Drug Resistance Genes and Molecular Epidemiological Characteristics of Carbapenem-Resistant Klebsiella pneumonia

Introduction

Klebsiella pneumoniae (K. pneumoniae) is one of the most common gram-negative opportunistic pathogens, which can cause respiratory tract infection, urinary tract infection, bloodstream infection and abdominal infection. Carbapenems and other β-lactam antibiotics are normally thought to be a good choice for the treatment of multi-drug resistant gram-negative bacterial infections. But due to the large or unreasonable use of β-lactam antibiotics, extended-spectrum beta-lactamase (ESBLs) and carbapenemase emerge which are capable of hydrolysis of carbapenems. Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged subsequently and become a threat to public health, presenting rapid spread over global,¹ which can cause higher morbidity and mortality.^2,3 There were big differences with the resistance and prevalence of CRKP in different regions of China,⁴ and the reports from Jiangxi China are limited, which brings great challenges to anti-infection treatment and nosocomial infection control. In our study, we analyzed the homology and the evolutionary relationship between isolates through genotyping and sequencing, and mastered the genome epidemiological characteristics of CRKP, so as to reveal the epidemic trend of CRKP and provide a basis for antibiotics selection, effectively reducing the nosocomial transmission of CRKP.

Materials and Methods

Data Collection

CRKP isolates were those that were resistant to at least one of the carbapenems, including imipenem, meropenem and ertapenem. We collected 42 non-repetitive clinical isolates of CRKP from the First Affiliated Hospital of Gannan Medical University (Ganzhou, China) from January 2018 to February 2021, which all were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, Bruker Daltonics, Germany). The patients were composed of 33 males and 9 females, aged from 12 to 91 years old, with an average age of (57.6±18.8) years old.

Antimicrobial Susceptibility Testing (AST)

Forty-two CRKP isolates were tested for antimicrobial susceptibility by the VitekII Compact system (bioMérieux, Marcy l’Etoile, Lyon, France) and the K-B disk diffusion method, with the results based on the standards of the Clinical and Laboratory Standards Institute (CLSI) 2021-M100.⁵ Quality control strains were Escherichia coli ATCC25922 and K. pneumoniae ATCC700603 (purchased from China National Health Inspection Center).

Detection of Resistance Genes

All of template DNAs were extracted by bacterial DNA Kit (Sangon Biotech, Shanghai, China) and used to detect carbapenemase genes (bla_KPC, bla_IMP, bla_NDM, bla_VIM, and bla_OXA-48) and ESBLs genes (bla_SHV, bla_TEM, and bla_CTX-M) by PCR. The primers⁶ are shown in Table 1. The PCR conditions were described as 95°C pre-denaturation for 5 mins, 94°C denaturation for 30s, 55°C annealing for 30s, 72°C extension for 60s, a total of 35 cycles; 72°C extension for 8 min at last. The positive products were sequenced by Shanghai Sangon Biotechnology, and the results were analyzed by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Table 1 Primers for Resistance Genes

Molecular Epidemiological Study

PFGE is a method to separate different-sized restricted fragments of bacterial DNA and analyzes the electrophoresis pattern to determine the strain type according to the typing standard.⁷ PFGE images were entered into BioNumerics (Version 5.1, Appliedmaths, Inc.) software to identify image bands. Based on the phase between the two images, the similarity coefficient is used for clustering with the unweighted pair group average method (UPGMA) to construct a clustering tree. MLST was performed to explore the genetic correlation of all strains. Seven housekeeping genes (gapA, mdh, pgi, phoE, infB, rpoB, and tonB) were amplified using oligonucleotides representing primer sequences provided by Institute Pasteur MLST (https://bigsdb.pasteur.fr/klebsiella/) (Table 2). The positive products were sequenced by Shanghai Sangon Biotechnology, and the results were submitted to MLST database (https://bigsdb.pasteur.fr/klebsiella/) for comparison.

Table 2 Primer Sequence of K. pneumoniae Housekeeping Genes

Statistical Analysis

The data were analyzed with SPSS for Windows version 22.0 (SPSS Inc., Chicago, IL, USA).

Results

Patients Data and Distribution of CRKP Isolates

The 42 CRKP isolates mainly originated from the sputum (17/42, 40.5%), followed by bronchoalveolar lavage fluid (8/42, 19.1%), urine (7/42, 16.7%), blood (3/42, 7.1%), and secretion (3/42, 7.1%) (Figure 1A). The patients were composed of 33 males (78.6%) and 9 females (21.4%), aged from 12 to 91 years old, with an average of (57.6±18.8) years old. Of them, 38.1% (16/42) were from intensive care unit (ICU) and 9.5% (4/42) each were from neurosurgery department and VIP department (Figure 1B), of which 70.8% of specimens (17/24) were from respiratory tract.

Figure 1 Distribution of 42 CRKP isolates: (A) source of isolates and (B) departments.

Antimicrobial Susceptibility Testing (AST)

The 42 isolates demonstrated multi-drug resistance to most of the antimicrobial agents tested, with resistance to amoxicillin-clavulanate, cefuroxime, ceftriaxone, and ceftazidime reaching 100%. The resistance to piperacillin-tazobactam, cefoperazone sulbactam, cefepime, ertapenem, imipenem, meropenem, amikacin, levofloxacin, and trimethoprim-sulfamethoxazole was 88.1%, 97.6%, 97.6%, 92.8%, 83.3%, 90.5%, 21.4%, 61.9%, and 81.0%, respectively. No tigecycline-resistant isolates were detected (Table 3).

Table 3 The Drug Resistance Rate of 42 CRKP Isolates

Major CRKP Resistance Genes

Among the 42 CRKP isolates, 36 (85.7%) produced carbapenemase, of which 83.3% of isolates (35/42) producing bla_KPC-2. 33.3% of isolates (14/42) produced bla_NDM, with 12 isolates (28.6%, 12/42) carrying bla_NDM-1 and 2 isolates (4.7%, 2/42) carrying bla_NDM-5. The detection rate of bla_IMP-4 and bla_OXA-48 was 2.4% (1/42), respectively. Twelve isolates (28.6%) carried both bla_KPC-2 and bla_NDM-1, one isolate (2.4%) carried both bla_KPC-2 and bla_IMP-4, and one isolate (2.4%) carried bla_KPC-2, bla_NDM-1, and bla_OXA-48. The bla_VIM gene was not detected in any isolate. A variety of other extended-spectrum beta-lactamases (ESBLs) were also detected. All 42 isolates carried bla_SHV, which were mainly bla_SHV-12 (61.9%, 26/42). Twenty-seven isolates (64.3%) carried bla_TEM, all were bla_TEM-1. All isolates carried bla_CTX-M-1, mainly bla_CTX-M-55 (59.5%, 25/42), and 12 isolates (28.6%) carried bla_CTX-M-9, mainly bla_CTX-M-65 (19.1%, 8/42) (Table 4). Some of the sequencing maps are shown in Figure 2.

Table 4 Resistance Genes of 42 CRKP Isolates

Figure 2 Sequencing map of blaKPC-2.

Molecular Typing Based on MLST and PFGE

The MLST results showed that 42 CRKP isolates were divided into 11 sequence types, which were mainly ST11, accounting for 61.9% (26/42), of which 92.3% of isolates (24/26) carrying bla_KPC-2. The remaining ST types were ST307 (5 strains), ST43 (2 strains), ST476 (2 strains), ST831 (1 strain), ST27 (1 strain), ST327 (1 strain), ST290 (1 strain), ST15 (1 strain), ST73 (1 strain), ST147 (1 strain). The 42 CRKP isolates were divided into 31 PFGE types, and 20 PFGE clusters A-T at the level of 85% similarity coefficient,⁷ with cluster A (26.2%, 11/42) and cluster H (21.4%, 9/42) dominating. The clonal subtypes of A₁, A₃, and H₆ were all ST11. However, 26 PFGE types contained only 1 isolate, that is, each of the 26 isolates had a unique PFGE type (Figure 3).

Figure 3 PFGE analysis.

Discussion

Carbapenems including imipenem and meropenem are usually used as effective antibiotics in the treatment of severe infections caused by multi-drug resistant K. pneumoniae. However, data from CARSS (China Antimicrobial Resistance Surveillance System) showed the resistance rate of K. pneumoniae to imipenem and meropenem has rapidly increased from 3.0% and 2.9% in 2005 to 25.0% and 26.3% in 2018.⁸ The number of CRKP isolates also has increased rapidly in the past decade. In China and the European countries, CRKP accounted for about 70%~90% of carbapenem-resistant enterobacteria (CRE).^9,10 In this study, the CRKP mainly isolated from sputum and bronchoalveolar lavage fluid, and the patients were mainly from ICU and neurosurgery, who had been treated with numerous antibiotics. The study showed that the CRKP isolates were extensively drug resistant, with resistance to third and fourth-generation cephalosporins highly reaching 100%, and the resistance to imipenem and meropenem were 83.3% and 90.5%, respectively. There were relatively consistent with the situation in Zhejiang, Guangdong, Sichuan China,^11–13 which extremely limited antibiotics selection in clinic. Clinical laboratories should jointly carry out more antibiotics sensitivity tests, including fosfomycin, chloramphenicol and sulbactam, etc, in order to find possible effective anti-infective antimicrobials, which are helpful to the accurate use of antibiotic and the prevention and control of CRKP infection in hospitals.

The resistance mechanisms mainly include producing carbapenemase, extended-spectrum beta-lactamases (ESBLs) or AmpC enzymes combined with structural mutations, such as the lack of outer membrane proteins and high expression of effluence pump genes or changes in penicillin-binding proteins.¹⁴ At present, carbapenemase production is the main mechanism and bla_KPC-2 is the most common resistance gene in K. pneumoniae in China.^10,15 Since the first report of K. pneumoniae producing bla_KPC-2 carbapenemase in Zhejiang China in 2007, CRKP has been reported all over the country, such as Beijing, Shanghai, Jiangsu, etc.¹⁶ Outbreaks of CRKP producing bla_KPC-2 carbapenemase also have been reported in the United States, Italy and Spain.^17,18 Our study depicted that 85.7% of isolates produced carbapenemase, of which bla_KPC-2 was detected in 83.3% of isolates, indicating that bla_KPC-2 production was the main resistant gene in our hospital, which was consistent with the research report that bla_KPC-2 is the main carbapenemase genotype carried by CRKP in China. bla_NDM-1 is a metallo-β-lactamase with strong hydrolytic activity in Enterobacter commonly, which can hydrolyze penicillin, cephalosporin, cephamycin, and other β-lactam antimicrobials. Isolates carrying bla_NDM-1 are multi-drug resistant, and it has been reported that bla_NDM-1 can be spread horizontally between different bacterial species through plasmids and integrons,¹⁹ becoming a “super bacteria”. In our study, the detection rate of bla_NDM-1 was 28.6%, it also indicated the widespread of bla_NDM-1 in our hospital, which was consistent with that bla_NDM-1 was the main genotype of CRKP in Hainan province, China,²⁰ but quite different from the reports that the detection rate of bla_NDM was only 1.7% in Changsha China⁶ and Shanghai China.²¹ So the present study indicated that there were regional differences in the prevalence of carbapenemase genes. Therefore, it is necessary to conduct multi-center and transverse investigations of drug resistance genes over the country.

Carbapenemase production isolates also carried multiple ESBLs (bla_SHV, bla_TEM, bla_CTX-M, etc.) simultaneously. Our study revealed that 61.9% and 64.3% of isolates carried bla_SHV-12 and bla_TEM-1 respectively, which was consistent with domestic research and abroad.^22,23 The subtypes of bla_CTX-M were mainly bla_CTX-M-55 (59.5%) and bla_CTX-M-65 (19.1%), which was quite different with the results from Shandong Province and Beijing China,^15,22 it may be related to clinical medication habits, the number of isolates and different regions. There were 64.3% of isolates carrying three types of ESBLs simultaneously, suggesting that the coexistence of multiple resistance genes may be an important cause of the extensive drug resistance of CRKP in our hospital.

The PFGE results showed that the 42 CRKP isolates were divided into 31 PFGE types and 20 PFGE clusters A-T, with cluster A (26.2%) and cluster H (21.4%) dominating; however, 26 isolates had a single and unique PFGE type, indicating the polymorphism and the complexity of the genetic background in our hospital. The result of MLST showed that 26 isolates (61.9%) were the dominant ST11 type, of which 92.3% of isolates (24/26) carrying bla_KPC-2, which was consistent with the domestic research result.²⁴ ST11 and ST258 belong to the CC258 clonal complex, with only one site difference between them. ST258 was less reported in China, also not found in our study, but experienced large-scale outbreaks in the United States and other countries,^4,25 which indicated there were differences in prevalence in China and abroad.

Conclusion

In summary, the CRKP in our hospital is resistant to most of common antibiotics and presents multi-drug resistance, so antibiotics selection needs to be rationalized and standardization. Mechanism of carbapenem resistance is related to different resistant genes, with bla_KPC-2 dominating and bla_NDM-1 needing to be focused on. ST11 is the main clonotype of CRKP. So ST11-bla_KPC-2 Klebsiella pneumoniae extremely needed to be focused on clinical management and effective measures to prevent and control further dissemination. Therefore, it is necessary to strengthen the antibiotic resistance monitoring and conduct epidemiological investigation termly.