Screening of Bacteriophages against different genotypes of Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolated from five hospitals in Cavite and Metro Manila, Philippines

Abstract

Background: Extended-spectrum β-lactamase (ESBL) K. pneumoniae infections are emerging health problem in the Philippines. Recently, bacteriophages have been explored to target several antibiotic resistant bacteria as a potential alternative therapeutic option to conventional antibiotics.

Objectives: This study isolated extended-spectrum β-lactamase (ESBL) producing K. pneumoniae harboring different β-lactamase genes to evaluate the host range specificity of an isolated bacteriophages.

Methodology: K. pneumoniae were isolated from five selected hospitals in Cavite and Metro Manila,
Philippines and its ESBL-production was determined through the Phenotypic Confirmatory Disc Diffusion Test (PCDDT). The identity of the isolates was then confirmed by amplification and sequencing of the 16 rRNA gene. The type of β-lactamase genes carried by the K. pneumoniae ESBL-positive strains was detected by amplification of the bla , bla , bla and bla genes. Meanwhile, bacteriophages were isolated from CTX-M TEM SHV OXA-1 water samples in Marikina River and its host range specificity was tested against the different ESBL-producing K. pneumoniae strains.

Results: From a total of 25 K. pneumoniae, 6 (24%) were found to be ESBL-producers by PCDDT. Genotyping of the β-lactamase genes showed that one of the phenotypically confirmed isolate contained the bla while CTX-M another possessed both the bla and bla genes. Furthermore, another isolate harbored the bla , bla , CTXM SHV CTX-M OXA-1 and bla genes while the remaining isolates contained all the four bla genes tested. Meanwhile, two virulent SHV phages namely, KP1 and KP2 that showed the largest clearing zones against K. pneumoniae were selected to determine its host range specificity against the different ESBL-positive K. pneumoniae strains. Both phages were able to infect and lyse all ESBL-positive K. pneumoniae regardless of the type or number of bla genes it possessed. Phage KP2 which showed the highest lytic capability was then subjected to Transmission Electron
Microscopy (TEM) and was found to belong to the Order Caudovirales under the Family Myoviridae.

Conclusion: This study showed that phage KP2 was host specific to the different ESBL-producing K.
pneumoniae harboring single or multiple bla genes suggesting that it might hold a great potential for possible phage therapy against ESBL-producing K. pneumoniae infections.