Molecular detection of core sensing genes of Klebsiella pneumoniae isolated from different clinical sources

Klebsiella pneumoniae is an important pathogenic Gram-negative, non-motile bacterium. The bacterium is considered an opportunistic pathogen that can harbor asymptomatically in the nasal cavity, throat, gastrointestinal tract and skin of healthy people. It is responsible for many common diseases, including urinary tract infections (UTIs), pneumonia, bacteremia, purulent liver abscesses and wound infections. The aim of this study was to isolate and characterize multidrug-resistant Klebsiella pneumoniae bacteria isolated from different clinical sources, to detect biofilm and quorum sensing genes and to study the effect of silver nanoparticles on gene expression of these genes. The study was conducted between September 2021 and September 2022. A total of 150 clinical Klebsiella pneumoniae strains isolated from sputum, urine and wound sources were cultured on MacConkey agar medium at 37℃ for 24 hours. VITEK-2 compact systems were used to identify the isolate and determine antibiotic susceptibility. Biofilm forming ability was determined by 96-well microplate method. Evaluation for the presence and expression of the quorum sensing gene (LuxS), biofilm formation gene (mrkD), Efflux gene (AcrAB), Rapid Amplified Polymorphic DNA (RAPD) Analysis and Enterobacterial Repetitive Intergenic Consensus (ERIC) Analysis genes was performed by PCR and RT-PCR. The toxicity of silver nanoparticles was tested by a hemolysis assay. Clinical strains of Klebsiella pneumoniae were isolated from 50% of sputum, 43.33% of urine and 6.66% of wounds. The strains were resistant to Extended spectrum beta lactamases (ESBL) 77%, Gentamicin 67%, Sulfamethoxazole/Trimethoprim 65%, Amoxicillin-clavulanic acid 55%, Ampicillin 39%, Cefotaxime 39%, Ceftazidime 39%, Cefixime 36%, Piperacillin/Tazobactam 33%, Meropenem 29%, Ciprofloxacin 29%, Imipenem 13% and Amikacin 13%. All strains were able to form biofilms, 13% of the total 150 isolates were strong biofilm producers, 40% were moderate biofilm producers and 47% were weak biofilm producers. Nucleic acid concentration and purity results were determined using a Qubit fluorometer. The analyzed samples contained DNA and RNA from 20 strong biofilm producer isolates, 20 moderate biofilm producer isolates and 20 weak biofilm producer isolates. The mean concentrations of nucleic acids were calculated as (21.95±1.30), (15.16±1.55), (23.33±3.23). Furthermore, the purity of the nucleic acids was calculated as the spectrophotometric absorption ratio at 260 nm/280 nm and was (1.85±0.08), (1.92±0.02), (1.90±0.02) for the three biofilm producing categories. RAPD PCR was dedicated to the determination of genetic polymorphism at the DNA level of thirty samples randomly and equally selected from the three groups, five primers, OP-V02, OP-V09, OP-V14, OP-V19 and OP-V04, were used to perform RAPD analysis in the present study. After PCR amplification with a specific primer and electrophoresis of the amplification bands obtained, the results were designated as (+), indicating the presence of the band, and (-), meaning the absence of the band. Thirty samples were randomly and equally selected from the three groups for ERIC PCR. ERIC profiles were compared using the Dice similarity matrix coefficient and clustered by the neighbor-joining method to prepare the phylogenetic tree. Isolates with two or more different bands in ERIC banding patterns were considered as different ERIC types. The dendrogram was drawn according to clusters. ERIC-PCR generated different banding patterns for each strong biofilm-producing K. pneumoniae isolate, with bands ranging in size from 175 to 4000 bp. The number of bands per isolate ranged from 3 to 11, indicating significant genetic diversity among the isolates. In K. pneumoniae isolates, which are moderate biofilm producers, the band size ranged between 175-2500 bp. The number of bands per isolate ranged from 3 to 11, indicating significant genetic diversity among isolates. Finally, the size of the amplified bands ranged from 175 to 4000 bp. The number of bands per isolate ranged from 4 to 10, indicating a less diverse genetic picture for weak biofilm producer K. pneumoniae compared to strong and moderate biofilm producer isolates. A real-time PCR assay was performed to determine the effect of nanoparticle treatment on the expression levels of genes involved in quorum sensing (LuxS) and biofilm formation (mrkD). The results of this study showed that the mean ∆∆ of the LuxS gene before treatment with nanoparticles (0.142±0.46) was not statistically significantly different from the mean ∆∆ of the same gene after treatment with nanoparticles (0.102±0.68) (p=0.961); when the fold changes of the same gene before and after treatment with nanoparticles were compared, the same comparison was not statistically significantly different (p=0.133). The results of this study revealed that the mean ∆∆ of the mrkD gene before nanoparticle treatment (0.68±0.23) was not statistically significantly different (p=0.063) from the value of the same gene after nanoparticle treatment (-0.03±0.34). However, a statistically significant difference (p=0.049) was observed when the fold changes of the same gene before and after exposure to nanoparticles were compared. The toxicity of silver nanoparticles was tested with a hemolysis assay. A 500 mg/mL solution of silver nanoparticles accompanied by hemolysis was 0.033%, which means that silver nanoparticles are non-hemolytic and biocompatible. This study provides valuable information about the antibiotic resistance and biofilm-forming ability of Klebsiella pneumoniae clinical isolates. High resistance rates were observed for ESBLs 77%, Gentamicin 67% and Sulfamethoxazole/Trimethoprim 65%. Resistance to carbapenems such as Meropenem (29%) and Imipenem (13%) was also recorded, emphasizing the difficulties in treatment. All isolates formed biofilms, with 13% being strong producers, 40% moderate producers and 47% weak producers, emphasizing the persistence and virulence of the pathogen. RAPD and ERIC-PCR analyses revealed significant genetic diversity among the isolates, especially in strong and moderate biofilm producers. Silver nanoparticles showed a statistically significant effect in reducing the expression of the biofilm-associated mrkD gene, while LuxS gene expression was not affected. The nanoparticles were biocompatible and exhibited negligible hemolysis at the concentration tested. This study highlights the public health threat posed by multidrug-resistant K. pneumoniae and the potential of silver nanoparticles as an adjunctive strategy to reduce biofilm-associated virulence