Abstract
Phage therapy involves the application of lytic bacteriophages for treatment of clinical infections but bacterial resistance may develop over time. Isolated from nosocomial infections, small colony variants (SCVs) are morphologically distinct, highly virulent bacterial strains that are resistant to conventional antibiotics. In this study, SCVs was derived from Pseudomonas aeruginosa exposed to the lytic bacteriophage PB1 and these cells were resistant to subsequent phage infection by PB1. To elucidate the mechanism of the SCV phage resistance, we performed phenotypic assays, DNA microarrays and whole-genome sequencing. Compared with wild-type P. aeruginosa, the SCV isolate showed impaired biofilm formation, decreased twitching motility, reduced elastase and pyocyanin production. The SCV is also more susceptible to the antibiotic ciprofloxacin and exhibited higher syrface hydrophobicity than the wild-type, indicative of changes to cell surface lipopolysaccharide (LPS) composition. Consistent with these results, transcriptomic studies of SCV revealed up-regulation of genes involved in O-specific antigen (OSA) biosynthesis, suggesting the regulation of surface moieties may account for phage resistance. Western blot analysis showed a difference in OSA distribution between the two strains. Simultaneously, genes involved in aromatic and branched chain amino acid catabolism were down-regulated. Whole genome sequencing of the SCV revealed multiple single nucleotide variations within the Pf1 prophage region, a genetic locus known to play a crucial role in biofilm formation and to provide survival advantage via gene transfer to a subpopulation of cells. Insights into phenotypic and genetic changes in SCV gained here should help direct future studies to elucidate mechanisms underpinning phage resistance, leading to novel counter resistance measures.
Highlights
The emergence of multidrug-resistant pathogens and the difficulties in developing new antibiotics have spurred the resurgence of interest in phage therapy, involving the use of lytic bacteriophages against specific pathogens as a form of treatment (DiMasi et al, 2003; Gilbert et al, 2003; Spellberg et al, 2004; Talbot et al, 2006; Keen, 2012; Ly-Chatain, 2014)
Since F1 was isolated under the selection pressure imposed by PB1 phage, it is possible that F1 is resistant to subsequent bacteriophage treatment
The generation time for F1 strain as determined by OD600 measurement is lower than F0 (Figure 1C), which may suggest that F1 has a faster growth rate, contradicting previous reports on SCVs being slow-growing (Proctor et al, 2006)
Summary
The emergence of multidrug-resistant pathogens and the difficulties in developing new antibiotics have spurred the resurgence of interest in phage therapy, involving the use of lytic bacteriophages against specific pathogens as a form of treatment (DiMasi et al, 2003; Gilbert et al, 2003; Spellberg et al, 2004; Talbot et al, 2006; Keen, 2012; Ly-Chatain, 2014). P. aeruginosa have been isolated from urinary tract and inner ear infections, burn wounds and from the surface of human epithelia in cystic fibrosis patients (Govan and Deretic, 1996; Lyczak et al, 2000; Lang et al, 2004; Taneja et al, 2004). In another, Khawaldeh et al (2011) reported the use of a lytic bacteriophage cocktail to treat a human patient suffering from P. aeruginosa urinary tract infection. These reports indicate that while phage therapy can be initially effective against P. aeruginosa, spontaneous phage resistance often occurs afterward, rendering the phage therapy ineffective
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