In Vitro Activity, Stability and Molecular Characterization of Eight Potent Bacteriophages Infecting Carbapenem-Resistant Klebsiella pneumoniae

Abstract

Background: Members 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, including bacteriophage therapy. Bacteriophages are considered very safe and effective in treating bacterial infections. In this study, we characterize eight lytic bacteriophages that were previously isolated by our team against carbapenem-resistant Klebsiella pneumoniae. Methods: The one-step-growth curves, stability and lytic ability of eight bacteriophages were characterized. Restriction fragment length polymorphism (RFLP), random amplification of polymorphic DNA (RAPD) typing analysis and protein profiling were used to characterize the microbes at the molecular level. Phylogenetic trees of four important proteins were constructed for the two selected bacteriophages. Results and conclusions: All eight bacteriophages showed high efficiency for reducing bacterial concentration with high stability under different physical and chemical conditions. We found four major protein bands out of at least ten 15–190 KDa bands that were clearly separated by SDS-PAGE, which were assumed to be the major head and tail proteins. The genomes were found to be dsDNA, with sizes of approximately 36–87 Kb. All bacteriophages reduced the optical density of the planktonic K. pneumoniae abruptly, indicating great potential to reduce K. pneumoniae infection. In this study, we have found that tail fiber protein can further distinguished closely related bacteriophages. The characterised bacteriophages showed promising potential as candidates against carbapenem-resistant Klebsiella pneumoniae via bacteriophage therapy.

Potential of an Isolated Bacteriophage to Inactivate Klebsiella pneumoniae: Preliminary Studies to Control Urinary Tract Infections

“…The adsorption profile of phage KP1LMA showed that after 10 and 60 min, approximately 60 and 75% of the phage particles were adsorbed to the host cells, respectively, with an adsorption constant of K= 4.23246 × 10 −10 mL −1 min −1 . Similar results have been obtained with phage K2a [42]. However, in general, the adsorption rate of phage KP1LMA is lower than in other studies for Klebsiella phages [24,42,43].…”

“…Similar results have been obtained with phage K2a [42]. However, in general, the adsorption rate of phage KP1LMA is lower than in other studies for Klebsiella phages [24,42,43]. Phage LASTA and SJM3 adsorption assays showed that approximately 97 and 94%, respectively, of the phage particles were adsorbed to K. pneumoniae after 20 min [24].…”

“…It has been observed that K. pneumoniae phages have a wide range of latent periods and burst sizes, ranging from high burst sizes (410 PFU/infected cell) and short latent periods (21 min) [32] to low burst sizes (31.7 PFU/infected cell) and long latent periods (30 min) [34]. Baqer et al (2022) demonstrated that K. pneumoniae-infecting phages K2a, K2b, K2w5, K2w6, Kp99, K9w5, K9w6, K9coc had burst sizes of 116, 41, 354, 106, 214, 66, 130 and 210 PFU/host cell, respectively, with latency periods of approximately 5, 20, 20, 25, 20, 30, 10 and 10 min, respectively [42]. Phage BL02, presented a burst size of 156 PFU/infected cell in an average lysis period of 50 min [48].…”

“…However, since this phage presented a low adsorption rate, the likelihood of secondary infections is unlikely, and this may be a characteristic that favors application in scenarios of bacterial biofilm formation, as is the case for UTIs [51]. A phage's burst size and latent period depend on the phage type, the host's physiological state, the growth medium's composition, the pH, and the temperature of the incubation [42]. A smaller burst size can be caused by the phage's large size and the host's small size.…”

Biopharmaceutical properties of polyvalent bacteriophage capsules

“…Однако при пероральном приеме жидких лекарственных форм фаговых препаратов происходит их значительная инактивация под воздействием кислой среды желудочного сока [3]. Существующая проблема недостаточной стабильности характеристик препаратов бактериофагов и снижение их титра обусловливают необходимость учитывать кислотно-щелочные условия, а также влияние иных факторов в желудочно-кишечном тракте при разработке состава пероральных форм препаратов бактериофагов [4]. С целью устранения указанных недостатков на предприятии АО «НПО «Микроген» (филиал в г. Пермь «Пермское НПО «Биомед») разработан лекарственный препарат Секстафаг® Пиобактериофаг поливалентный в форме капсул, обеспечивающих сохранение литической активности бактериофага [5].…”