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.

Immunoinformatics Driven Prediction of Multiepitopic Vaccine Against Klebsiella pneumoniae and Mycobacterium tuberculosis Coinfection and Its Validation via In Silico Expression.

Klebsiella pneumoniae and Mycobacterium tuberculosis coinfection is one of the most lethal combinations that has been becoming frequent yet, not diagnosed and reported properly. Due to the simultaneous occurrence of both infections, diagnosis is delayed leading to inadequate treatments and mortality. With the rise of MDR Klebsiella and Mycobacterium, a prophylactic and an immunotherapeutic vaccine has to be entailed for preemptive and adroit therapeutic approach. In this study, we aim to implement reverse vaccinology approach that encompasses a comprehensive evaluation of vital aspects of the pathogens to explore immunogenic epitopes against Omp A of Klebsiella and Rv1698, Rv1973 of Mtb that may help in vaccine development. The designed multi-epitopic vaccine was assessed for antigenicity, allergenicity and various physiochemical parameters. Molecular docking and simulations were executed to assess the immunogenicity and complex stability of the vaccine. The final multi-epitopic vaccine is validated to be highly immunogenic and can serve as a valuable proactive remedy for subject pathogens.

Draft genome sequence of an extensively drug-resistant neonatal Klebsiella pneumoniae isolate harbouring multiple plasmids contributing to antibiotic resistance.

OBJECTIVES: Klebsiella pneumoniae is a notorious nosocomial pathogen that has become a significant cause of neonatal infections causing morbidity and mortality. A multidrug-resistant K. pneumoniae isolate (K184) was isolated from a 5-day-old infant admitted to the neonatal intensive care unit of a local hospital in Rawalpindi, Pakistan. Whole-genome analysis of the isolated strain was performed to gain a better understanding of the genetic basis of antimicrobial resistance and virulence determinants. METHODS: K. pneumoniae isolate K184 was sequenced on an Illumina HiSeq 2500 platform. The genome was assembled using SPAdes with 30× coverage and was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v.4.3. Characterisation of the strain was performed using MLST 2.0 server. Plasmids, antimicrobial resistance determinants and virulence factors were identified using PlasmidFinder v.2.0, the Comprehensive Antibiotic Resistance Database (CARD) and Virulence Factors Database (VFDB), respectively. RESULTS: Neonatal K. pneumoniae isolate K184 has a considerably large genome with a size of 6,686,067 bp and a GC content of 55.6%. The isolate possesses three plasmids actively contributing to antimicrobial resistance, which classifies it as heavily loaded genome, along with three prophage regions. With 15 antimicrobial resistance determinants and various virulence factors, the neonatal isolate belongs to ST2096. CONCLUSION: The genome of neonatal isolate K184 studied here provides an insight into antibiotic resistance and virulence determinants. This draft genome can be used to compare antimicrobial-resistant K. pneumoniae strains isolated from the neonatal population.

Effectiveness of a Lytic Phage SRG1 against Vancomycin-Resistant Enterococcus faecalis in Compost and Soil.

Nosocomial infections caused by vancomycin-resistant Enterococcus have become a major problem. Bacteriophage therapy is proposed as a potential alternative therapy. Bacteriophages are viruses that infect bacteria and are ubiquitous in nature. Lytic bacteriophage was isolated from sewage water that infects VREF, the causative agent of endocarditis, bacteraemia, and urinary tract infections (UTIs). The phage produced clear plaques with unique clear morphology and well-defined boundaries. TEM results of phage revealed it to be 108 ± 0.2 nm long and 90 ± 0.5 nm wide. The characterization of bacteriophage revealed that infection process of phage was calcium and magnesium dependent and phage titers were highest under optimum conditions for VREF, with an optimal temperature range of 37-50°C. The maximum growth was observed at 37°C, hence having 100% viability. The latent period for phage was small with a burst size of 512 viral particles per bacterial cell. The phage was tested against various clinical strains and results proved it to be host specific. It can be used as a potential therapeutic agent for VREF infections. The phage efficiently eradicated VREF inoculated in cattle compost, poultry compost, and a soil sample which makes it a potential agent for clearing compost and soil sample.