Isolation And Characterization Of Newly Lytic Bacteriophages Active Against Klebsiella Pneumoniae Clinical Isolates

Klebsiella pneumoniae, a gram-negative enteric bacterium, is one of commonpathogens that cause nosocomial infection. K. pneumoniae has been documented tocause invasive infections leading to abscesses at other sites (eg, kidney, spleen, brain,and prostate). Moreover, community-acquired pyogenic liver abscess caused by K.pneumonia has increasingly emerged recently, and multi-drug resistant K. pneumoniaeisolates carrying extended-spectrum beta-lactamases (ESBLs) encoding plasmids arebecoming increasingly associated with nosocomial infection. The high incidence ofmultidrug resistant bacteria has led to limited efficacy of treatment with currentantibiotics and a high probability of patient colonization by resistant strains.Bacteriophages, or phages, are viruses that infect bacteria, and these viruses areubiquitous throughout the environment. Phage therapy is referred to application ofphage as an agent against bacterial infection. This manuscript focuses on characterizingthe biology of lytic bacteriophages active against K. pneumoniae strains and theirpotential as alternative antimicrobials against drug-resistant K. pneumoniae isolates.Using25clinical K. pneumoniae strains as the host,4lytic bacteriophages(φKp-lyy15, φKp-lyy17, φKp-lyy19and φKp-lyy22) were isolated from waste waterdischarged by hospitals. Transmission electron microscopy revealed that these isolatedphages were classified in the order Caudovirales on the basis of their morphologicalfeatures. Phage φKp-lyy15and φKp-lyy19belong to member of Siphoviridae family,phage φKp-lyy17was classified into Podoviridae family and phage φKp-lyy22exhibited characteristics typical of member of Guttaviridae family. Analysis of phagecharacteristics was performed and growth activities included the following,(1) thelatent period of the φKp-lyy22was15min, followed by rise period of nearly80min,and plateau phase. The burst size was60PFU/ml. The phage φKp-lyy22was shown tolyse3of25(12%) clinical K. pneumonia strains, exhibited a narrow host range.(2) thephage φKp-lyy15was shown having a latent period of10min and rise period of30 min. The burst size was65particles per one infected cell.(3) φKp-lyy15had a doublestranded DNA, the genome of φKp-lyy15is sensitive to EcoRV、NdeI and BglII.(4)SDS-PAGE analysis of phage φKp-lyy15revealed that3major protein bands and8minor protein bands were found, with molecular weights ranging from15to170kDa.(5) the phage φKp-lyy15was shown to specifically infect and lyse8of25(32%) K.pneumonia strains, exhibiting a broad range of lytic activity against host cells.(6)phage φKp-lyy15was capable of lysing host cells effectively in a range of pH4～10and at temperature of50℃～60℃.In conclusion,4isolated lytic phages can be assigned to different families of orderCaudovirales. Phage φKp-lyy15and φKp-lyy19were classified as member ofSiphoviridae family. Phage φKp-lyy17belonged to Podoviridae family, whileφKp-lyy22belonged to Guttaviridae family. Given that phage φKp-lyy15exhibiteda strong virulence, high specificity and resistance to both pH and temperature, phageφKp-lyy15may be candidate therapeutic agent to control infectious diseases caused bydrug-resistance K. pneumoniae.