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Characterization and Full Genome Sequence of Novel KPP-5 Lytic Phage against Klebsiella pneumoniae Responsible for Recalcitrant Infection.

ABSTRACT: Klebsiella pneumoniae is a hazardous opportunistic pathogen that is involved in many serious human diseases and is considered to be an important foodborne pathogen found in many food types. Multidrug resistance (MDR) K. pneumoniae strains have recently spread and increased, making bacteriophage therapy an effective alternative to multiple drug-resistant pathogens. As a consequence, this research was conducted to describe the genome and basic biological characteristics of a novel phage capable of lysing MDR K. pneumoniae isolated from food samples in Egypt. The host range revealed that KPP-5 phage had potent lytic activity and was able to infect all selected MDR K. pneumoniae strains from different sources. Electron microscopy images showed that KPP-5 lytic phage was a podovirus morphology. The one-step growth curve exhibited that KPP-5 phage had a relatively short latent period of 25 min, and the burst size was about 236 PFU/infected cells. In addition, KPP-5 phage showed high stability at different temperatures and pH levels. KPP-5 phage has a linear dsDNA genome with a length of 38,245 bp with a GC content of 50.8% and 40 predicted open reading frames (ORFs). Comparative genomics and phylogenetic analyses showed that KPP-5 is most closely associated with the Teetrevirus genus in the Autographviridae family. No tRNA genes have been identified in the KPP-5 phage genome. In addition, phage-borne virulence genes or drug resistance genes were not present, suggesting that KPP-5 could be used safely as a phage biocontrol agent.

SUBMITTER: Sofy AR

PROVIDER: S-EPMC8066614 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major challenge for clinical management worldwide with limited antimicrobial options. Phages are considered an alternative option. Here, we isolated and identified a phage able to lyse ST11-K64 CRKP, the major type in China. This phage has a narrow host range, only lysing ST11-K64 CRKP, and inhibits the growth of host strains for 3 h forming large clear plaques (3.0 to 6.0 mm in diameter) with a surrounding halo. This phage exhibited excellent stability in different temperatures and pH and did not contain any virulence, lysogenic, antimicrobial resistance genes nor tRNA, meeting the criteria for phage therapy. Genomic analysis revealed that it represents a novel species of the Przondovirus genus according to ICTV standards. However, phage-resistant bacterial mutants emerged after 4-h exposure. Compared to the parental strain, phage-resistant mutants showed nonmucoid appearance and exhibited significantly reduced virulence for Galleria mellonella larva. Three randomly selected phage-resistant mutants were genome sequenced. Interruption of capsular polysaccharide biosynthesis-associated gene wcaJ or wbaZ by IS903B alone or an IS903-formed composite transposon was identified. Interruption of wcaJ is a known phage resistant mechanism, while that of wbaZ is not. By complementing the intact wbaZ, the phage susceptibility was restored, confirming the role of wbaZ interruption in phage resistance. This highlights that alteration in the capsular polysaccharide biosynthesis gene cluster, which could be due to transposable elements, is a major mechanism for resistance to Przondovirus phages in CRKP. Noncapsule-targeting phages may be combined for improving phage therapy against CRKP. IMPORTANCE Phage therapy is an alternative approach against multidrug resistant microorganisms such as carbapenem-resistant Klebsiella pneumoniae (CRKP), which represents a major challenge for treatment due to very limited options of antimicrobial agents. For optimizing phage therapy, more new lytic phages are needed. Here, we isolated and characterized a phage of a novel species able to rapidly lyse a major type of CRKP without carrying any virulence, lysogenic, antimicrobial resistance genes. This phage is therefore suitable for clinical treatment. However, phage-resistant mutants of CRKP strains were observed after exposure. We found a new mechanism, i.e., interruption of a capsular polysaccharide biosynthesis gene wbaZ by an insertion sequence-formed composite transposon. Our study demonstrates the capsular polysaccharide biosynthesis gene cluster as a major source of resistance to certain lytic phages in CRKP. This requires more studies to counter phage resistance. Our studies also highlight the critical role of insertion sequences in phage resistance.

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Characterization and Full Genome Sequence of Novel KPP-5 Lytic Phage against Klebsiella pneumoniae Responsible for Recalcitrant Infection.

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