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.

T Regulatory Cells in Rheumatoid Arthritis with Reference to Anti-Citrullinated Peptide Antibody and TNF-alpha Inhibitor Therapy.

T regulatory cells (Tregs) plays an important role in maintaining self-tolerance and preventing autoimmune diseases by inhibiting proliferation and cytokine production of self-reactive T cells. Controversy was reported regarding the frequency of CD4+CD25+ Tregs in the peripheral circulation of rheumatoid arthritis (RA) patients compared to normal controls. Also, some showed that treatment with TNF-α inhibitor restored the capacity of Tregs. This work aimed to study Tregs in the peripheral blood of RA patients versus control in addition to those on TNF-α inhibitor therapy compared to those who have not received it and to correlate with status of anti-cyclic citrullinated peptide antibody (ACPA). Two groups of RA patients were studied; one on TNF-α inhibitor therapy and the other not. Additionally, age-matched apparently healthy controls were studied. The percentage of CD4+CD25+ T cells in the total lymphocytic cell population was determined by flow cytometry analysis while ACPA concentration was measured by a second-generation peptide-based ELISA. Mean level of Tregs was significantly lower in the studied RA patients compared to the control group. Patients in early disease (0-5 years) had low mean Tregs percentage compared to patients with long duration of disease (> 10 years) (P=0.044). Patients on TNF-α blocker therapy had elevated Tregs percentage relative to patients on methotrexate (MTX) (P=0.022) and other therapies. No effect of gender or age was found on Tregs levels. In RA patients, 85.4% were ACPA seropositive and 65.9% of seropositive patients have concentration of > 100U/ml. The mean Treg percentage was significantly lower in ACPA seronegative group compared to the seropositive group (P=0.013). In conclusion, the studied RA patients have low Treg, and TNF-α blocker therapy increased its number, compared to other therapies.