Isolation and characterization of phages against pan-resistant Klebsiella pneumoniae.

PUJATO, S.; MERCANTI, D.; QUIBERONI, A.; PUJATO, N.; RADISIC, M.; FERNANDEZ, A.; BRIGGILER MARCÓ, M.

Congreso; 32 Congresso Brasileiro Microbiología 2023; 2023

Antibiotic resistance is a growing public health challenge worldwide. In particular, infections caused by the bacterium Klebsiella pneumoniae have become increasingly difficult to treat due to their ability to develop resistance to multiple antibiotics. In September 2022, a carbapenemase-producing strain of Klebsiella pneumoniae was isolated in Argentina. When determining the Minimum Inhibitory Concentration (MIC) and/or the disk diffusion test, phenotypic resistance to penicillins, cephalosporins, monobactams, carbapenems, aminoglycosides, fosfomycin, lipopeptides, tetracyclines/glycylcyclines, quinolones, rifamycins, folate inhibitors, phenicols, and nitrofurans available in Argentina was confirmed. Due to its high resistance, the use of bacteriophages was considered as an alternative for its treatment.The objective of this study was to isolate, characterize, and sequence bacteriophages capable of lysing antibiotic-resistant strains of Klebsiella pneumoniae and to select those that do not possess antibiotic resistance genes or lysogeny-associated genes for use in phage therapy.Wastewater samples from meat locker and sewage near a hospital were collected to isolate Klebsiella pneumoniae bacteriophages. Phages were propagated in LB-Ca-Mg broth. DNA was extracted with phenol-chloroform, and its restriction pattern was analyzed using BioNumerics™V6 software. DNA sequencing was performed with the DNA Nextera XT kit (Illumina). Sequences were assembled and quality assessed using FastQ software. Genome assembly was confirmed by PCR, and genes were predicted using GeneMark and ORF Finder. Sequences were compared to other phage genomes using NCBI blast.It was possible to isolate a total of 8 highly specific phages for the pan-resistant strain of Klebsiella pneumoniae. All demonstrated the ability to lyse the strain in a liquid medium in 3 hours, obtaining a titer of 1010 PFU/mL. When cut with enzymes, 5 different patterns were observed. The sequencing analysis revealed that the selected phages did not have antibiotic resistance genes or genes associated with lysogeny.The results obtained indicate that the specific bacteriophages isolated in this study could be a promising alternative as adjuvants to antibiotic therapy in the treatment of infections caused by the antibiotic-resistant strain of Klebsiella pneumoniae. The ability of phages to selectively kill target bacteria, without affecting healthy human cells, and their ability to adapt to constantly evolving bacteria make them a potentially effective option against resistant strains