Development of a Phage Cocktail to Control Proteus mirabilis Catheter-associated Urinary Tract Infections

Abstract

Proteus mirabilis is an enterobacterium that causes catheter-associated urinary tract infections (CAUTIs) due to its ability to colonize and form crystalline biofilms on the catheters surface. CAUTIs are very difficult to treat, since biofilm structures are highly tolerant to antibiotics. Phages have been used widely to control a diversity of bacterial species, however, a limited number of phages for P. mirabilis have been isolated and studied. Here we report the isolation of two novel virulent phages, the podovirus vB_PmiP_5460 and the myovirus vB_PmiM_5461, which are able to target, respectively, 16 of the 26 and all the Proteus strains tested in this study. Both phages have been characterized thoroughly and sequencing data revealed no traces of genes associated with lysogeny. To further evaluate the phages' ability to prevent catheter's colonization by Proteus, the phages adherence to silicone surfaces was assessed. Further tests in phage-coated catheters using a dynamic biofilm model simulating CAUTIs, have shown a significant reduction of P. mirabilis biofilm formation up to 168 h of catheterization. These results highlight the potential usefulness of the two isolated phages for the prevention of surface colonization by this bacterium.

Keywords: Proteus mirabilis; bacteriophage therapy; bacteriophages; biofilms; phage cocktail; urinary tract infection.

FIGURE 1

Transmission electron micrographs of Proteus mirabilis phages: (A) Pm5460; (B) Pm5461. Scale bars represent 100 nm.

FIGURE 2

Screening for biofilm formation of fifteen P. mirabilis isolates on 96-well microtiter plates using crystal violet staining. Data points represent an average of three independent experiments performed in triplicate. Error bars indicate standard deviation.

FIGURE 3

Number of phages coating silicone surfaces per surface area. Data points represent an average of three independent experiments performed in triplicate and error bars indicate standard deviation. Statistical differences (p < 0.05) between the phage initial concentration and adsorbed phages were determined by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni post hoc test.

FIGURE 4

Number of phages released from silicone surfaces per surface area. Data points represent an average of three independent experiments performed in triplicate and error bars indicate standard deviation. The approximate concentration of adhered 5460 phage is presented in the graphic as a black dot intermittent gray line, while approximate concentration of adhered 5461 phage is presented as an intermittent black line. Statistical differences (p < 0.05) between the approximate initial phage titer with released phage were determined by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni post hoc test.

FIGURE 5

Phage cocktail effect on P. mirabilis biofilms under dynamic conditions. Representative SEM and epifluorescence images (A) and relative number of CFU (B) of P. mirabilis biofilms formed in normal and phage-coated Foley catheter show a decrease on the biofilm population, as well as on the overall biomass. SEM images are not representative of the overall biofilm as they are focused on spaces where biofilm is present. Relative CFU values were normalized using the higher concentration of biofilm cells obtained in each independent experiment. Magnification of 100x and 1000x, were used for epifluorescence and SEM images, respectively. Data points represent an average of four independent experiments and error bars indicate standard deviation. Statistical differences (p < 0.05) between control biofilms and phage-cocktail- -treated biofilms (^∗) were determined by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni post hoc test.