Antibiotic Resistance and Transcriptional Analysis of Klebsiella pneumoniae to Fluoroquinolones.

Abstract

Multi Drug Resistant K. pneumoniae is considered as a serious health concern worldwide. Infections caused by K. pneumoniae are of prime importance among elderly, neonates and immunocompromised patients in hospital settings, however, this pathogen is also credited with various community-acquired diseases in which sepsis and pneumonia are blatant ones. Fluoroquinolone antibiotics are broad-spectrum and potent antimicrobial agents frequently opted by the clinicians to manage various infectious diseases. Several mechanisms like modifying enzymes, efflux pumps, target site mutation and target protective proteins are responsible for resistance against these antimicrobial agents. Emergence of fluoroquinolone-resistant bacteria is elevating with a worrying rate. In Pakistan, this important subject has been neglected so far, as little data is available about molecular insights of MDR K. pneumoniae especially with reference to Fluoroquinolone, which is rapidly being used as the drug of choice to treat bacterial infections caused by the Gram-negative bacteria, in different clinical and health care settings. Henceforth, present study was designed with the manifesto to study the molecular insights and transcriptional analysis of K. pneumoniae against the Fluoroquinolones. Clinical samples (n = 889) including urine (UTI Patients n = 278), Tracheal aspirates (n = 222), Wounds (n = 174) and Blood (n = 215) were collected from hospital settings. Isolation and identification of K. pneumoniae was done with the help of conventional microbiological procedures. Characterization of K. pneumoniae was done based on phenotypic and genetic characteristics. Phenotypic characterization was done based on cultural and morphological properties. Moreover, biochemical characterization was completed by employing the biochemically characterized samples to API® 20E strip (bioMérieux, France). Polymerase Chain Reaction (PCR) was then performed to target a portion of 16S rDNA for molecular identification of K. pneumoniae. Both, Disc-Diffusion Assay and Broth Micro-Dilution Assay were performed to study the antibiotic susceptibility profiling of K. pneumoniae isolates against various antibiotics including β-lactams and Penicillin, Quinolones, Cephalosporins, Tetracyclines, Aminoglycosides, Sulpha drugs and others like Chloramphenicol and Tigecycline. Minimum Inhibitory Concentration (MIC) of the drug Ciprofloxacin against K. pneumoniae was determined by broth dilution method. Afterwards, molecular insights of Fluoroquinolone resistance were studied by targeting various genes of chromosomal origin, responsible for resistance which include Quinolone Resistant Determining Regions i.e. QRDRs (gyrA & gyrB) and then from the plasmid origin, Plasmid Mediated Quinolone Resistant determinants i.e. PMQRs (qnrA, qnrB & qnrS). Lastly, transcriptional analysis of K. pneumoniae was observed under Ciprofloxacin stress in time depended manner using qPCR by targeting genes like gyrA, lexA, eu-Tu, mutS, dnaK, ftsZ and clpB. Findings of the present study revealed out that among 889 clinical samples that were collected from various sample sources, 200 (22.49 %) were confirmed as K. pneumoniae. Whereas, distribution of K. pneumoniae from confirmed (200) isolates was as follow Urine (UTI patients); 86 (43%), Tracheal aspirates; 51 (25.5 %), Purulent wounds; 33 (16.5 %) and Blood; 30 (15 %). Isolates of K. pneumoniae has shown resistance to almost all the used antibiotics (19) in the study except for Cefotaxime and Tigecycline. Additionally, significant resistance was found against Fluoroquinolones. Out of 200 confirmed isolates of K. pneumoniae 179 (89.5 %) showed resistance to Ciprofloxacin. MIC of 64 µg/ml was recorded for 42 % isolates. Total 81 (45.5 %) out of 179 isolates were found to harbor qnrB gene. Sequence analysis of qnrB revealed that it is qnrB1 variant and has been assigned the Accession number MF953600. The gyrA was found with a strong mutation for Ciprofloxacin resistance i.e. Ser83→Ile. Out of 179 ciprofloxacin resistant isolates in 98 (54.75 %) gyrA gene was present whereas 41 (22.9 %) isolates harbor qnrB1 gene. In 40 (22.5 %) isolates both gyrA and qnrB1 genes were present. Transcriptional analysis of K. pneumoniae isolates has revealed that basal gene expression is altered against Ciprofloxacin stress in a time depended manner. With the finding of current study, it was concluded that MDR K. pneumoniae is an urgent public health pathogen in Pakistan. It has been found that it is resistant to Ciprofloxacin which is considered as one of the most important and frequently used antibiotics in the country. Indigenous K. pneumoniae has fluoroquinolone resistance due to qnrB1 variant of PMQR and mutation in gyrA gene of QRDRs. In addition, Ciprofloxacin has influenced the gene expression of indigenous K. pneumoniae. Mounting antibiotic resistance and emerging MDR superbugs like K. pneumoniae pose a significant threat worldwide especially in developing countries, and it could only be contained by devising comprehensive studies and strategies to control the menace of infectious disease caused by resistant pathogens.