Genetic and Functional Analysis of the pks Gene in Clinical Klebsiella pneumoniae Isolates

ABSTRACT The pks gene cluster encodes colibactin, which can cause DNA damage and enhance the virulence in Escherichia coli. However, the role of the pks gene in Klebsiella pneumoniae has not been fully discussed. The aim of this study was to analyze the relationship between the pks gene cluster and virulence factors, as well as to assess antibiotic resistance and biofilm formation capacity in clinical isolates of Klebsiella pneumoniae. Thirty-eight of 95 clinical K. pneumoniae strains were pks positive. pks-positive strains usually infected emergency department patients, and pks-negative strains often infected hospitalized patients. The positive rates of K1 capsular serotype and hypervirulence genes (peg-344, rmpA, rmpA2, iucA, and iroB) were significantly higher in the pks-positive isolates than the pks-negative isolates (P < 0.05). The biofilm formation ability of pks-positive isolates was stronger than that of pks-negative isolates. Antibacterial drug susceptibility test showed the resistance of pks-positive isolates was weaker than that of pks-negative isolates. In conclusion, patients with pks-positive K. pneumoniae infection might have worse treatment outcomes and prognosis. pks-positive K. pneumoniae might have stronger virulence and pathogenicity. Clinical infection with pks-positive K. pneumoniae needs further attention. IMPORTANCE The infection rate with pks-positive K. pneumoniae has been increasing in recent years. Two previous surveys in Taiwan reported 25.6% pks gene islands and 16.7% pks-positive K. pneumoniae strains in bloodstream infections, and Chinese scholars also did a survey of K. pneumoniae bloodstream infections in Changsha, China, and found 26.8% pks-positive K. pneumoniae. In addition, it was found that the pks gene cluster might encode colibactin, which could be related to the virulence of K. pneumoniae. Studies confirmed that the prevalence of colibactin-producing K. pneumoniae was increasing. It is necessary to consider the clear relationship between the pks gene cluster and high pathogenicity in K. pneumoniae.

characteristics of their pathogenicity and the expression of virulence genes are increasingly attracting attention.
The polyketide synthase (pks) gene cluster, which encodes the synthetic genotoxin colibactin, has been primarily found in Enterobacteriaceae, such as Escherichia coli, K. pneumoniae, and Citrobacter (7,8). The pks gene cluster encodes a synthetic genotoxin of colibactin that induces DNA damage in eukaryotic cells, which is associated with other bacterial virulence factors (adhesins, toxins, and siderophores) (7,9). Studies have found that pks-positive K. pneumoniae infection exacerbated lymphopenia in septic mouse models (10), promotes the development of meningitis (11), and significantly increases mortality in patients (12). Therefore, there might be a potential correlation between pks gene clusters and virulence.
In this study, we collected a total of 98 clinical isolates of K. pneumoniae from our hospital and analyzed the prevalence of the pks gene cluster and virulence genes, antimicrobial susceptibility, and biofilm formation. This study aimed to evaluate the effect of pks gene cluster on pathogenicity and virulence in order to provide new insights for the clinical treatment of K. pneumoniae infection.

RESULTS
Clinical characteristics of pks-positive and pks-negative K.pneumoniae isolates. A total of 95 strains of K. pneumoniae were collected and divided into two groups based on the pks gene identification results: the pks-positive group and the pks-negative group. The determination of pks gene-positive results was based on the positivity of clbA, clbB, clbN, and clbQ (Fig. 1). pks-positive K. pneumoniae strains were more likely to infect patients from the emergency department than pks-negative K. pneumoniae strains. pks-positive K. pneumoniae strains were more often isolated from blood samples (P , 0.05). Besides, we found that patients with pks-positive K. pneumoniae infection had fewer concomitant underlying diseases than patients with pks-negative K. pneumoniae infection ( Table 1).
Biofilm formation. Our data revealed that 98% of K. pneumoniae isolates were biofilm producers. In this study, 38% and 60% of isolates were weakly and moderately biofilmproducing strains, respectively. The prevalence of moderate biofilm formation in pks-positive K. pneumoniae was significantly higher than in pks-negative K. pneumoniae (84.2% compared to 43.8%; P , 0.05) ( Table 4).

DISCUSSION
Klebsiella pneumoniae is a potential hospital superbug that has attracted clinical attention (13). hvKP frequently exhibits hypermucoviscous phenotypes and carries a variety of hypervirulence genes (14,15). Worryingly, hvKP has been spreading worldwide and causing severe metastatic infections, particularly in immunologically active populations (16). Furthermore, the emergence of multidrug-resistant (MDR) highly pathogenic strains has created significant challenges in the clinical field (17,18). Therefore, the research on K. pneumoniae could help the clinical treatment of K. pneumoniae-related infection and avoid unnecessary treatment and improper use of medicine.
Two previous surveys pointed out that the proportion of pks genes in 207 strains of K. pneumoniae was 25.6% (19) and the proportion of pks-positive K. pneumoniae isolates in bloodstream infections was 16.7% (20) in Taiwan. A survey of pks-positive K. pneumoniae bloodstream infections in Changsha indicated a prevalence of 26.8% in China (21). Furthermore, the analysis of clinical characteristics showed that pks-positive isolates were more frequently encountered in community-acquired infection (21). In this study, the analysis of clinical characteristics showed that pks-positive isolates more frequently infected emergency department patients. Compared to pks-negative strains, more pks-positive strains were collected from blood specimens. These results suggested that the infection of pks-positive strains might reflect severe clinical infection.
In this study, we found that the virulence genes were mainly present in pks-positive K. pneumoniae isolates, and the positivity rate of the virulence genes (peg-344, rmpA, rmpA2, iucA, and iroB) was higher than that in the pks-negative K. pneumoniae isolates. peg-344, rmpA, rmpA2, iucA, and iroB were considered to be the biomarkers of highly virulent strains (4). The peg-344 gene encodes an endometrial transporter and is one of the markers of K. pneumoniae virulence screening (22). rmpA and rmpA2 are regulatory genes for polysaccharide expression in the capsule of K. pneumoniae, which reduce the yield and virulence of the capsule of the strain if missing (23). Iron absorption enhances bacterial virulence. iucA and iroB are important genes for the expression of K. pneumoniae siderophores, which are major virulence determinants of systemic infection (24). The above findings supported that the pks gene cluster might be associated with highly virulent strains.
In this study, the detection rate of highly virulent capsular serotypes in the pks-positive K. pneumoniae was 69.5%, with 14 strains not detected. This showed that the positive sero-  type of the K1 type was significantly higher in pks-positive K. pneumoniae strains than in pks-negative ones (P , 0.05). The presence of capsular serotypes is one of the main virulence factors of hvKP, which can protect the organism against phagocytosis by host phagocytes and damage by lysosomes via their complement (25). Currently, K1 and K2 serotypes of K. pneumoniae can acquire drug-resistant phenotypes by mediating various drug-resistant genes through mobile genetic elements, posing a great challenge for clinical treatment (26). This suggested that the strains carrying a pks gene cluster might be more closely associated with virulent capsular serotype K1, and this group of strains might be highly virulent or more likely to acquire a drug-resistant phenotype.
The pks-positive isolates were found to be associated with low antimicrobial drug resistance. In this study, statistical analysis showed that pks-positive isolates were significantly less resistant to the 10 tested antimicrobial drugs than the pks-negative group. This situation might be due to the fact that pks-positive isolates have a high proportion of highly pathogenic serotypes and virulence genes, as the acquisition of virulence is usually accompanied by a decrease in resistance. However, we also observed highly resistant strains within the pks-positive group, which presents a concerning scenario for the future as it combines genotoxicity and drug resistance. Additionally, our analysis of the data revealed that most pks-positive K. pneumoniae isolates exhibited a high capacity for biofilm formation. This biofilm formation might protect bacteria from host immune attack and antibiotics.
Therefore, it is possible that pks-positive K. pneumoniae isolates have stronger virulence and pathogenicity, which could result in worse treatment outcomes and prognosis for individuals infected with these strains. To prevent K. pneumoniae infections, there is a need for epidemiological surveillance that targets virulence factors, as well as effective infection control measures and the development of new therapeutic approaches.

MATERIALS AND METHODS
Bacterial isolates. A total of 95 nonrepetitive K. pneumoniae isolates were collected for this study. Relevant clinical data were also retrieved. These isolates were identified, handled, and preserved using standard microbiological laboratory procedures (27).
Detection of pks gene cluster, virulence genes, and capsular serotypes. The presence of the pks gene cluster and virulence genes was detected by PCR as previously described. The clinical isolates were screened for the presence of pks gene cluster using primers for the four representative genes (clbA, clbB, clbN, and clbQ) of the genomic cluster in order to document the presence of a complete cluster (28). After overnight culture, K. pneumoniae was suspended in 300 mL of sterile distilled water, heated at 95°C for 10 min, and then centrifuged at 12,000 Â g for 5 min to remove cellular debris. The supernatant was stored at 4°C and used as the template for amplification. The PCR amplification procedure included predenaturation at 94°C for 5 min, denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and 72°C extension for 1 min for 30 cycles, and finally 72°C extension for 10 min. The PCR products were visualized by 2% agarose gel electrophoresis.
To investigate the association of pks and hypervirulence, the presence of five hypervirulence genes (peg-344, rmpA, rmpA2, iucA, and iroB) and capsular serotypes was determined by PCR following previously published protocols (4,29). The primers used in this study are listed in Table 5.
Detection of biofilm formation. Biofilm-forming ability was detected by crystal violet staining. The strains were incubated in LB broth medium, shaken overnight at 37°C, prepared in 0.5 MacConkey's turbidity solution, and diluted 1:100 with LB broth. The diluted broth was added to a 96-well microtiter plate at 200 mL/well, and 3 wells were inoculated with 200 mL sterile LB broth as a negative control. The plate was washed three times with phosphate-buffered saline (PBS [pH 7.0]), dried at room temperature, fixed with methanol solution for 20 min, the methanol was discarded, and then the plate was stained using 1% crystal violet solution. After 15 min, the plate was washed with PBS until colorless. After drying, 200 mL of anhydrous ethanol was used to fully dissolve the crystal violet, the mixture was transferred to a new microplate, and absorbance was measured at 570 nm. Each assay was performed in triplicate and repeated four times.

pks-Positive K. pneumoniae
Microbiology Spectrum Statistical analysis. Categorical variables were analyzed by using the chi-square test or Fisher's exact test. For continuous variables, Student's t test or the Mann-Whitney U test was used to analyze the data, as appropriate. All data analysis was performed with SPSS software (version 25.0). A P value of ,0.05 was considered statistically significant.