Carbapenem-resistant Klebsiella pneumoniae (CRKP) has received increasing attention worldwide because of the widespread misuse of carbapenem antibiotics, with CRKP difficult to treat (Gong et al., 2018; Yungyuen et al., 2021). Several factors contribute to the development of CRKP resistance, among which carbapenemase-producing enzymes are the most predominant resistance mechanism (Conte et al., 2016; El-Badawy et al., 2020; Zhang et al., 2021). In 1996, the bla _KPC gene was first identified in the United States (Yigit et al., 2001). The CRKP producing KPC enzyme was the most common CRKP isolate in the global outbreak, causing serious endemic epidemics in Europe, America, Asia, and the Middle East (Munoz-Price et al., 2013; Aires-De-Sousa et al., 2019; Rodrigues et al., 2019; Yu et al., 2019). In China, the detection rate of bla _KPC-2 is approximately 73% (Zhang et al., 2017). Previous studies have shown that the highest detection rate of bla _NDM among K. pneumoniae isolates was in China, where the detection rate was as high as 44.1% (Safavi et al., 2020). The first detection of the OXA-48 enzyme in CRKP was found in a urine sample from a Turkish patient (Poirel et al., 2004), and more than 50 countries reported the OXA-48 outbreak, including Taiwan, Zhejiang, and other areas in China (Lu et al., 2018; Shu et al., 2019).

A case of liver abscess caused by hypervirulent K. pneumoniae (hvKP) infection was first reported in 1986 in Taiwan Province, China (Liu et al., 1986). Since then, hvKP has been reported in many countries, including China, South Korea, Japan, Spain, Madagascar, Cambodia, and Senegal (Cubero et al., 2016; Zhang Y. et al., 2016; Harada et al., 2019; Yoon et al., 2019; Huynh et al., 2020; Parrott et al., 2021). A defining feature of hvKP is its hypermucoid appearance on agar plates. With its unique phenotypic (such as high mucus type and special serotype) and genotypic (such as carrying special virulence genes) characteristics, hvKP has a strong clinical pathogenicity. It can cause infection in patients with low immune function and severe community-acquired infection in young people with normal immune function, and can present migratory spread (Russo and Marr, 2019). HvKP has emerged because of several mechanisms, including pili, lipopolysaccharides, capsular polysaccharides, virulence factors and iron ingestion (Cheng et al., 2010; Lee et al., 2017; Chen et al., 2020). Patients infected with hvKP often show rapid symptom onset and an insidious disease course, making treatment more difficult (Shon and Russo, 2012; Zhang R. et al., 2016; Rahim et al., 2019). Resistance due to CRKP carrying virulence factors increases the failure rate of patient treatment. Therefore, understanding the virulence of CRKP in hospital isolates is vital.

Ceftazidime-avibactam (CAZ-AVI) is currently an effective treatment for carbapenem-resistant Enterobacteriaceae infection and has been approved for treating adults with complicated urinary tract infections (including pyelonephritis), complicated intra-abdominal infections, hospital-acquired pneumonia (including ventilator-associated pneumonia), and other infections caused by aerobic gram-negative bacteria. Avibactam (AVI), an enzyme inhibitor of triethylenediamine, does not contain a beta-lactam ring, and its enzyme inhibition spectrum is broad. AVI acylates the serine residue of beta-lactamase via noncovalent bonding with the beta-lactamase binding region, thus forming an active covalent compound, and the generated product does not undergo hydrolysis. Thus, the intervention has a long-acting enzyme inhibitory effect (Zhanel et al., 2013). In the CAZ-AVI combination, AVI protects the CAZ from degradation via various serine beta-lactamases, thus promoting CAZ-AVI activity. However, many cases of CAZ-AVI resistance have been reported since CAZ-AVI was approved in 2015 (Shields et al., 2016; Wang et al., 2020). The isolates producing metallo-β-lactamases such as bla _NDM are not active against CAZ-AVI (Zhang et al., 2020), and the detection rates of bla _NDM are different in different populations. Therefore, detecting the in vitro sensitivity of CAZ-AVI to CRKP would aid in understanding the clinical application of CAZ-AVI in CRKP treatment and predicting the application prospects of CAZ-AVI in future clinical treatments.

Sequence type 258 (ST258), an international high-risk epidemic CRKP lineage, is frequent in European and American countries (Wyres et al., 2020), whereas Sequence type 11 is the most common CRKP clone in China, and is part of the clonal complex 258 (CC258) and is a single-locus variant of ST258 (Liao et al., 2020).

We aimed to study a CRKP collection isolated at the Yongchuan Hospital of Chongqing Medical University, revealing their clinical characteristics, determining their resistance and virulence factors, and investigating the possible underlying resistance mechanisms.

As expected, the department with the highest prevalence of CRKP was the ICU, and the major source was sputum (Zhang et al., 2021). The duration of hospital stay in the same department might indicate that the isolates were disseminated within the departments. All mortality occurred in the ICU (10/11) or respiratory and critical care medicine departments; however, the patients rarely had underlying diseases. We hypothesized that mortality was closely associated with complications.

Among the 13 children and 114 adults, the resistance rate to CAZ-AVI was significantly higher in children than in adults. Therefore, in the clinical use of CAZ-AVI, attention must be paid to this result. In addition, the detection rate of bla _NDM in children was significantly higher than that in adults. Our results are similar to those of a previous study indicating that acquiring a bla _NDM-5-harboring plasmid led to resistance to CAZ-AVI in KPC-2-producing K. pneumoniae during treatment (Huang et al., 2021). With the CAZ-AVI combination, AVI protects CAZ from degradation by various serine beta-lactamases, thus promoting CAZ-AVI activity. Therefore, the two isolates that contained only bla _KPC and bla _TEM but were resistant to CAZ-AVI require further investigation.

Polymyxins (including colistin and polymyxin B) are the last resort for treating carbapenem-resistant Enterobacteriaceae infections (Macesic et al., 2020); therefore, the resistance in 42.5% of isolates in our study was concerning. A plasmid-mediated polymyxin resistance gene, mcr-1, was first reported in 2015 (Liu et al., 2016), and China had the highest prevalence of mcr-positive isolates (Nang et al., 2019). However, mcr-1 was not detected in any of the isolates. The reasons for this resistance require further study.

Regarding resistance genes, most (98.4%) isolates had more than two genes, indicating that the multiple resistance of CRKP may be because of multiple gene interactions. In the present study, bla _KPC-2 was the main carbapenem resistance gene, which agrees with the current prevalence (Hu et al., 2020). Furthermore, the environment surrounding bla _KPC-2 as IS26-tnpR-ISkpn27-KPC-2-ISkpn6-IS26-Tn3 was different from the transposon Tn4401 popular abroad (Bowers et al., 2015) and the transposon Tn1721 popular in china (Wang et al., 2015; Yang et al., 2020). It is a typical plasmid-mediated antibiotic resistance gene that is widely distributed in plasmids of different sizes and types (Jain et al., 2013; Pitout et al., 2015), including IncF, IncI, IncA/C, IncN, IncX, IncR, IncP, IncU, IncW, IncL/M, and ColE (Chen et al., 2014). Based on the differences in plasmid replication, bla _KPC exists mainly in the IncF plasmid in eastern China (Hu et al., 2020). Our results showed that when bla _KPC-2 and plasmids were detected simultaneously, the plasmid was IncF; therefore, the results were consistent with those from previous studies. IncF + IncFIIA appeared in all isolates carrying bla _KPC-2, indicating that plasmids have strong mobility and can be transferred between isolates, causing that spread and diffusion of antibiotic resistance.

KPC resistance plasmids simultaneously contain various virulence and retention factors, thus significantly improving the adaptability of KPC-producing strains to the external environment and facilitating the spread of KPC (Dong et al., 2018). uge, mrkD, kpn, and fim-H were present in almost all CRKP isolates in the present study. A previous study showed that fim-H and mrkD encode distinct types of fimbriae that often cause respiratory and urinary tract infections (Yan et al., 2016). Our clinical data showed that 79.0% of the patients had lung diseases, and 71.7% of isolates were from the respiratory tract. Several studies have indicated that isolates coexpressing the mucus phenotype coding gene (rmpA) and aerobin gene (aero) can be considered hvKP (Siu et al., 2012). Two isolates in our study, belonging to ST1373 and ST23 can be defined as hvKP based on this condition. The detection rate of hvKP in the present study was only 1.6%, indicating that the CRKP isolates in this hospital were not yet highly virulent. This result is consistent with those of our previous study in the ICU (Zeng et al., 2021). Several studies have observed that hvKP over time has been susceptible to antibiotics and related mainly to community-acquired infections (Klaper et al., 2021). However, there are reports where this has been reversed and hvKP with antibiotic resistance and involved in infections associated with hospital care are gradually beginning to appear (Gu et al., 2018).

Based on a comparison of MLST and ERIC-PCR, we concluded that all isolates from the hospital had a high degree of homology, particularly in ST11 (81.1%), in agreement with the prevalence in China (Qi et al., 2011; Liao et al., 2020). ST11 has recently been reported elsewhere in China, causing fatal infections and high mortality rates in other hospitals (Hu et al., 2020; Qiao et al., 2020). We concluded that all isolates had high homology based on the similarity coefficients of different clusters by ERIC-PCR. One novel allele of locus infB in MLST was found in the present study, which was defined as infB-236. In addition, three new STs were identified. ST1887 CRKP is a ST commonly found in our hospital, accounting for 3.1% of cases. All four ST1887 isolates expressed the bla _NDM and bla _TEM resistance genes, and the virulence genes of uge, fim-H, mrkD, and allS, which were higher than those in ST11 CRKP, particularly bla _NDM (p < 0.0001) and allS (p < 0.0001) ( Table 1 ).

In summary, ST11 CRKP isolates with bla _KPC-2 remain widespread in western Chongqing, southwestern China, and transmission within the hospital should be monitored, and effectively controlled. Effective measures should be taken to prevent further expansion of multi-drug resistant bacteria.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, MZ156798.

The studies involving human participants were reviewed and approved by The Ethics Committee of Yongchuan Hospital of ChongQing Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

BL and XZ conceived of and designed the study. WH and JZ wrote this paper and contributed equally to this work. LZ, CY, LY, and KH performed the experiments. JW, JL, and XL analyzed the data. All authors contributed to the article and approved the submitted version.

This work was supported by General projects of Chongqing Natural Science Foundation (cstc2020jcyj-msxm0067), Yongchuan Natural Science Foundation (2021yc-jckx20053) and Talent introduction project of Yongchuan Hospital of Chongqing Medical University (YJYJ202005, YJYJ202004).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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