Introduction
Klebsiella pneumoniae is not only a common pathogen causing nosocomial infections but also an important cause of community-acquired infections, that colonizes human mucosal surfaces such as the nasopharynx and the gastrointestinal tract.1 In recent years, with the prevalence of multidrug-resistant and hypervirulent K. pneumoniae in the world, the incidence rate of K. pneumoniae infections has risen dramatically, such as urinary tract infection, pneumonia, liver abscess, and so on.1 Compared with the classical K. pneumoniae (cKp), hypervirulent K. pneumoniae (hvKp) possesses higher toxicity, which can cause severe infection in immunocompromised people, with high pathogenicity and mortality.2 Although many factors contribute to the high virulence of the hvKp, virulence factors, including capsule, siderophores, lipopolysaccharide, and fimbriae, play an essential role in the pathogenesis of several diseases.3–6 Numerous reports have shown that K1 and K2 serotypes are strongly associated with hvKp among 79 serotypes of K. pneumoniae.7,8 Additionally, some genes, rmpA, iutC, and ybtA, which are responsible for the production of high viscosity, iron-acquiring factors, aerobactin and yersinia actin, respectively, have been associated with the hypervirulence of K. pneumoniae.5,9 Recently, the pks (polyketide synthase) gene cluster, as a new virulence factor, has aroused great public concern.10
The pks gene cluster is a genetic locus that was first described in some Escherichia coli strains from the B2 phylogroup by Nougayrede in 2006.11 It contains 19 genes (clbA to clbS) with 54 kb and encodes a multi-enzyme complex capable of producing a genotoxin called colibactin. Previous studies have shown that colibactin can cleave host DNA double strands, resulting in cell cycle arrest, DNA damage, and mutations.12,13 Moreover, it increases the likelihood of serious complications of bacterial infections. For instance, production of colibactin by pks+ E. coli exacerbates lymphopenia associated with septicemia and increases the morbidity and mortality of urosepsis and meningitis in immunocompromised mice.14,15 Additionally, pks-positive E. coli has been associated with mutations in colorectal cancer.13,16,17 Subsequently, the pks island has also been found in several other members of the Enterobacteriaceae family, such as Citrobacter koseri, K. pneumoniae, and Enterobacter aerogenes, but was found to be relatively infrequent.18–20 A study in Europe showed that the prevalence of the pks gene cluster was 34% in E. coli strains of phylogenic lineage B2, but only 3.5% in K. pneumoniae clinical isolates.18 While the predominance of pks genes in bloodstream-sourced K. pneumoniae is approximately 25.6% and 26.8% in Taiwan and Changsha, respectively,21,22 little is known about its epidemiology in clinical isolates from cancer patients in China.
Given the potential role of the pks gene cluster in cancer and its association with hypervirulence, it is crucial to investigate the prevalence and molecular characteristics of pks-positive K. pneumoniae in patients with cancer. This study aimed to address this gap by examining the presence of the pks gene cluster and analyzing the clinical and molecular features of pks-positive K. pneumoniae isolates from patients with cancer in China. Understanding the distribution and characteristics of these isolates will provide valuable insights into their pathogenic potential, and inform clinical practice and epidemic surveillance.
Materials and Methods
Bacterial Isolates Collection
A total of 279 non-repetitive clinical K. pneumoniae isolates were obtained from all cancer patients in China at Cancer Hospital Chinese Academy of Medical Sciences, Shenzhen Center between January 2022 and June 2024. All cases were diagnosed according to the International Classification of Diseases, 10th Revision (ICD-10) and presented with clinical evidence of infection (including clinical symptoms, laboratory indicators, and microbiological evidence). These strains were isolated from diverse specimens, including sputum, blood, urine, drainage fluid, bile, catheter, gastric juice, vaginal secretion, and wound secretion. The collection, isolation, and culture of all clinical specimens must be performed under aseptic conditions and comply with the standards of CLSI (Clinical and Laboratory Standards Institute) guidelines and WHO Laboratory Biosafety Manual. After being isolated and purified, these strains were preserved at −80 °C in a tube containing 20% glycerol for a long time. The full 10 μL loop of colonies after balancing to room temperature were spread onto the Columbia blood agar (Oxoid, Brno, Czech Republic) and incubated at 37 °C for 24 h in 5% CO2 atmosphere. At the same time, the information of these patients was also collected. This study was approved by the hospital ethics committee (Approval No: JS2024-18-1).
Identification and Antimicrobial Susceptibility Testing
Isolates were identified by by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS; bioMerieux SA, Lyons, France) according to the manufacturer’s protocol. Antimicrobial susceptibility testing was performed using automatic microbial identification and the antibiotic sensitivity analysis system, Vitek 2 Compact (bioMerieux SA, Lyons, France). The results of the antibiotic sensitivity test were determined based on the breakpoints recommended in the guidelines of the 2023 Clinical and Laboratory Standards Institute (CLSI).
Identification of the Pks Gene Cluster in Clinical K. pneumoniae Isolates
Genomic DNA was extracted from 279 clinical isolates using a bacterial DNA extraction kit (Tiangen Biochemical Technology, Beijing, China) and quantified using Qubit 4.0 according to the manufacturer’s instructions. PCR was used to detect pks genes (clbA, clbB, clbN, and clbQ). The primers and amplification conditions used in the present study for pks detection are listed in Table 1.11 The PCR products were visualized using 2% agarose gel electrophoresis.
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Table 1 Primers Used for Amplification of the Tested Pks Genes
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The positive of pks gene clusters were verified by blasting whole genomic coding ORFs against E. coli clb reference genes (GenBank accession: AM229678.1)11 with both identity and coverage threshold greater than 80%.
Whole-Genome Sequencing and Analysis
A total amount of 0.2 μg of DNA per sample was used as input material for DNA library preparations using the Rapid Plus DNA Lib Prep Kit (RK20208) (Beijing Baiao Innovation Technology, China). Subsequently, the library quality was assessed on the Agilent 5400 system (AATI) and quantified by real-time PCR (1.5 nM). The qualified libraries were pooled and sequenced on Illumina platforms (Illumina, San Diego, CA, USA). Sequencing reads were assembled using Shovill (1.1.0) (https://github.com/tseemann/shovill), and the contamination and completeness of the assembled genome were assessed using CheckM (v1.2.2).23 Whole-genome annotation was performed using the Prokka software (1.14.6).24
SNP distance and phylogenetic tree construction were performed for pks-positive strains. Phylogenetic analysis was conducted using IQ-TREE software (version 2.3.5) and visualized with the ggtree package in R (version 4.4.2). The K159 strain was used as the reference genome, and core genomic SNPs (cgSNPs) were identified using Snippy (v4.6.0) (https://github.com/tseemann/snippy).
Sequence types (ST) and serotypes were determined from whole-genome data using Kleborate (2.2.0)25 against pubMLST database26 and Kaptive database.27
Virulence genes and antibiotic resistance genes were identified using the ABRicate (1.0.1)28 and AMRFinderPlus (3.11.14)29 from genome assembly, respectively.
Statistical Analysis
All analyses were performed with the Statistical Package for the Social Sciences version 28.0 (SPSS, Chicago, IL, USA). Significance of differences in frequencies and proportions was tested by the χ2 test or Fisher’s exact test. A P-value <0.05 was considered statistically significant.
Results
Clinical Characteristics of Pks-Positive K. pneumoniae
Among 279 K. pneumoniae isolates, 35 (12.54%) pks gene cluster positive representatives were identified, which were mainly isolated from the sputum (20, 57.14%). The clinical characteristics of the patients who isolated K. pneumoniae isolates are presented in Tables S1 and S2. The average age of patients with pks-positive K. pneumoniae was 59, and most of them were male (27, 77.14%). And the diagnosis of lung cancer (15, 42.86%) was predominant in patients harbouring pks-positive isolates, followed by gastric cancer (3, 8.57%). But comparing with patients infected by pks-negative K. pneumoniae, there was no significant difference in age, specimen source, infections position, and sexes in patients harbouring pks-positive isolates (P > 0.05) (Table 2).
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Table 2 Clinical Data of Patients Infected with Pks-Positive and Pks-Negative K. pneumoniae
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Antimicrobial Susceptibility of Pks-Positive Isolates
There was no significant difference in rates of susceptibility between the pks-positive and pks-negative K. pneumoniae isolates to most antibiotics, including β-lactam/β-lactamase inhibitors, fluoroquinolones, cephamycin, aminoglycosides, and carbapenems, except for sulfonamides (Tables S3 and S4). For example, the susceptibility rates of cefoperazone sulbactam, piperacillin tazobactam, cefuroxime, ceftazidime, ceftriaxone, cefepime, amikacin were 100%, 85.71%, 74.29%, 91.43%, 85.71%, 88.57%, and 100% in the pks-positive K. pneumoniae, and compared with the pks-negative K. pneumoniae, where the respective rates for these antibiotics were 95.90%, 88.52%, 72.95%, 84.02%, 77.05%, 84.02%, and 98.36% (Table 3). Although there was a tendency that the pks+ K. pneumoniae isolates were less resistant to carbapenem agents tested versus pks-isolates (100% vs 98.36%), the difference was insignificant. Sulfamethoxazole was the only agent to which pks-positive isolates were significantly more susceptible than pks-negative isolates (100% vs 75.82%, P<0.001) (Table 3).
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Table 3 Susceptibility of Pks-Positive and Pks-Negative K. pneumoniae to Antimicrobials
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Molecular Characteristics of Pks-Positive K. pneumoniae
In this study, whole-genome sequencing of 35 pks+ K. pneumoniae isolates was performed, and the detailed quality assessment results are shown in Table S5. The average genome size of 35 pks+ K. pneumoniae isolates was 6.02 Mbp, and the average GC content was 57.38%. The average largest were 0.72 Mbp, and N50 scaffolds were 0.29 Mbp in length, indicating the high assembling quality. The result of genome sequencing showed that virulence associated serotype K1 (17, 48.57%) was the predominant serotype, and K2 accounted for 25.71% in pks-positive K. pneumoniae (Figure 1). Six other K serotypes (K116 (3), K113 (2), K20 (1), K25 (1), K57 (1), and K62 (1)) accounted for 25.72% of isolates.
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Figure 1 Phylogenetic tree based on SNP sites in core genes of 35 pks-positive strains.
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Among the 35 pks-positive K. pneumoniae, the multilocus sequence typing showed that the predominant sequence types were ST23 (19, 54.29%) and ST65 (8, 22.86%), while another six STs each had no more than 3 strains, ST133 (3, 8.57%), ST268 (1, 2.86%), ST348 (1, 2.86%), ST380 (1, 2.86%), ST592 (1, 2.86%), and ST792 (1, 2.86%) (Figure 1). The whole genomic phylogeny and SNP distance were inferred, and we found that there is no direct and recent transmission (cgSNP differences less than 20) among ST23 and ST65 isolates (Figure 1).
Virulence genes were prevalent in pks-positive isolates, particularly the siderophore systems (aerobactin, enterobactin, salmochelin, and yersiniabactin) which played different roles in infection within the host. In 35 pks-positive isolates, Enterobactin synthase genes (entAB, fepC) and yersiniabactin siderophore system genes (ybtA/E/P/Q/S/T/U/X, irp1, irp2) were at least 97.14%, meanwhile the aerobactin siderophore synthesis system genes (iucA/B/C/, iutA) and salmochelin genes (iroB/C/D/N) were at least 85.71% (Table 4). Furthermore, rmpA genes, which were the positive regulator of the mucoid phenotype, and peg-344, which could encode an intracellular transporter protein, were, respectively, found in 62.86% and 54.29% of pks-positive isolates (Table 4).
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Table 4 Virulence Genes and Drug Resistance Genes of Pks-Positive K. pneumoniae
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As for antibiotic resistance genes, pks-positive isolates harbored some β-lactamase genes, including blaCTX-M, blaTEM, and blaSHV. Only four isolates proved positive for CTX-M-1 group, and two isolates proved positive for CTX-M-9 group. Additionally, the screen of SHV β-lactamase genes showed that the frequencies of SHV-11, SHV-75, SHV-26, and SHV-207 were 30 (85.71%), 3 (8.57%), 1 (2.86%), and 1 (2.86%), respectively. And only two isolates were blaTEM-1 positive. However, no pks-positive isolates proved positive for the genes that confer resistance towards carbapenems.
Discussion
The pks gene island, encoding the genotoxin colibactin, has garnered significant attention due to its ability to induce DNA double-strand breaks and transient G2-M cell cycle arrest in host cells.12 This genotoxic activity suggests that colibactin may contribute to various disease entities, including newborn meningitis, urinary tract infections, bloodstream infections, and potentially cancer development.15,22,30 In addition, some studies reported that the pks-positive E. coli was more highly represented in CRC patients and could promote human CRC development.17,31 Our study is the first to investigate the prevalence and molecular characteristics of K. pneumoniae harboring the pks island in Chinese cancer patients, providing valuable insights into its epidemiology and clinical significance in this specific population.
Up to now, there have been few epidemic reports on emerging pks-positive K. pneumoniae. In Europe and Iraq, the occurrence of pks-positive K. pneumoniae was 3.5%18 and 7.14%,20 respectively. In this study, the prevalence of the pks gene cluster among K. pneumoniae isolates was 12.54%, which was higher than those reported in the literature. But in two previous studies conducted in Taiwan and Changsha, the positive rates of pks-positive K. pneumoniae isolated from blood was 16.8%32 and 26.8%,22 respectively. And some studies revealed that the prevalence of pks gene in E. coli was high, ranging from 29.2% to 72.7%.31,33,34 Therefore, we found that the epidemiological distribution of pks-positive strains exhibits regional and interspecies differences, which may be associated with environmental, host, and pathogen factors.
Colibactin encoded by the pks gene cluster has been shown to induce host DNA damage, thus may contribute to higher mutation rates that drive the occurrence of tumors. By analyzing 3668 Dutch samples of different cancer types, a study found that the colibactin was present in a variety of tumors.35 Our findings backed up the above results, which documented pks-positive K. pneumoniae had been isolated from different types of cancer patients. Jens Puschhof et al proved that the pks gene cluster was present at a higher frequency in colorectal cancer compared to other types of cancer.35 And the presence of pks-positive K. pneumoniae has been found in 4–27% colon cancer patients.18,21,32,36 However, our findings revealed that pks-positive K. pneumoniae isolates were predominantly associated with lung cancer patients (42.86%), followed by gastric cancer, which was different from the above researches that reported higher prevalence in colorectal cancer patients. This may be due to the specific patient population and sampling bias, as only parenteral specimens were collected. However, this highlights the potential role of pks-positive K. pneumoniae in various types of cancer, not limited to colorectal cancer. Further studies are needed to elucidate the specific mechanisms by which pks-positive K. pneumoniae contributes to cancer development and progression.
There are many similarities between pks-positive K. pneumoniae and hvKp. Firstly, previous studies have revealed that hvKp were almost exclusively of serotype K1 or K2, and ST23 and ST65 were predominant sequence types.5,7 On the other hand, the hvKp K1 strains were strongly associated with ST23, while the hvKp K2 strains belong to different STs (ST65, ST86, and others).5,8 In our study, the great majority (74.28%) of pks-positive isolates belonged to K1 or K2 serotype. And all K1 strains belong to ST23, whereas K2 strains were divided into two major clades, ST65 and ST380. To investigate whether there is transmission or possible outbreaks among single ST isolates, whole-genomic phylogeny and SNP distance were inferred, and we found that there is no direct and recent transmission (cgSNP differences less than 20) among ST23 and ST65 isolates, suggesting the patients get these infections from different sources. Two ST133 isolates, k130 and k131, showed almost no cgSNP differences (Figure 1), suggesting direct transmission among their host patients. However, the mechanism of transmission still needs further study. Secondly, another study suggested that hvKp were positive for several virulence factors, such as iucA, iroB, peg-344, rmpA, and so on.5,7 Our study found that pks-positive isolates generally carried several virulence genes. Additionally, the high prevalence of rmpA and peg-344 genes indicates that these isolates may exhibit a mucoid phenotype, which is associated with increased resistance to phagocytosis and host immune responses.5 Therefore we assumed that the emerging pks genotoxic trait is associated with the virulence genes of hvKp. We also found that the pks-positive strains in this study showed high sensitivity to most antibiotics, which is likely due to the fact that most of these isolates belong to K1 and K2 serotype to protect bacteria from phagocytosis and inhibit the host immune response. And compared with pks-negative strains, pks-positive strains showed higher sensitivity to sulfamethoxazole (P<0.05), which provided an important reference for antibiotic treatment. Although the rate of MDR in pks-positive isolates is low at present, the presence of β-lactamase genes, such as blaCTX-M, blaTEM, and blaSHV, indicates that these isolates have the potential to develop multidrug resistance. Therefore, continued surveillance of antimicrobial resistance patterns in pks-positive K. pneumoniae is essential to guide appropriate treatment strategies and prevent the emergence of multidrug-resistant strains.
While our study provides important insights into the prevalence and molecular characteristics of pks-positive K. pneumoniae in cancer patients, several limitations should be acknowledged. The sample size was relatively small, and only parenteral specimens were included, which may limit the generalizability of our findings. Additionally, the study was conducted in a single center, and further multicenter studies with larger sample sizes are needed to confirm our results.
Recently, it was described that the exposure to pks-positive E. coli is responsible for mutational signature in colorectal cancer, so it seems that pks-positive bacteria can induce mutation of CRC driver genes and, therefore, pks may become a marker of CRC carcinogenesis and therapy.31 Future research should focus on elucidating the specific mechanisms by which pks-positive K. pneumoniae contributes to cancer development and progression. Additionally, longitudinal studies are needed to monitor the evolution of antimicrobial resistance in these isolates and to develop targeted therapeutic strategies.
Conclusion
Our study highlights the potential pathogenicity of pks-positive K. pneumoniae in cancer patients in China, emphasizing the need for close clinical attention and epidemic tracking. The findings underscore the importance of continued surveillance and research to better understand the role of this genotoxic pathogen in cancer-associated infections.
Ethics Statement
This study was approved by the ethics committee of Cancer Hospital Chinese Academy of Medical Sciences, Shenzhen Center (Approval No. JS2024-18-1). This study was retrospective and associated with bacterial drug susceptibility and the genetic information of the specimens, hence our ethical petition for exemption from informed consent was accepted. All patients have been informed that their samples will be used for research and have signed informed consent for sample collection. The data of all patients in this study were collected anonymously and ensured the confidentiality of their information. This study was conducted in accordance with the guidelines set out in the Declaration of Helsinki.
Acknowledgments
We gratefully acknowledge the support and resources provided by the Microbiology Laboratory, Cancer Hospital Chinese Academy of Medical Sciences, Shenzhen Pathogen Infection Research Alliance (SPIRA) and Department of Clinical Laboratory, Shenzhen Third People’s Hospital.
Funding
This research was supported by Sanming Project of Medicine in Shen zhen (No.SZSM202311002) and Science and Technology Program of Shenzhen (Grant Nos. KCXFZ20230731100901003, KJZD20230923115116032, JCYJ20210324131212034).
Disclosure
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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