A Genotypic Analysis of Five P. aeruginosa Strains after Biofilm Infection by Phages Targeting Different Cell Surface Receptors

Abstract

Antibiotic resistance constitutes one of the most serious threats to the global public health and urgently requires new and effective solutions. Bacteriophages are bacterial viruses increasingly recognized as being good alternatives to traditional antibiotic therapies. In this study, the efficacy of phages, targeting different cell receptors, against Pseudomonas aeruginosa PAO1 biofilm and planktonic cell cultures was evaluated over the course of 48 h. Although significant reductions in the number of viable cells were achieved for both cases, the high level of adaptability of the bacteria in response to the selective pressure caused by phage treatment resulted in the emergence of phage-resistant variants. To further investigate the genetic makeup of phage-resistant variants isolated from biofilm infection experiments, some of these bacteria were selected for phenotypic and genotypic characterization. Whole genome sequencing was performed on five phage-resistant variants and all of them carried mutations affecting the galU gene as well as one of pil genes. The sequencing analysis further revealed that three of the P. aeruginosa PAO1 variants carry large deletions (>200 kbp) in their genomes. Complementation of the galU mutants with wild-type galU in trans restored LPS expression on the bacterial cell surface of these bacterial strains and rendered the complemented strains to be sensitive to phages. This provides unequivocal evidence that inactivation of galU function was associated with resistance to the phages that uses LPS as primary receptors. Overall, this work demonstrates that P. aeruginosa biofilms can survive phage attack and develop phage-resistant variants exhibiting defective LPS production and loss of type IV pili that are well adapted to the biofilm mode of growth.

Phage therapy as an alternative or complementary strategy to prevent and control biofilm-related infections

“…In a study developed by Oechslin et al, the genomic profile of two P. aeruginosa phage-resistant strains was analysed and revealed mutations in genes encoding phage receptors, namely pilT and galU, when compared to the wild-type strain [36]. The same was reported by Pires et al in which the authors described that mutations affecting the galU gene and the pil genes were responsible for bacterial resistance to phages [13 ]. This fact can be a consequence of the endogenous oxidative stress suffered by biofilm cells that leads to DNA damage within biofilms resulting in the development of genetic variants with high adaptability to external conditions [37,38].…”

“…It is well known that phages are currently considered as promising antimicrobial agents for biofilm prevention and control [7,8]. Nonetheless, although several studies have reported significant reductions in mono and dual-species biofilms after phage treatment [2,3,[9][10][11][12], the complete eradication of biofilms is an almost impossible task [13 ]. Phages need to reach their host bacteria and attach to specific receptors located at cell surfaces before infection and replication inside their hosts is initiated.…”

Control ofSalmonellaEnteritidis on food contact surfaces with bacteriophage PVP-SE2

“…As expected, higher numbers of resistant colonies emerged at 22 C than at 4 C, with percentages varying between 0 and 33.3% in cells recovered from biofilms formed on polystyrene and 0-100% in cells from SS surfaces, respectively. Since biofilms formed on SS were thicker compared to those formed on polystyrene, cells will be subjected to more stress, and the existence of cellular metabolites and oxygen reactive species surrounding biofilm cells may induce mutations at a higher rate due to the diffusional limitations (Pires et al 2017). At 4 C, most of the surviving cells recovered from polystyrene and SS surfaces as well as from poultry skins (control and pretreatment experiments) were susceptible to all four phages.…”

Effective Pro-Inflammatory Induced Activity of GALT, a Conserved Antigen in A. Pleuropneumoniae, Improves the Cytokines Secretion of Macrophage via p38, ERK1/2 and JNK MAPKs Signal Pathway

“…The gal T gene is located in the gal gene cluster, which is involved in the metabolism of galactose. Genes of this cluster are widely distributed in various pathogenic agents and these genes have been demonstrated to be associated with the virulence factors such as formation of LPS, CPS, and biofilm formation (Priebe et al, 2004 ; Wong and Akerley, 2005 ; Ramjeet et al, 2008 ; Chai et al, 2012 ; Caboni et al, 2015 ; Meyer et al, 2015 ; Oechslin et al, 2017 ; Pires et al, 2017 ). However, the mechanism of GALT-mediated pathogenesis is still unknown.…”