Isolation and Characterization of a Novel Lytic Bacteriophage against the K2 Capsule-Expressing Hypervirulent Klebsiella pneumoniae Strain 52145, and Identification of Its Functional Depolymerase

Abstract

Klebsiella pneumoniae is among the leading bacteria that cause nosocomial infections. The capsule of this Gram-negative bacterium is a dominant virulence factor, with a prominent role in defense and biofilm formation. Bacteriophages, which are specific for one bacterial strain and its capsule type, can evoke the lysis of bacterial cells, aided by polysaccharide depolymerase enzymes. In this study, we isolated and characterized a bacteriophage against the nosocomial K. pneumoniae 52145 strain with K2 capsular serotype. The phage showed a narrow host range and stable lytic activity, even when exposed to different temperatures or detergents. Preventive effect of the phage in a nasal colonization model was investigated in vivo. Phlyogenetic analysis showed that the newly isolated Klebsiella phage B1 belongs to the Webervirus genus in Drexlerviridae family. We identified the location of the capsule depolymerase gene of the new phage, which was amplified, cloned, expressed, and purified. The efficacy of the recombinant B1dep depolymerase was tested by spotting on K. pneumoniae strains and it was confirmed that the extract lowers the thickness of the bacterium lawn as it degrades the protective capsule on bacterial cells. As K. pneumoniae strains possessing the K2 serotype have epidemiological importance, the B1 phage and its depolymerase are promising candidates for use as possible antimicrobial agents.

Topically Applied Bacteriophage to Control Multi-Drug Resistant Klebsiella pneumoniae Infected Wound in a Rat Model

“…The titer of phage suspension was diluted 10 folds in a 96-well plate, where each lane contained exactly 180 μL of SM buffer and 20 μL of phage suspension. Only 10 μL aliquots were spotted in triplicate onto bacterial lawns [ 52 ].…”

“…The titer of phage suspension was diluted 10 folds in a 96-well plate, where each lane contained exactly 180 µL of SM buffer and 20 µL of phage suspension. Only 10 µL aliquots were spotted in triplicate onto bacterial lawns [52] Pulsed-field gel electrophoresis was used to determine the genomic size of phage ZCKP8 (10 10 PFU/mL) DNA [53]. In brief, phage was suspended in agarose plugs and digested with lysis buffer (0.2% w/v SDS [Sigma Aldrich, Gillingham, UK]; 1% w/v N-Lauryl sarcosine [Sigma Aldrich, Gillingham, UK]; 100 mM EDTA; 1 mg/mL Proteinase K [ThermoFischer Scientific], Waltham, MA, USA), overnight at 55 • C for 18 h with gentle shaking to lyse the phage capsids and digest protein components.…”

Characterization and Functional Studies of a Novel Depolymerase Against K19-Type Klebsiella pneumoniae

“…They are also found to be safer since phage therapies may result in the development of bacterial resistance to phages and new antimicrobial resistance, which would not be a concern when using depolymerases ( Loc-Carrillo and Abedon, 2011 ; Principi et al, 2019 ). So far, 34 distinct depolymerases that targeted a range of 22 K. pneumoniae capsular types were discovered ( Wu et al, 2019 ; Liu et al, 2020 ; Squeglia et al, 2020 ; Volozhantsev et al, 2020 ; Dunstan et al, 2021 ; Gorodnichev et al, 2021 ; Li J. et al, 2021 ; Pertics et al, 2021 ). These depolymerases had great potential for capsular typing of K. pneumoniae , and they were found to be effective in raising the susceptivity of bacteria to serum killing ( Pan et al, 2019 ; Li J. et al, 2021 ).…”

“…For example, depolymerases can increase K. pneumoniae ’s susceptibility to gentamicin at a lower concentration ( Bansal et al, 2014 ); Depolymerase Dpo42 and Dpo43 of K47-type K. pneumoniae make host bacteria fully susceptible to the killing effect of serum complement ( Liu et al, 2020 ; Volozhantsev et al, 2020 ); Depolymerase KP32gp37 and KP32gp38 of K3-type and K21-type K. pneumoniae , respectively, can efficiently decrease K. pneumoniae resistance to phagocytosis by macrophages ( Majkowska-Skrobek et al, 2018 ). Till now, depolymerases targeting 23 K. pneumonia capsular types, namely, K1, K2, K3, K5, K8, K11, K13, K21, K23, K25, K30, K35, K47, K56, K57, K63, K64, K69, KN1, KN2, KN3, KN4, and KN5, have been identified and reported ( Wu et al, 2019 ; Liu et al, 2020 ; Squeglia et al, 2020 ; Volozhantsev et al, 2020 ; Dunstan et al, 2021 ; Gorodnichev et al, 2021 ; Li J. et al, 2021 ; Pertics et al, 2021 ).…”

Application of Bacteriophages for Human Health: An Old Approach against Contemporary “Bad Bugs”

“…At the same time, this collection reports many descriptions of newly characterized phages, which could be good candidates for the treatment of human infections due to Enterococcus faecalis [ 8 , 9 ], Klebsiella pneumoniae [ 10 ], Escherichia coli [ 11 ] and Pseudomonas aeruginosa [ 12 ] or to combat phytopathogens [ 13 , 14 ] or bacteria responsible for foodborne diseases [ 15 , 16 , 17 ]. It is relevant to underscore that, besides the use of standard techniques for general characterization of phages, some contributions evaluated their effective activity on bacterial biofilm [ 8 , 11 , 12 ] or on animal infection models [ 9 , 10 ], while others took original experimental approaches tailored to the future intended usage of the investigated phages [ 8 , 13 , 16 ]. Altogether, these models constitute major steps forward for the applicability of phages for therapeutic purposes or, more generally, as biocontrol agents.…”