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Small Colony Variants and Single Nucleotide Variations in Pf1 Region of PB1 Phage-Resistant Pseudomonas aeruginosa.

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.

SUBMITTER: Lim WS

PROVIDER: S-EPMC4783410 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Small Colony Variants and Single Nucleotide Variations in Pf1 Region of PB1 Phage-Resistant Pseudomonas aeruginosa.

Lim Wee S WS Phang Kevin K S KK Tan Andy H-M AH Li Sam F-Y SF Ow Dave S-W DS

Frontiers in microbiology 20160309

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 elu ...[more]

PMID: 27014207

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Project description:From May 1997 to December 2001, a serotype O:6 multidrug-resistant strain of Pseudomonas aeruginosa colonized or infected 201 patients in the University Hospital of Besançon (France). The susceptibility profile of this epidemic clone to fluoroquinolones and aminoglycosides was relatively stable during the outbreak but showed important isolate-to-isolate variations (up to 64-fold) in the MICs of beta-lactams. Analysis of 18 genotypically related isolates selected on a quarterly basis demonstrated alterations in the two DNA topoisomerases II and IV (Thr83-->Ile in GyrA and Ser87-->Leu in ParC) and production of an ANT(2")-I enzyme. Although constitutively overproduced in these bacteria, the MexXY efflux system did not appear to contribute significantly to aminoglycoside resistance. beta-Lactam resistance was associated with derepression of intrinsic AmpC beta-lactamase (with isolate-to-isolate variations of up to 58-fold) and sporadic deficiency in a 46-kDa protein identified as the carbapenem-selective porin OprD. Of the 18 isolates, 14 were also found to overproduce the efflux system MexAB-OprM as a result of alteration of the repressor protein MexR (His107-->Pro). However, complementation experiments with the cloned mexR gene demonstrated that MexAB-OprM contributed only marginally to beta-lactam and fluoroquinolone resistance. Of the four isolates exhibiting wild-type MexAB-OprM expression despite the MexR alteration, two appeared to harbor secondary mutations in the mexA-mexR intergenic region and one harbored secondary mutations in the putative ribosome binding site located upstream of the mexAB oprM operon. In conclusion, this study shows that many mechanisms were involved in the multiresistance phenotype of this highly epidemic strain of P. aeruginosa. Our results also demonstrate that the clone sporadically underwent substantial genetic and phenotypic variations during the course of the outbreak, perhaps in relation to local or individual selective drug pressures.

Project description:UnlabelledPseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086 The information obtained in this study should be useful for further development of safe and efficient phage therapy.ImportancePseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed.

Dataset Information

Small Colony Variants and Single Nucleotide Variations in Pf1 Region of PB1 Phage-Resistant Pseudomonas aeruginosa.

Publications

Small Colony Variants and Single Nucleotide Variations in Pf1 Region of PB1 Phage-Resistant Pseudomonas aeruginosa.

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