Exploring the resistome, virulome, and mobilome of multidrug-resistant Klebsiella pneumoniae isolates: deciphering the molecular basis of carbapenem resistance.

BACKGROUND: Klebsiella pneumoniae, a notorious pathogen for causing nosocomial infections has become a major cause of neonatal septicemia, leading to high morbidity and mortality worldwide. This opportunistic bacterium has become highly resistant to antibiotics due to the widespread acquisition of genes encoding a variety of enzymes such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases. We collected Klebsiella pneumoniae isolates from a local tertiary care hospital from February 2019-February 2021. To gain molecular insight into the resistome, virulome, and genetic environment of significant genes of multidrug-resistant K. pneumoniae isolates, we performed the short-read whole-genome sequencing of 10 K. pneumoniae isolates recovered from adult patients, neonates, and hospital tap water samples. RESULTS: The draft genomes of the isolates varied in size, ranging from 5.48 to 5.96 Mbp suggesting the genome plasticity of this pathogen. Various genes conferring resistance to different classes of antibiotics e.g., aminoglycosides, quinolones, sulfonamides, tetracycline, and trimethoprim were identified in all sequenced isolates. The highest resistance was observed towards carbapenems, which has been putatively linked to the presence of both class B and class D carbapenemases, blaNDM, and blaOXA, respectively. Moreover, the biocide resistance gene qacEdelta1 was found in 6/10 of the sequenced strains. The sequenced isolates exhibited a broad range of sequence types and capsular types. The significant antibiotic resistance genes (ARGs) were bracketed by a variety of mobile genetic elements (MGEs). Various spontaneous mutations in genes other than the acquired antibiotic-resistance genes were observed, which play an indirect role in making these bugs resistant to antibiotics. Loss or deficiency of outer membrane porins, combined with ESBL production, played a significant role in carbapenem resistance in our sequenced isolates. Phylogenetic analysis revealed that the study isolates exhibited evolutionary relationships with strains from China, India, and the USA suggesting a shared evolutionary history and potential dissemination of similar genes amongst the isolates of different origins. CONCLUSIONS: This study provides valuable insight into the presence of multiple mechanisms of carbapenem resistance in K. pneumoniae strains including the acquisition of multiple antibiotic-resistance genes through mobile genetic elements. Identification of rich mobilome yielded insightful information regarding the crucial role of insertion sequences, transposons, and integrons in shaping the genome of bacteria for the transmission of various resistance-associated genes. Multi-drug resistant isolates that had the fewest resistance genes exhibited a significant number of mutations. K. pneumoniae isolate from water source displayed comparable antibiotic resistance determinants to clinical isolates and the highest number of virulence-associated genes suggesting the possible interplay of ARGs amongst bacteria from different sources.

Characterization of exclusively non-commensal Neisseria gonorrhoeae pangenome to prioritize globally conserved and thermodynamically stable vaccine candidates using immune-molecular dynamic simulations.

Neisseria gonorrhoeae (Ngo) has emerged as a global threat leading to one of the most common sexually transmitted diseases in the world. It has also become one of the leading antimicrobial resistant organisms, resulting in fewer treatment options and an increased morbidity. Therefore, in recent years, there has been an increased focus on the development of new treatments and preventive strategies to combat its infection. In this study, we have combined the most conserved epitopes from the completely assembled strains of Ngo to develop a universal and a thermodynamically stable vaccine candidate. For our vaccine design, the epitopes were selected for their high immunogenicity, non-allergenicity and non-cytotoxicity, making them the ideal candidates for vaccine development. For the screening process, several reverse vaccinology tools were employed to rigorously extract non-homologous and immunogenic epitopes from the selected proteins. Consequently, a total number of 3 B-cell epitopes and 6 T-cell epitopes were selected and joined by multiple immune-modulating adjuvants and linkers to generate a promiscuous immune response. Additionally, the stability and flexible nature of the vaccine construct was confirmed using various molecular dynamic simulation tools. Overall, the vaccine candidate showed promising binding affinity to various HLA alleles and TLR receptors; however, further studies are needed to assess its efficacy in-vivo. In this way, we have designed a multi-subunit vaccine candidate to potentially combat and control the spread of N. gonorrhoeae.