34	NDM-1	SHV+CTX-M	DHA		entB+fimH+mrkD		ISCR1	ST4495
36	NDM-1	SHV+CTX-M	DHA		entB+fimH+mrkD		ISCR1	ST4495
37	NDM-1	SHV+CTX-M	DHA		entB+fimH+mrkD		ISCR1	ST4495
39	NDM-1	SHV	DHA		entB+mrkD		ISCR1	ST4495
40	NDM-1	SHV	DHA+ACC		entB+fimH		ISCR1	ST37
45	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
46	NDM-1	SHV+CTX-M	DHA+ACC		entB+fimH+mrkD		ISCR1	ST4495
49	NDM-1	SHV+CTX-M	DHA+ACC		entB+fimH+mrkD		ISCR1	ST4495
51	KPC-2	SHV+CTX-M	ACC		entB+fimH+mrkD			ST11
56	NDM-1	SHV+CTX-M	DHA+ACC		entB+fimH+mrkD		ISCR1	ST4495
57	NDM-1	SHV+CTX-M	DHA+ACC		entB+fimH+mrkD		ISCR1	ST4495
58	NDM-1+IMP-4	SHV+CTX-M	DHA+ACC		entB+fimH			ST4495
101	NDM-1	SHV	ACC		entB+fimH+mrkD			ST-37
131	KPC-2	SHV+TEM	ACC	IntI1	entB+fimH+mrkD+ rmpA/rmpA2+ iucA+terB+aerobactin+HI1B+iroN+iutA	addA2	ISCR1	ST-11
131	KPC-2	SHV+TEM	ACC	IntI1		addA2	ISCR1	ST-11
210	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
211	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
214	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
215	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
221	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2	ISCR1	ST-11
223	KPC-2	SHV+TEM+CTX-M	ACC		entB+fimH+mrkD	dfrA12, addA2		ST-147
227	KPC-2	SHV+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
228	KPC-2	SHV+CTX-M	ACC		entB+fimH+mrkD			ST-11
230	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
232	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
233	KPC-2	SHV	ACC	IntI1	entB+fimH+mrkD	dfrA12, addA2		ST-147
234	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
241	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
244	KPC-2	SHV	ACC	IntI1	entB+fimH+mrkD	ddfrA12		ST-23
245	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
251	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
252	KPC-2	SHV+TEM+CTX-M		IntI1	entB+fimH+mrkD	addA2		ST-11
257	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
258	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
267	NDM-1	SHV	ACC	IntI1	entB+fimH+mrkD	addA2	ISCR1	ST-11
271	KPC-2	SHV+TEM+CTX-M	ACC		entB+fimH+mrkD			ST-11
285	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
354	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
406	KPC-2	SHV+TEM	DHA	IntI1	fimH+mrkD	OXA-10, addA1, aacA4	ISCR1	ST-318
436	KPC-2	SHV+TEM	DHA	IntI1	entB+fimH+mrkD	addA2		ST-17
440	NDM-1	SHV+TEM	ACC	IntI1	mrkD	addA2		ST-307
445	NDM-1	SHV+TEM	ACC		mrkD			ST-307
473	NDM-1+IMP-4	SHV+TEM	ACC	IntI1	entB+fimH+mrkD	addA2	ISCR1	ST-152
475	NDM-1	SHV+TEM	ACC	IntI1	fimH+mrkD	addA2	ISCR1	ST-15
478	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2	ISCR1	ST-11
479	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH	addA2		ST-11
480	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
483	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
489	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	dfrA12, addA2		ST-11
490	KPC-2	SHV	ACC	IntI1	entB+fimH+mrkD	dfrA27, arr3		ST-467
494	NDM-5	SHV+CTX-M	DHA	IntI1	entB+fimH+mrkD	addA2	ISCR1	ST-2407
495	KPC-2	SHV+TEM+CTX-M		IntI1	entB+fimH+mrkD	ddfrA12	ISCR1	ST-11
496	KPC-2	SHV+TEM	ACC	IntI1	entB+fimH+mrkD	gcuF, dfrA12		ST-405
497	KPC-2	SHV+TEM		IntI1	entB+fimH+mrkD+rmpA/rmpA2+ iucA+ terB+wcaG+ aerobactin+ HI1B+iroN+iutA	addA2		ST-11
497	KPC-2	SHV+TEM		IntI1		addA2		ST-11
498	KPC-2	SHV+CTX-M	ACC	IntI1	entB+mrkD	ddfrA12		ST-11
499	NDM-5	SHV+CTX-M	DHA	IntI1	entB+fimH+mrkD	addA2	ISCR1	ST-2407
500	KPC-2	SHV+TEM+CTX-M	ACC		entB+fimH+mrkD			ST-11
502	NDM-5	SHV+CTX-M	DHA	IntI1	entB+fimH+mrkD	addA2		ST2407
504	NDM-5+IMP-4	SHV+TEM+CTX-M	DHA	IntI1	entB+fimH+mrkD	addA2		ST-11
505	KPC-2	SHV+TEM+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11
506	NDM-1	SHV+CTX-M	DHA	IntI1	entB+fimH+mrkD	addA2		ST4495
507	KPC-2	SHV+CTX-M	ACC	IntI1	entB+fimH+mrkD	addA2		ST-11

Table 3 The allelic profile and STs of 66 CRKP strains detected in this study.

Number of isolates	ST	gapA	infB	mdh	pgi	phoE	ropB	tonB
37	11	3	3	1	1	1	1	4
13	4495^a	4	1	99	1	9	5	5
3	2407	2	1	99	1	9	5	5
2	147	3	4	6	1	7	4	38
2	307	4	1	2	52	1	1	7
2	37	2	9	2	1	13	1	16
1	15	1	1	1	1	1	1	1
1	17	2	1	1	1	4	4	4
1	23	2	1	1	1	9	4	12
1	405	2	1	62	3	10	4	110
1	318	16	24	21	31	68	22	67
1	467	16	24	21	27	76	22	67
1	152	2	3	2	1	1	4	56

^aNovel ST detected in this study.

Table 4. Univariable analysis of risk factors and outcomes associated with CRKP infections.

Variable	CRKP (n=66)	CSKP (n=66)	P-value
Demographic, n (%) or IQR
age (years)	44.5 (0,60)	46.5 (0.61)	0.754
sex (male)	47 (71.2%)	46 (69.7%)	0.894
length of hospital stay	15 (7,27)	25 (10,38)	0.1
admission to ICU	14 (21.1%)	7 (10.6%)	0.096
Co-morbidity, n (%)
malignant disease	7 (10.6%)	15 (22.7%)	0.062
diabetes mellitus	11 (16.7%)	12 (18.2%)	0.819
hypertension	16 (24.2%)	16 (24.2%)	1
heart disease	9 (13.6%)	11 (16.7%)	0.627
hepatobiliary disease	12 (18.2%)	15 (22.7%)	0.517
respiratory disease	45 (68.2%)	33 (50%)	0.034
renal disease	7 (10.6%)	16 (24.2%)	0.039
urinary tract infection	10 (15.2%)	9 (13.6%)	0.804
craniocerebral disease	15 (22.7%)	9 (13.6%)	0.176
hematological disease	32 (48.5%)	19 (28.8%)	0.02
gastrointestinal disease	8 (12.1%)	5 (7.6%)	0.381
Invasive procedures and devices
tracheal cannula	24 (36.4%)	7 (10.6%)	<0.001
central venous catheter	5 (7.6%)	12 (18.2%)	0.069
foreign material in the body	14 (21.1%)	7 (10.6%)	0.096
Surgical operations after admission	9 (13.6%)	16 (24.2%)	0.12
colostomy	3 (4.5%)	1 (1.5%)	0.612
gastrostomy	2 (3%)	0	0.476
Prior antibiotic use, n (%).
penicillins	2 (3%)	5 (7.6%)	0.437
first, second-generation cephalosporins	7 (10.6%)	15 (22.7%)	0.21
third, fourth-generation cephalosporins	17 (25.6%)	19 (28.8%)	0.696
aminoglycosides	4 (6.1%)	1 (1.5%)	0.362
quinolones	20 (30.3%)	21 (31.8%)	0.851
metronidazole	0	1 (1.5%)	1
glycopeptides	0	0	NA
carbapenems	30 (45.5%)	32 (48.5%)	0.727
β-lactams and β-lactamase inhibitor combination	40 (60.1%)	25 (37.9%)	0.009
Clinical outcomes, n (%)
patient outcome: mortality	5 (7.6%)	0	0.068

Note: Bold face indicates values that are significant (P < 0.05). CR, carbapenem-resistant; CS, carbapenem-susceptible; interquartile range; ICU, intensive care unit.

Table 5. Multivariate analysis of risk factors for CRKP infections.

Factors	Odds ratio	95%, Confidence interval	P-value
hematological disease	2.568	(1.106,5.568)	0.028
tracheal cannula	4.883	(1.797,13.265)	0.002
prior β-lactams and β-lactamase inhibitor combination use	4.271	(1.760,10.365)	0.001

Supplement.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Carbapenem-Resistant Klebsiella Pneumoniae in Southwest China: Molecular Characterizations and Risk Factors Caused by NDM and KPC Producers

Status:

Version 1

Abstract

Figures

Introductin

Materials And Methods

Statistical analysis.

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Tables

Supplementary Files

Status:

Version 1