Efficacy of phage vB_Ps_ZCPS13 in controlling Pan-drug-resistant Pseudomonas aeruginosa from urinary tract infections (UTIs) and eradicating biofilms from urinary catheters

Abstract

Background: Pan-drug resistance (PDR) is a ticking time bomb, as it causes high human hospitalizations and mortality rates. For instance, Pseudomonas aeruginosa is associated with significant rates of urinary tract infections (UTIs) due to several reasons including antibiotic resistance, biofilm formation and the presence of various virulence factors. Consequently, there is an urgent need for safe and effective alternative antibacterials. Phage therapy is a promising alternative that uses naturally occurring bacteriophages (phages). Therefore, our present study investigated the isolation and characterization of a novel virulent phage (vB_Ps_ZCPS13) against the PDR Pseudomonas aeruginosa strain (Ps13).

Methods: Phage vB_Ps_ZCPS13 was isolated from raw sewage water in Egypt during the springtime. The isolated phage was purified and amplified, followed by estimating its purity and genome size using pulsed-field gel electrophoresis (PFGE), morphology using transmission electron microscopy (TEM), antibacterial activity against other P. aeruginosa hosts, physiochemical stability studies, whole genome sequencing, antibiofilm activity on urinary catheters using scanning electron microscopy (SEM), and cytotoxicity assays against normal human skin fibroblast (HSF) cell lines.

Results: Based on vB_Ps_ZCPS13 morphology under TEM, the phage has been classified as a myovirus. In consistent with the PFGE results, DNA sequencing revealed a phage genome size of 92,443 bp, with lytic-associated genes and no antimicrobial resistance or virulence factors. Phage vB_Ps_ZCPS13 presented a wide host range of over 93% of tested clinical isolates having different multiple antibiotic resistance (MAR) indices. Furthermore, phage vB_Ps_ZCPS13 exhibited high efficiency in plaque formation (EOP ≥ 1) against 13% of the strains and exhibited low frequencies of bacteriophage insensitive mutants (BIM). The physical stability test against harsh environmental conditions revealed phage stability within a pH range of 3.0-11.0 and stable at temperatures below 70 °C. Phage vB_Ps_ZCPS13 also exposed a significant antibacterial activity in vitro across different MOIs, with the highest reduction in bacterial growth observed at lower MOIs. Furthermore, vB_Ps_ZCPS13 demonstrated potent biofilm inhibition and clearance capabilities, effectively eradicating P. aeruginosa from the urinary catheter surface. Moreover, the phage presented no cytotoxicity against normal human skin fibroblast (HSF) cell lines at high titer.

Conclusions: Our study offers an effective phage as a therapeutic candidate against PDR Gram-negative P. aeruginosa infections, including catheter-associated urinary tract infections.

Keywords: Myovirus; Pseudomonas aeruginosa; Bacteriophage; Biofilm; Catheter-associated urinary tract infections (CAUTIs); Catheters; Pan-drug resistance (PDR); Phage therapy; Urinary tract infections (UTIs).

Fig. 1

Antibiotic sensitivity profile for 20 UTI P. aeruginosa isolates over 10 antibiotics (sensitivity is indicated in green, intermediate in yellow, and resistance in pink) with their MAR index values

Fig. 2

Transmission electron micrograph of phage vB_Ps_ZCPS13, scale bar; 100.0 nm

Fig. 3

Heatmap of host range and EOP for different P. aeruginosa isolates tested against phage vB_Ps_ZCPS13. (a) The host range results represent light green for lysed bacteria and red for resistant bacteria. (b) The EOP values are represented as green (≥ 1), light red (≥ 0.001 and < 1), and dark red (< 0.001)

Fig. 4

Phage vB_Ps_ZCPS13 physicochemical stability at different temperatures (a), pH values (b), and under a UV lamp (c)

Fig. 5

The phage-bacterial dynamics of phage vB_Ps_ZCPS13 at 37 °C. (a) Phage infection with MOI 0.1, (b) Phage infection with MOI 1, and (c) Phage infection with MOI 10. CFU: Colony-forming unit; PFU: Plaque-forming unit

Fig. 6

Genomic circular map of phage vB_Ps_ZCPS13. Coding sequences (CDSs) are represented in different colors according to the predicted functional category. The GC content skew is represented in the middle circle. The map was constructed by Proksee [38]

Fig. 7

Predicted topology of ORF 32 (putative class II holins) by DeepTMHMM. The top part of the chart represents the topology of the predicted domain. The probability of the predicted topology was 100%

Fig. 8

Proteomic tree of phage vB_Ps_ZCPS13. (a) Circular proteomic tree based on genome-wide similarities of phage vB_Ps_ZCPS13 (highlighted with a red star), along with its closely related reference phage genomes. (b) Rectangular proteomic tree comparing phage vB_Ps_ZCPS13 with 40 phages that have the highest ViPTree S_G scores

Fig. 9

Phylogenetic analysis of phage vB_Ps_ZCPS13. (a) VIRIDIC heatmap for the intergenomic similarity between phage vB_Ps_ZCPS13 and closely related phages from ViPTree. (b) VIRIDIC heatmap for the intergenomic similarity between phage vB_Ps_ZCPS13 and all Pakpunavirus listed in the ICTV database.The alignment capacity is determined on the basis of three factors: intergenomic similarities (indicated by hues from green to blue), the percentage of aligned genome sequence in a pair (represented by shades of pink), and their length ratio (depicted by shades of black)

Fig. 10

Phylogenetic tree based on a conserved viral signature protein (i.e., terminase large subunit) between phage vB_Ps_ZCPS13 and other phages. The tree was constructed by MEGA11

Fig. 11

Antibiofilm activity of phage vB_Ps_ZCPS13. (a) Biofilm formation inhibition at different MOIs. (b) Disruption of pre-formed mature biofilm at different MOIs. Statistically significant difference marked by asterisks, where *** indicates P < 0.0001

Fig. 12

Representative SEM images of silicone urinary catheter surfaces treated with phage vB_Ps_ZCPS13. (a1 and b1) Untreated control for biofilm inhibition and biofilm clearance, respectively, (a2 and b2) phage-treated after 48 h for biofilm inhibition and biofilm clearance, respectively at magnification of 5000x. The relative number of CFU of P. aeruginosa biofilms formed in treated and untreated catheters for biofilm inhibition (c1) and biofilm clearance (c2). Statistically significant difference marked by asterisks, where *** indicates P < 0.0001

Fig. 13

Phage vB_Ps_ZCPS13 cytotoxicity against normal human skin fibroblast (HSF) cell lines, as assessed by the MTT assay. (a) normal HSF cell lines, (b) treated HSF cell lines, (c) percentage of cell viability for both control and treated HSF cell lines