Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae

Abstract

BackgroundMembers of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment. Bacteriophage therapy forms one of these alternate strategies.MethodsElectron microscopy, burst size, host range, sensitivity of phage particles to temperature, chloroform, pH, and restriction digestion of phage DNA were used to characterize Klebsiella phages.Results and conclusionsOf the 32 isolated phages eight belonged to the family Myoviridae, eight to the Siphoviridae whilst the remaining 16 belonged to the Podoviridae. The host range of these phages was characterised against 254 clinical Enterobacteriaceae strains including multidrug resistant Klebsiella isolates producing extended-spectrum beta-lactamases (ESBLs). Based on their lytic potential, six of the phages were further characterised for burst size, physicochemical properties and sensitivity to restriction endonuclease digestion. In addition, five were fully sequenced. Multiple phage-encoded host resistance mechanisms were identified. The Siphoviridae phage genomes (KP16 and KP36) contained low numbers of host restriction sites similar to the strategy found in T7-like phages (KP32). In addition, phage KP36 encoded its own DNA adenine methyltransferase. The φKMV-like KP34 phage was sensitive to all endonucleases used in this study. Dam methylation of KP34 DNA was detected although this was in the absence of an identifiable phage encoded methyltransferase. The Myoviridae phages KP15 and KP27 both carried Dam and Dcm methyltransferase genes and other anti-restriction mechanisms elucidated in previous studies. No other anti-restriction mechanisms were found, e.g. atypical nucleotides (hmC or glucosyl hmC), although Myoviridae phage KP27 encodes an unknown anti-restriction mechanism that needs further investigation.

Capsule-Targeting Depolymerase, Derived from Klebsiella KP36 Phage, as a Tool for the Development of Anti-Virulent Strategy

“…While studying the phage KP36, we noticed the appearance of hazy, expanding halo zones surrounding plaques with a small clear center formed by it on a bacterial lawn [21]. This observation indicated that phage KP36 may produce a virion-associated polysaccharide-degrading enzyme.…”

“…We originally isolated Klebsiella phage KP36 from biologically-treated sewage in Poland using the K. pneumoniae clinical isolate 486 as the host strain [21]. This phage and bacterial strain have been deposited in the Polish Collection of Microorganisms (Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland) under accession numbers F/00068 and B/F/00068, respectively.…”

“…Bacteriophage KP36 genomic DNA was extracted and purified as previously described [21]. The gp50 open reading frame (ORF; NCBI acc.…”

“…In our previous study, the phage KP36 (vB_KpnS_KP36), whose genome encompasses 49,797 base pairs (bp) encoding 80 gene products, was characterized [21]. Transmission electron microscopy and genome sequence analyses revealed that this phage belongs to the family Siphoviridae , and is currently classified as a member of the “ KP36likevirus ” genus within subfamily Tunavirinae [22].…”

Tradeoffs in bacteriophage life histories

“…24 A recent, largescale study of 32 environmental phages and almost 150 Klebsiella strains also found faster replication to be associated with a narrower host range, with slowerreplicating phages typically exhibiting the broadest spectrum of lytic activity. 25 A tradeoff between replication rate and host range is plausible both evolutionarily (e.g., broader host range as compensation for decreased replication; specialism vs. generalism) and energetically, since producing the structures that physically enable a broader host range (e.g., displaying multiple types of receptorbinding tail fibers on a single virion) 26,27 requires a greater energy expenditure. All in all, it seems that versatility has its drawbacks-even at the edge of life.…”