Probiotic Lacticaseibacillus paracasei from human gut microbiome against colistin-resistant Klebsiella pneumoniae: in vitro, in vivo and probiogenomic approaches

Antibiotic treatment regimens fail to address Klebsiella pneumoniae exhibiting resistance to multiple drugs, including the last resort antibiotic, colistin. The use of probiotics as candidates for alternative antimicrobial therapy or as a source of new antibiotics is considered as an emerging trend in therapeutics. Rejuvenating the human gut with probiotics offers an intriguing therapeutic approach in various enteric diseases. However, the precise role of probiotics in non-enteric infections, particularly those caused by colistin-resistant Klebsiella pneumoniae remains unresolved, prompting further comprehensive research. Therefore, we propose an innovative prophylactic approach using Lacticaseibacilli of human gut origin against this pathogen. Probiotic characterisation like tolerance to acid, bile and sodium chloride were performed to evaluate its gastric survival. In vitro experiments revealed that non-neutralised cell-free supernatant (CFS) of Lacticaseibacillus has the potential to inhibit pathogenic K. pneumoniae. The observed growth reduction is suggestive of the cumulative effect of organic acids and other antimicrobial substances in CFS. The two Lacticaseibacillus paracasei isolates exhibited promising activity (with suspected proteinaceous heat labile molecules) against K. pneumoniae and those with better adhesion to CaCo-2 cell lines were selected for downstream studies. Scanning electron microscopic analysis of CFS treated pathogen cells revealed cell surface distortions and pore formations. The prophylactic potential of Lacticaseibacillus (live and heat-inactivated forms) in Balb/c mice model showed a reduction in histopathological and microbiological alterations caused by K. pneumoniae, when compared with untreated pathogen control. Whole genome analysis of the potential probiotic isolate revealed the genome is devoid of any antibiotic resistance genes and other virulence markers indicating its safety in vivo. Furthermore, the in vitro pathogen inhibition results were reinforced by antiSMASH and BAGEL analysis, which predicted the presence of putative bacteriocin genes. Hence, this multiapproach research study has revealed a promising prophylactic probiotic from human gut microbiome against multi-drug resistant K. pneumoniae.