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04 June 2024: Lab/In Vitro Research  

Molecular Prevalence and Geographical Variations of Carbapenem-Resistant ST15 Isolates in a Tertiary Hospital in Ningbo, China

Hui Gao1ACEFG*, Yanye Tu1BCDFG, Qingcao Li1BCF, Qiaoping Wu1ACEF

DOI: 10.12659/MSM.943596

Med Sci Monit 2024; 30:e943596

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Abstract

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BACKGROUND: In China, the most prevalent type of CRKP is ST11, but the high-risk clone ST15 has grown in popularity in recent years, posing a serious public health risk. Therefore, we investigated the molecular prevalence characteristics of ST15 CRKP detected in a tertiary hospital in Ningbo to understand the current potential regional risk of ST15 CRKP outbreak.

MATERIAL AND METHODS: We collected and evaluated 18 non-duplicated CRKP strains of ST15 type for antibiotic resistance. Their integrons, virulence genes, and resistance genes were identified using polymerase chain reaction (PCR), and their homology was determined using MALDI-TOF MS.

RESULTS: The predominant serotype of 18 ST15 CRKP strains was K5. ST15 CRKP exhibited the lowest antimicrobial resistance to Cefoperazone/sulbactam (11.1%), followed by trimethoprim/sulfamethoxazole (22.2%). Resistance gene testing revealed that 14 out of 18 ST15 CRKP strains (77.8%) carried Klebsiella pneumoniae carbapenemase 2 (KPC-2), whereas all ST15 CRKP integrons were of the intI1 type. Furthermore, virulence gene testing revealed that all 18 ST15 CRKP strains carried ybtS, kfu, irp-1, and fyuA genes, followed by the irp-2 gene (17 strains) and entB (16 strains). The homology analysis report showed that 2 clusters had closer affinity, which was mainly concentrated in classes C and D.

CONCLUSIONS: The ST15 CRKP antibiotic resistance rates demonstrate clear geographical differences in Ningbo. Additionally, some strains carried highly virulent genes, indicating a possible evolution towards carbapenem-resistant highly virulent strains. To reduce the spread of ST15 CRKP, we must rationalize the clinical use of antibiotics and strengthen resistance monitoring to control nosocomial infections.

Keywords: Carbapenems, Klebsiella pneumoniae, beta-Lactamase KPC-2, Virulence

Introduction

Klebsiella pneumoniae is an opportunistic Enterobacteriaceae bacterium that causes severe clinical infections, such as pneumonia, liver abscess, and bloodstream and urinary tract infections [1]. In the past 20 years, the carbapenem-resistant Enterobacteriaceae (CRE) has become more prevalent because of antibiotic abuse; consequently, the rapid spread of carbapenem-resistant K. pneumoniae (CRKP) has become a major public health problem [2]. According to the report of China’s antibiotic resistance surveillance system (http://www.carss.cn/), the rate of carbapenem resistance in China increased to 11.3% in 2021, and the mortality rates caused by K. pneumoniae was as high as 40–50%. Carbapenemase production is a crucial factor in the mechanisms of carbapenem resistance in CRKP. Furthermore, the activation of the efflux pump system and the loss of outer membrane protein (OMP) expression may be responsible for resistance mechanisms to carbapenems [3,4]. According to the Ambler classification, carbapenemases are classified as Ambler class A β-lactamases (blaKPC), class B metallo-βlactamases (blaVIM, blaIMP, and blaNDM) and class D β-lactamases (blaOXA-48) [5]. The most common carbapenemase gene of CRKP is blaKPC in China; meanwhile, most CRKP strains are ST11 [6]. Recent reports on the dominant sequence type of CRKP bloodstream infection have shifted from ST11 to ST15 at a hospital in Northeast China between 2015 and 2020, whereas ST15 CRKP isolates exhibited more amikacin sensitivity and serum resistance than ST11 CRKP isolates, which may have had a stronger virulence [7]. The ST15 CRKP – a high-risk clone with global distribution – is responsible for the dissemination of ESBLs and carbapenemases together with the clones ST11, ST258, and ST147. The emergence of ST15 CRKP strains carrying ESBL genes and virulence-resistant plasmids has coincided with the increase in ST15 CRKP infection [8]. Recent studies have identified ST15 CRKP isolates that produce blaVIM-1 and blaCTX-M-15 in various hospitals in Southern Spain [9]. ST15 CRKP found in China contained the ColKP3-type plasmid that carried the blaOXA-232 gene [10].

Studies on the ST15 CRKP infection in Zhejiang are scarce. Considering the regional difference of ST15 CRKP, it is essential to investigate its distribution, drug resistance, and molecular biological features of ST15 CRKP in this region. The present study studied the prevalence and molecular epidemiological characteristics of ST15 CRKP by detecting capsule serotype genes, antimicrobial resistance genes, and virulence-associated genes comprehensively in a tertiary hospital in Ningbo. This investigation will shed light on the biological epidemiology of ST15 CRKP in Ningbo.

Material and Methods

BACTERIAL STRAINS AND PATIENT DATA:

Eighteen non-duplicated ST15 CRKP strains were collected from clinical isolates in our hospital from January to December 2021. These strains were confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, EXS3000, Zybio, China). All clinical isolates were identified for resistance to imipenem and meropenem with antimicrobial susceptibility testing, which was tested using the VITEK 2 system (BioMérieux, Marcy-l’Étoile, France). K. pneumoniae NUTH-K2044 (ST23-KL1) was used as a positive control strain. The clinical data of patients were collected and included age, gender, antibiotics, underlying conditions, and invasive operations. The study was approved by our hospital’s ethics committee.

DRUG SUSCEPTIBILITY ANALYSIS:

The minimum inhibitory concentrations (MICs) of Amikacin, Ampicillin/sulbactam, Aztreonam, Ertapenem, Nitrofurantoin, Trimethoprim/Sulfamethoxazole, Ciprofloxacin, Piperacillin/Tazobactam, Gentamicin, Cefepime, Cefoperazone/Sulbactam, Ceftriaxone, Ceftazidime, Cefotetan, Cefotaxime, Tobramycin, Imipenem, and Levofloxacin were determined using the VITEK-2 compact system, which was verified using the disk diffusion method (Kirby-Bauer method) [11]. The results of the antimicrobial susceptibility tests were interpreted according to the Clinical Laboratory Standards Institute (CLSI) [12]. The quality control strains Escherichia coli (ATCC25922) and K. pneumoniae (ATCC 700603) were donated by Ningbo Clinical Laboratory Center.

DETECTION OF RESISTANCE GENES AND VIRULENCE GENES:

The DNA of CRKP strains was extracted using the boiling method, as previously described. The carbapenem-related resistance genes were amplified using polymerase chain reaction (PCR), including blaIMP, blaNDM, blaVIM-1, blaVIM-2, blaOXA-23, and blaOXA-48 [9]. In addition, PCR was used to detect 6 capsular serotype-specific genes (K1, K2, K5, K20, K54, and K57) and 18 representative virulence-associated genes (rmpA, magA, aerobactin, entB, ybtS, kfu, fyuA, irp-1, irp-2, uge, WabG, rmpA2, peg-344, iroB, iucA, terB, silS, and repA) [13–16]. In addition, the integrase genes (intI1, intI2, and intI3) of class 1, 2, and 3 integrons in CRKP were amplified [17]. After staining with EB (ethidium bromide; 0.5 mg/mL) visualized in a gel document system (BioRad, UK), the amplified products were electrophoresed on 1.2% agarose gel.

MLST TYPING ASSAY:

Multilocus sequence typing (MLST) was performed using the available protocol from the Pasteur Institute MLST website (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html). The 7 housekeeping genes of K. pneumoniae isolates (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were amplified, and the sequences were then submitted to the MLST database to compare the sequence type of CRKP strains.

MALDI-TOF MS STRAIN PROTEIN HOMOLOGY ANALYSIS:

The screened ST15 CRKP strain was inoculated on Columbia sheep blood agar plates and cultured for 18 h at 37°C in a 5% CO2 incubator. After applying a solitary colony straight onto the target plate and adding 1 μL of the matrix solution (α-cyano-4-hydroxycinnamic acid; CHCA), the plate was left to air dry at room temperature. Quality control was performed with K. pneumoniae NUTH-K2044. The EXS3000 instrument (Zybio, China) was used for the tests, and all procedures were conducted in accordance with the manufacturer’s instructions [18].

STATISTICAL PROCESSING:

Data were processed using WPS Office 10.1 software. The data are expressed as means ± standard deviation (SD). Principal components analysis (PCA) was generated using the respective functionality of the EX-Accuspec V1.0.21.6 and EXS 3000 database v1.2 software.

ETHICAL APPROVAL:

All aspects of the work are the authors’ responsibility, and they must make sure that any concerns about the integrity or accuracy of any portion of the work are duly examined and addressed. This study was approved by the Ethics Committee of Ningbo Medical Centre Lihuili Hospital, Ningbo University (No. KY2022SL281-01). The investigation was conducted in accordance with the guidelines of the Declaration of Helsinki.

Results

CLINICAL CHARACTERISTICS:

Based on MLST analysis, 18 isolates of ST15 CRKP were screened out. ST15 CRKP was isolated from sputum (12 isolates), drainage fluid (3 isolates), blood (2 isolates), and urine (1 isolate). Twelve (66.7%) of the 18 patients were men, and 6 (33.3%) were women. They were all older than 52 years (mean age: 73.0±11.0 y). The clinical departments where the patients were treated were the ICU (9 strains), neurology (7 strains), and orthopedics (1 strain) departments. Table 1 summarizes various clinical features of 18 patients infected with ST15 CRKP isolates.

ANTIMICROBIAL SUSCEPTIBILITY:

Eighteen isolates of ST15 CRKP exhibited different drug resistances. All ST15 CRKP were resistant to ertapenem and imipenem. Cefoperazone/sulbactam had the lowest antimicrobial resistance rate (11.1%), followed by Trimethoprim/sulfamethoxazole (22.2%) and amikacin (38.9%). Table 2 shows the antimicrobial resistance profiles of the 18 drugs.

ANTIMICROBIAL RESISTANCE GENES, VIRULENCE-ASSOCIATED GENES, AND CAPSULE SEROTYPE GENES:

Among the 18 ST15 CRKP isolates, K5 was the most common serotype (n=14), followed by K2 (n=2) and K20 (n=2). Figure 1 displays the positive rates of the 18 virulence genes in 18 ST15 CRKP isolates. The virulence-associated genes ybtS, irp1, kfu, fyuA, and wabG were found in all 18 ST15 CRKP isolates, followed by irp2 (n=17), entB (n=16), silS (n=14), uge (n=13), iucA (n=6), terB (n=6), rmpA2 (n=5), peg344, and repA (n=1). Fourteen ST15 CRKP isolates carried carbapenemase gene blaKPC-2, whereas the other carbapenem genes (blaIMP, blaNDM, blaVIM-1, blaVIM-2, blaOXA-23, and blaOXA-48) were undetected in the 18 ST15 CRKP isolates. The integrons of all ST15 CRKP isolates were the intI1 type.

MALDI-TOF MS-BASED TYPING:

MALDI-TOF MS was used to analyze the homology of 18 ST15 CRKP isolates. Cluster analysis was performed on the 18 strains of ST15 CRKP. The cluster dendrogram was divided into 4 clusters at a distance of >0.55 from the horizontal line, with 1 strain in cluster A, 1 strain in cluster B, 10 strains in cluster C, and 7 strains in cluster D. This result indicates that 10 strains in cluster C and 7 strains of ST15 CRKP in cluster D are more closely related and had higher homology, whereas 1 strain of CP58 in cluster A was more distantly related to other strains (Figure 2).

Discussion

Recently, various antibiotics have been widely used in clinical practice, contributing to a yearly increase in the detection rate of CRKP. According to the China Network for Surveillance of Bacterial Drug Resistance (CHINET) (2021), the detection rate of K. pneumoniae among gram-negative bacteria was 20.7%, whereas the detection rate of CRKP rose from 6.4% in 2014 to 11.3% in 2021. The most common isolate in China was previously assumed to be KPC-2-producing ST11 CRKP. According to a recent study, the types of ST sequences in China are presently transitioning from ST11 to ST15. ST15 CRKP has also evolved in various countries in recent years, including China, Bulgaria, Vietnam, and Iran [19–25]. In the present study, 18 ST15 CRKP strains were collected from Ningbo tertiary hospitals, 14 of which were primarily isolated from sputum (12 strains, 66.7%), and the patients were mainly from ICU and neurology departments, with most having long-term hospitalization, invasive surgery, and antibiotic treatment. This finding increases the possibility of ST15 CRKP colonization substantially. Hospitals must improve sterile hygiene and ward supervision to avoid transmission between patients and healthcare workers.

ST15 CRKP detected in our region was resistant to most clinical antimicrobial drugs, including 100.0% for carbapenems ertapenem and imipenem, 94.4% for ampicillin/sulbactam and furantoin, and more than 80% for levofloxacin, ciprofloxacin, gentamicin, tobramycin, piperacillin/tazobactam, ceftazidime, ceftriaxone, cefadroxil, and cefotaxime. Cefoperazone/sulbactam had the lowest resistance rate of ST15 CRKP antimicrobial drugs (11.1%), followed by trimethoprim-sulfamethoxazole (22.2%) and amikacin (38.9%), which was significantly different from the full sensitivity of ST15 CRKP to amikacin in Nanjing and the 100% resistance rate to amikacin in Shanghai [22,26]. The mechanisms of CRKP resistance are complicated, including carbapenemase generation, high levels of AmpCase or extended-spectrum-lactamase (ESBL), deletion or mutation of genes producing outer membrane proteins, changed drug targets, and high expression of the efflux pump system; the most common mechanism is carbapenemase production [6]. The detection of common clinical carbapenemase genes in this study revealed that 77.8% (14/18) of ST15 CRKP carried the KPC-2 gene, but no blaIMP, blaNDM, blaVIM-1, blaVIM-2, blaOXA-23 or blaOXA-48 drug resistance genes were found, which was consistent with other regions in China where ST15 CRKP was dominated by KPC-2 genes [27]. Four of the ST15 CRKP strains did not harbor blaKPC-2 but were resistant to carbapenems, which might be connected to the efflux pump’s activity [28]. All 18 ST15 CRKP strains were tested for intI1 integrons, but neither intI2 nor intI3 integrons were found, consistent with earlier research indicating that Klebsiella pneumoniae carried class I integrons [14].

Virulence factors influence the pathogenesis of CRKP. In this study, virulence gene detection of 18 ST15 CRKP strains indicated 100% carriage of ybtS, kfu, irp-1, and fyuA genes, which was compatible with Hang Zhao’s findings [27]. entB was not found in 2 of the ST15 CRKP strains, suggesting that they received iron through distinct pathways [14]. Furthermore, rmpA and rmpA2 may modulate mucoviscosity at the transcriptional level by regulating the cps locus. RmpA was not found in any of the 18 ST15 CRKP strains tested; however, the detection rate of rmpA2 was 22.2%, which is significantly higher than the rate reported in Vietnam [21]. With 14 strains carrying silS and 6 carrying terB, tellurite and silver resistance played a significant part in CRKP infection [15,28]. Four of the 18 ST15 CRKP strains, which may be ST15 Hv-CRKP strains, harbored the blaKPC, rmpA2, silS, and terB genes. The results suggest that ST15 has become another emerging international high-risk clone. We will further explore its plasmid type in our next study.

Currently, clinical infections with ST15 CRKP are gradually increasing, and there are significant regional differences in the drug resistance and virulence characteristics of ST15 CRKP. Therefore, it is important to understand the molecular epidemiological characteristics and drug resistance distribution of CRKP in the region, strengthen drug resistance monitoring, and control the use of clinical antibiotics to reduce nosocomial infections and transmission of CRKP.

Conclusions

The ST15 CRKP’s characterization of antibiotic resistance rate revealed notable regional variations in Ningbo, with Cefoperazone/sulbactam exhibiting the highest sensitivity, followed by Trimethoprim/sulfamethoxazole and amikacin. Furthermore, ST15 CRKP may be evolving toward extremely virulent and carbapenem-resistant strains, as evidenced by the presence of virulence genes (iucA, terB, rmpA2, peg344, and repA) in some strains. Accordingly, prudent administration of antibiotics in the clinic and improved surveillance of nosocomial infections with ST15 CRKP is required to reduce the spread of ST15 CRKP and its progression into Hv-CRKP.

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