Development and Evaluation of Bacteriophage Cocktail to Eradicate Biofilms Formed by an Extensively Drug-Resistant (XDR) Pseudomonas aeruginosa

Abstract

Extensive and multiple drug resistance in P. aeruginosa combined with the formation of biofilms is responsible for its high persistence in nosocomial infections. A sequential method to devise a suitable phage cocktail with a broad host range and high lytic efficiency against a biofilm forming XDR P. aeruginosa strain is presented here. Out of a total thirteen phages isolated against P. aeruginosa, five were selected on the basis of their high lytic spectra assessed using spot assay and productivity by efficiency of plating assay. Phages, after selection, were tested individually and in combinations of two-, three-, four-, and five-phage cocktails using liquid infection model. Out of total 22 combinations tested, the cocktail comprising four phages viz. φPA170, φPA172, φPA177, and φPA180 significantly inhibited the bacterial growth in liquid infection model (p < 0.0001). The minimal inhibitory dose of each phage in a cocktail was effectively reduced to >10 times than the individual dose in the inhibition of XDR P. aeruginosa host. Field emission-scanning electron microscopy was used to visualize phage cocktail mediated eradication of 4-day-old multi-layers of XDR P. aeruginosa biofilms from urinary catheters and glass cover slips, and was confirmed by absence of any viable cells. Differential bacterial inhibition was observed with different phage combinations where multiple phages were found to enhance the cocktail's lytic range, but the addition of too many phages reduced the overall inhibition. This study elaborates an effective and sequential method for the preparation of a phage cocktail and evaluates its antimicrobial potential against biofilm forming XDR strains of P. aeruginosa.

Keywords: Pseudomonas aeruginosa; bacteriophage; biofilm; extensively drug resistant (XDR); phage cocktail.

Figure 1

Heat map depicting host lytic spectrum of P. aeruginosa phages against various Pseudomonas strains.

Figure 2

Transmission electron micrographs of (a) φPA170 and (b) φPA180. Both the phages belong to family Myoviridae, with a thick short tail, prominent base plate, tail pins, and tail fibres.

Figure 3

(a) Temperature sensitivity assay and (b) pH stability assay of phages φPA170, φPA172, φPA173, φPA177, and φPA180. Error bars depict standard deviation.

Figure 4

Inhibition of XDR P. aeruginosa VTCCBAA1047 growth by individual phages (a) φPA170, (b) φPA172, (c) φPA173, (d) φPA177, and (e) φPA180 in liquid infection models using time-kill assay over a time period of 10 h. Bacteriophages were used at MOIs ranging from 1 to 0.00001. Each point represents the mean of three replicates. The values of optical density used to plot graphs are blank (negative control) subtracted values. Each point represents the mean of three replicates. Error bars in the graph depict standard deviation from mean.

Figure 5

Lytic activity of bacteriophage cocktails against XDR P. aeruginosa VTCCBAA1047. A total twenty-two cocktails were designed with five phages in different combinations. Cocktail comprising (a) two phages, (b) three phages, (c) four phages, and (d) five phages were used to study inhibition of bacteria growth in liquid infection model using time-kill assay. The change in bacterial turbidity was measured at a 15 min interval successively for 10 h. All of the phages were used at a less-than-minimum inhibitory MOI. The values of optical density used to plot graphs are blank (negative control) subtracted values. Each point represents the mean of three replicates. Error bars in the graph depict standard deviation from mean.

Figure 6

Biofilm eradication ability of phage cocktail comprising φPA170, φPA172, φPA173, and φPA180. Panels (a–c) represent 4-day-old biofilms developed on the inner walls of urinary catheter. Multilayered protrusions of tightly packed biofilm cells (a), and gelatinous matrix of EPS surrounding the cells (c) are observed. Arrows in (b,c) indicate cracks in biofilm multilayer and cell to cell EPS junctions respectively. Panels (d–f) represent eradicated biofilms after treatment with bacteriophage cocktail for 12 h. Arrows in (f) indicate cell debris. Biofilms were visualized in a JSM-7610F Plus Scanning electron microscope, Jeol, Akishima, Japan, after glutaraldehyde fixation and ethanol gradient dehydration.

Figure 7

Biofilm eradication ability of phage cocktail comprising φPA170, φPA172, φPA173, and φPA180. Panels (a–c) represent 4-day-old biofilms developed on the borosilicate glass cover slips. Swivelled cords as indicated by arrows (a,b) and monolayers (c) of compactly adhered P. aeruginosa cells can be observed. Panels (d–f) represent eradicated biofilms and burst cell debris (indicated by arrows) after treatment with bacteriophage cocktail for 12 h. Biofilms were visualized in a JSM-7610F Plus Scanning electron microscope, Jeol, Akishima, Japan, after glutaraldehyde fixation and ethanol gradient dehydration.