Extended genomic analyses of the broad-host-range phages vB_KmiM-2Di and vB_KmiM-4Dii reveal slopekviruses have highly conserved genomes

Abstract

High levels of antimicrobial resistance among members of the Klebsiella oxytoca complex (KoC) have led to renewed interest in the use of bacteriophage (phage) therapy to tackle infections caused by these bacteria. In this study we characterized two lytic phages, vB_KmiM-2Di and vB_KmiM-4Dii, that were isolated from sewage water against two GES-5-positive Klebsiella michiganensis strains (PS_Koxy2 and PS_Koxy4, respectively). ViPTree analysis showed both phages belonged to the genus Slopekvirus. rpoB gene-based sequence analysis of 108 presumptive K. oxytoca isolates (n=59 clinical, n=49 veterinary) found K. michiganensis to be more prevalent (46 % clinical and 43 % veterinary, respectively) than K. oxytoca (40 % clinical and 6 % veterinary, respectively). Host range analysis against these 108 isolates found both vB_KmiM-2Di and vB_KmiM-4Dii showed broad lytic activity against KoC species. Several hypothetical homing endonuclease genes were encoded within the genomes of both phages, which may contribute to their broad host range. Differences in the tail fibre protein may explain the non-identical host range of the two phages. Pangenome analysis of 24 slopekviruses found that genomes within this genus are highly conserved, with more than 50 % of all predicted coding sequences representing core genes at ≥95 % identity and ≥70 % coverage. Given their broad host ranges, our results suggest vB_KmiM-2Di and vB_KmiM-4Dii represent attractive potential therapeutics. In addition, current recommendations for phage-based pangenome analyses may require revision.

Lytic bacteriophage vB_KmiS-Kmi2C disrupts biofilms formed by members of theKlebsiella oxytocacomplex, and represents a novel virus family and genus

“…The phage was screened against 56 clinical and 28 animal isolates representing a range of Klebsiella spp. ( K. michiganensis n=49, K. oxytoca n=25, K. grimontii n=9, K. huaxiensis n=1) [9, 21]. It did not infect the closely related K. michiganensis strains PS_Koxy1 and PS_Koxy4 [9].…”

“…vB_KmiS-Kmi2C did not infect animal isolates of Klebsiella spp. [21], but did infect some of the clinical K. oxytoca ( n =2) and K. michiganensis ( n =4) strains within our extended in-house collection [21] ( Table 1 ). From strain information provided by the Pathogen Bank (EUCAST testing) all these strains were resistant to amoxycillin, and sensitive to amikacin, ceftazidime, ceftazidime, ceftriaxone, cefuroxime, ciprofloxacin, ertapenem, gentamicin, meropenem, piptazobactam and trimethoprim.…”

“…Filter-sterilised sewage samples (0.45 µm cellulose acetate filter; Millipore) collected from mixed-liquor tanks at Mogden Sewage Treatment Works (March 2017) were screened against K. michiganensis strain PS_Koxy2 as described previously [21]. Host range analysis was carried out as described previously [21].…”

“…Filter-sterilised sewage samples (0.45 µm cellulose acetate filter; Millipore) collected from mixed-liquor tanks at Mogden Sewage Treatment Works (March 2017) were screened against K. michiganensis strain PS_Koxy2 as described previously [21]. Host range analysis was carried out as described previously [21]. After overnight incubation, overlay assay plates were inspected for lysis, with results recorded according to a modification of [22]: ++, complete lysis; +, hazy lysis; 0, no visible plaques.…”

“…Based on recently updated guidelines for the classification of phage based on nucleotide sequence identity, and subsequent protein based phylogenetic analyses, we show here that phage vB_KmiS-Kmi2C represents a new genus. # Strain on which phage vB_KmiS-Kmi2C was isolated; all other strains used in host range analysis but not infected by the phage are described in [21]. PTU, plasmid taxonomy unit; -, no data available.…”