Epidemiological and Molecular Characteristics of bla_NDM-1 and bla_KPC-2 Co-Occurrence Carbapenem-Resistant Klebsiella pneumoniae

Introduction

Klebsiella pneumoniae is an important clinical pathogen. With the widespread use of antibiotics, its multidrug resistance has gradually increased, particularly to carbapenems.¹ The first carbapenemase in K. pneumoniae was discovered in 1996, which was encoded by bla_KPC gene.² Subsequently, other carbapenemase genes were discovered, including bla_NDM, bla_OXA-48, bla_VIM, and bla_IMP.^3–6 Among them, KPC-2, one of class A carbapenemase, is relatively common in China. With the widespread popularity of carbapenem and wide distribution of carbapenemase genes, carbapenem-resistant K. pneumoniae (CRKP) isolation rates are gradually increasing globally. At present, K. pneumoniae carbapenemase (KPC) is one of the most important carbapenemases in clinical setting, and its rapid spread has posed a huge challenge to global public health.⁷ In China, CRKP isolates mainly carry bla_KPC-2 or bla_NDM-1 genes for carbapenem resistance, and multilocus sequence typing is mainly ST11.^8,9

K. pneumoniae exhibits two pathogenic types: hypervirulent K. pneumoniae (hvKP) and classical K. pneumoniae (cKP).¹⁰ These two types cause a great challenge globally in terms of hospital infections.¹¹ cKP forms multidrug-resistant (MDR) and extensively drug-resistant strains by acquiring various antimicrobial resistance genes.¹ A typical representative is CRKP, which is related with high morbidity and mortality rates.¹² hvHP easily causes liver abscess, pneumonia, meningitis, and endophthalmitis in healthy individuals and has been reported to be closely related to iconic virulence factors including regulators of myxoid phenotypes (rmpA and rmpA2) and virulence genes (yersiniabactin and aerobactin).¹⁰ For a long time, K. pneumoniae did not encode both multidrug resistance and high virulence phenotypes simultaneously.¹³ However, in recent years, reports are emerging continuously regarding the simultaneous emergence of carbapenem resistance and virulence in a single epidemic clone, which has become a serious public health threat.^14–16 ST11-CR-hvKp is the most representative clade detected in China.^15,17,18 Therefore, the genomic characterization of CRKP should be urgently studied to prevent, diagnose, and treat infections caused by K. pneumoniae.

In this study, we obtained 60 CRKP isolates from inpatients in a tertiary hospital in China, among which the NDM and KPC co-producing ST11-KL64 CRKP clone is a major threat in terms of nosocomial infections at this hospital, and these

strains displayed hypervirulence potential since all of them carried pLVPK-like plasmid. In addition, the transmission routes and genetic characterizations of these strains were investigated as well.

Materials and Methods

Patients and Isolates

From January 2019 to June 2021, 60 CRKP isolates were isolated from sputum, Tracheal aspiration, BAL, blood, urine, pus, and ascites fluid samples from the general ward and intensive care unit (ICU) of a tertiary hospital in Yangzhou, Jiangsu Province, China. There are 35 beds in the ICU of the hospital. The clinical data of patients were reviewed from the patient management system, including gender, age, in-patient department, underlying disease, clinical diagnosis, prognosis, duration of hospital stay, invasive procedures performed before sample collection, and antibiotic exposure. The underlying diseases included diseases of immune, respiratory, urinary, digestive, and blood systems, as well as hypertension, diabetes, and tumors. Invasive operations included invasive ventilator, tracheotomy and intubation, and drainage. The study was approved by the Research Ethics Committee of the Affiliated Hospital of Yangzhou University and was followed the Declaration of Helsinki. No identifiable patient information was collected in this study. All isolates were isolated for routine clinical experiment.

Bacterial Identification and Antimicrobial Susceptibility

The samples were plated onto 5% sheep blood agar and were cultured at 37°C for bacterial isolation. All isolates were identified using the VITEK-2 system. A multiplex PCR method was used to assess whether these isolates contained carbapenemase genes including bla_NDM, bla_KPC, bla_SIM, bla_GIM, bla_SPM, bla_AIM, bla_OXA-48, bla_BIC, bla_DIM, bla_IMP, bla_VIM (Table S1), referring to the primer sequence.¹⁹ The isolates carrying the corresponding carbapenemase-encoding gene were used as positive controls. Antimicrobial susceptibility was investigated using VITEK-2 system and the broth microdilution methods. The susceptibility breakpoint was interpreted according to 2018 Clinical and Laboratory Standards Institute guideline except for tigecycline and polymyxin E, which followed the criteria of European Committee on Antimicrobial Susceptibility Testing (version 12.0). Escherichia coli ATCC25922 was used as the quality control strain for antimicrobial susceptibility testing.

Filter Mating Assay

The transferability of carbapenem-resistant phenotypes was measured using filter-mating assay. K. pneumoniae YZ6 Hyg^r was used as the recipient strain, and the 60 CRKP isolates were used as the donor strains. Transconjugants were selected on LB agar plates supplemented with hygromycin (200 mg/L) and meropenem (2 mg/L).²⁰ The transconjugants carrying the gene encoding carbapenemase were confirmed using multiplex PCR and antimicrobial susceptibility testing.

Genome Extraction, Bioinformatics Analysis, and Phylogenetic Tree Construction

The epidemiological and molecular features of bla_KPC and bla_NDM co-occurrence were further studied using 18 CRKP isolates with bla_KPC-2 and bla_NDM-1 co-occurrence. FastPure Bacteria DNA Isolation Mini Kit (Vazyme, Nanjing, China) was used to extract the genomic DNA from all CRKP isolates with bla_KPC-2 and bla_NDM-1 co-occurrence. The extracted genomic DNA was subjected to 1% agarose gel electrophoresis, quantified using the Qubit fluorometer, and further subjected to short-read sequencing (2 × 150 bp) on the Illumina HiSeq 2500 platform. The short-read Illumina raw sequences of 18 CRKP isolates with bla_KPC-2 and bla_NDM-1 co-occurrence were screened for quality and assembled through SPAdes. The contigs smaller than 500 bp were purposefully removed. The sequence types (STs), capsule types, insertion sequences (IS), and multiple antimicrobial-resistance and virulence genes of these isolates were identified using CGE server (https://cge.cbs.dtu.dk) and Kleborate. The phylogenetic trees for the 18 CRKP isolates with bla_KPC-2 and bla_NDM-1 co-occurrence were constructed using Roary and FastTree. Further, visualization and modification were performed using iTOL5.

Results

Clinical Characteristics of CRKP Isolates

From January 2019 to June 2021, 60 strains of CRKP were isolated from 54 inpatients in a tertiary hospital in Yangzhou (Table 1). Among the 54 inpatients, 39 were males (72.22%) and 15 were females (27.78%), with an average age of 65.0 years. Clinical isolates were obtained from sputum (35/60, 58.33%), Tracheal aspiration (8/60,13,33%), BAL (5/60,8.34%), blood (6/60, 10.00%), urine (4/60, 6.67%), pus (1/60, 1.67%), and ascites (1/60, 1.67%) samples. The inpatients were from ICU (38/54, 70.37%), respiratory medicine (3/54, 5.56%), neurosurgery (3/54, 5.56%), general surgery (3/54, 5.56%), emergency (2/54, 3.70%), thoracic surgery (1/54, 1.85%), neurology (1/54, 1.85%), gastroenterology (1/54, 1.85%), cardiology (1/54, 1.85%), and nephrology (1/54, 1.85%) departments. Most patients were exposed to antibiotics, mainly cephalosporins, carbapenems, and enzyme inhibitors, and 45 patients had undergone invasive operation before sample collection.

Table 1 Characteristics of the 60 CRKP Isolates from the 54 Inpatients

Antimicrobial Susceptibility and Transferability

According to the drug sensitivity test data (Table 2), all CRKP isolates exhibited multiple-drug resistance. Moreover, most strains displayed resistance to doxycycline (58/60, 96.7%), chloramphenicol (59/60, 98.3%), and aztreonam (59/60, 98.3%) but remained susceptible to polymyxin (59/60, 98.3%). Multiple PCR revealed that a total of 18 isolates simultaneously carried bla_NDM-1 and bla_KPC-2. The results of conjugation assay (Figure 1) revealed that most of the bla_KPC-2-carrying CRKP isolates could not successfully transfer their carbapenemase genes into the recipient strain. By contrast, among the 18 strains with bla_KPC-2 and bla_NDM-1 co-occurrence, 16 strains could successfully transfer the carbapenem resistance phenotype to the recipient strain YZ6 Hyg^r, indicating that their carbapenem encoding genes were located on the conjugate plasmids.

Table 2 MICs (Mg/L) of All CRKP Strains in This Study

Figure 1 The results of conjugation assay. Red and blue colors, respectively, indicate positive and negative PCR results for blaNDM and blaKPC. Red color of NDM-1 and KPC-2 indicates successful conjugation transfer test results, blue color indicates failure results.

STs, Capsular Types, Virulence Genes, and Phylogenetic Analysis of CRKP Strains with bla_NDM-1 and bla_KPC-2 Co-Occurrence

MLST is a well-known gene typing method that can be used to monitor and control the spread of pathogens in hospitals.²¹ According to the K. pneumoniae MLST database, 18 CRKP strains with bla_NDM-1 and bla_KPC-2 co-occurrence were all identified as ST11, and the capsular type were KL64, suggesting that there was nosocomial infection of ST11 CRKP at this hospital. In addition, phylogenetic analysis revealed that 18 CRKP isolates with bla_NDM-1 and bla_KPC-2 co-occurrence were divided into two clades (Figure 2). Clade 1 included 9 strains (KP72, KP74, KP37, KP60, KP52, KP63, KP53, KP64 and KP62), whereas clade 2 contained 9 strains (KP58, KP44, KP43, KP80, KP78, KP20, KP81, KP73, and KP57).

Figure 2 Phylogenetic analysis of 18 CRKP strains with blaNDM-1 and blaKPC-2 co-occurrence. Distribution of antibiotic resistance genes and virulence genes in CRKP isolates. The cells of different colors indicate the presence of different genes. Blue represents virulence gene; remaining each color represents a type of drug resistance gene, and the blank cells represent the deletion of genes.

Identification of Antimicrobial Resistance Genes and Virulence Genes

According to the WGS results, the antibiotic-resistance genes and virulence genes of 18 CRKP strains with bla_NDM-1 and bla_KPC-2 co-occurrence were identified (Figure 2). All isolates contained resistance genes against β-lactams (bla_CTX-M-65, bla_KPC-2), tetracyclines [tet(A)], aminoglycosides (rmtB), sulfonamides (sul2), trimethoprim (dfrA14), fosfomycin (fosA), and quinolones (qnrS1). The β-lactam resistance gene bla_TEM-1B was present in 17 CRKP isolates; however, it was not present in KP74 isolates. The resistance genes against β-lactams (bla_ADC-25, bla_OXA-23, and bla_OXA-66), aminoglycosides [aph(3′)-Ic, strB, and strA], tetracyclines [tet(B)], macrolides [mph(E), msr(E)] were observed in KP62 isolates. However, these resistance genes were not observed in the remaining 17 isolates. Analysis combined with clinical data (Table 1), KP62 strains were isolated from ICU, and the patients had undergone invasive operations before sample collection. In addition, all CRKP strains with bla_NDM-1 and bla_KPC-2 co-occurrence contained yersiniabactin and aerobactin, which represented the potential of CR-hvKP phenotype. Except KP62, the regulatory factors of mucus phenotype gene (rmpA2) were detected in all CRKP isolates with bla_NDM-1 and bla_KPC-2 co-occurrence.

To determine the type of virulence plasmid harbored by these strains, plasmid pLVPK (GenBank accession AY378100), a classical virulence plasmid carrying a set of virulence genes, including iroBCDN, iucABCD, rmpA, and rmpA2, was used as reference plasmid. Surprisingly, we found that the virulence plasmids carried by all strains were aligned well with pLVPK based on Illumina-based contigs analysis (Figure 3), suggesting the identified virulence genes in these strains might be closely related to pLVPK-like plasmids.

Figure 3 Sequence comparison of 18 CRKP strains plasmids with pLVPK virulence plasmids. Sequence comparison revealed that region 1 of pLVPK plasmids was very similar to 18 blaNDM-1 and blaKPC-2 co-occurrence strains. Virulence factors, such as iroBCDN, iucABCD, iutA, and rmpA2, and a potassium tellurite resistance operon, such as terA, terE, terY, located on regions 2 and 3, respectively. The outermost circle annotates the genetic information, and different plasmids are assigned different colors.

Discussion

ICU is the main site of nosocomial infections and has been generally considered as a suitable place to study the epidemic characteristics of MDR strains,^22,23 particularly K. pneumoniae.²⁴ The phenomenon of drug resistance in bacterial isolates from ICU is becoming more and more serious in recent years. It is closely related to factors such as inpatients suffering from various basic diseases, the use of invasive surgical treatment, prolonged hospitalization, and extensive use of broad-spectrum antibiotics, causing a great challenge in terms of clinical antibacterial treatment.^25,26 In the study, among the 60 CRKP strains, 42 (70.00%) were isolated from the ICU. This exhibited a high prevalence rate in inpatients, which was consistent with previous studies.^27,28

According to MLST typing, all 18 CRKP strains with bla_NDM-1 and bla_KPC-2 co-occurrence belonged to ST11, indicating that K. pneumoniae positive for ST11 bla_KPC-2 was the dominant strain in this hospital. This was consistent with previous studies in China.^27,29 ST11 is the single locus variant of ST258, and they belong to the same clonal members of CG258,⁷ which has greatly contributed to the global spread of KPC-producing CRKP during the past 20 years.^9,30 In contrast, the ST258 is prevalent in North America and Europe, whereas ST11 is the main type in Asia.^31,32 Additionally, ST11 is the main sequence type of KPC producing CRKP in China and has been reported globally, including the United States, Europe, and Asia.^9,12,31–37

Previously, K. pneumoniae with the ST11 phenotype was a widely and commonly occurring MDR clone, exhibiting resistance to carbapenems; however, it was not highly virulent. However, ST11 has recently attracted considerable attention because of its feature of co-occurrence of resistance and hypervirulence genes in a single strain.¹² In our study, the analysis by Kleborate revealed that all 18 CRKP strains with bla_NDM-1 and bla_KPC-2 co-occurrence exhibited synthesis of aerobactin, which has been considered as the major siderophore system in the hvKP. The rmpA and rmpA2 virulence genes have been thought to control the capsular polysaccharide biosynthesis and symbolize a hypermucoviscous phenotype, which also existed in most of the 18 strains. Therefore, 18 CRKP strains with bla_NDM-1 and bla_KPC-2 co-occurrence isolated in this study exhibited hypervirulence phenotype and deserved our attention.

In addition, CRKP strains with bla_NDM-1 and bla_KPC-2 co-occurrence belonged to serotype KL64 in this study, which was different from previously reported KL1, KL2, and KL62 serotypes.⁹ Clinically, the ST11-KL64 CRKP isolates carrying the rmpA and rmpA2 virulence genes and producing KPC-2 were more survivable in the environment and could cause more severe infection.³⁴ It is reported that the inpatients infected with ST11-KL64 CRKP had a higher mortality. The results of this study revealed that one patient died among 16 patients with the infection of ST11-KL64 CRKP carrying rmpA and rmpA2 genes, indicating a high mortality rate of ST11-KL64 CRKP. This revealed the highly virulent nature of these ST11-KL64 CRKP isolates, and targeted surveillance was urgently needed in this regard. It is necessary to further conduct genomic epidemiological and evolutionary analyses throughout the country to elucidate the genetic basis and evolutionary characteristics of the widely spread carbapenem-resistant and highly virulent ST11-KL64 K. pneumoniae in China.

The results of phylogenetic tree revealed that 18 ST11-KL64 CRKP strains with bla_NDM-1 and bla_KPC-2 co-occurrence could be divided into two evolutionary clades, indicating two independent transmission events. Some patients of the two evolutionary clades overlapped in terms of strain isolation time or inpatient departments, which may be the main reason for the spread of K. pneumoniae in this hospital. For example, KP43, KP53, KP57, KP60, KP62, and KP64 were isolated from 8 hospitalized ICU patients with similar sampling time, indicating that transmission events occurred in a short period of time. The spread of ST11-CRKP in different departments or different wards of the same department in the hospital has been frequently reported.^6,9,11,32,38 The results revealed two independent outbreaks of ST11-KL64 CRKP strains with bla_NDM-1 and bla_KPC-2 co-occurrence in the ICU and respiratory ward from 2019 to 2021. These results confirmed that ST11-CRKP strains were prone to transfer. Further, we should further analyze the drug-resistant gene transfer of coexisting strains, and analyze the genetic environment of bla_NDM-1 and bla_KPC-2 in combination with the long read sequencing results. Therefore, practical approaches must be implemented to control transmission and reduce the occurrence of nosocomial infections.

Multiplex PCR revealed that among 60 CRKP isolates, 54 strains produced carbapenemase and contained bla_KPC gene. This was the most common mechanism of carbapenem resistance in K. pneumoniae. In China, it is reported that KPC-producing K. pneumoniae was the main strain causing outbreak.^39,40 Among the 54 CRKP strains with KPC-producing gene, 18 strains carried bla_NDM gene at the same time. In addition, the Illumina sequencing analysis revealed that apart from the many types of β-lactam-resistance genes, other resistance genes such as tet(A), rmtB, sul2, dfrA14, fosA, and qnrS1 were present in all 18 ST11-KL64 CRKP isolates with bla_NDM-1 and bla_KPC-2 co-occurrence, conferring resistance to tetracyclines, aminoglycosides, sulfonamides, trimethoprim, fosfomycin, and quinolones, respectively. The co-existence of carbapenemase, β-lactamases, and many types of drug resistance genes led to the multidrug resistance. Undoubtedly, the existence of resistance genes enables K. pneumoniae isolates to survive the attack of antibacterial drugs. Thus, treatment of infections caused by these multi-resistant CRKP strains is a great challenge because of limited availability of antimicrobials. Fortunately, polymyxin was effective in vitro, suggesting that it might be a valuable therapeutic option for ST11-KL64 CRKP infections.

Conclusion

Our study confirmed that the CRKP strains isolated from the hospital mainly had ST11-KL64 phenotype and mostly carried bla_KPC-2 resistance genes. The ICU was the main site of nosocomial infection and rapid transmission of CRKP. The strains exhibited high pathogenicity by acquiring various drug-resistance genes and virulence genes, causing a major challenge to public health. Therefore, it is urgent to develop effective strategies to control and prevent further nosocomial infection.