Assessment of Phage-Mediated Inhibition and Removal of Multidrug-Resistant Pseudomonas aeruginosa Biofilm on Medical Implants

Figures & data

Table 1 Clinical Bacterial Isolates and Their Antimicrobial Resistance Patterns

Isolates	Source	Antibiotic Resistance
PA 1095	Wound abscess	AMP, AMC, CTX, CXM, TZP, TET
PA 1098	Sputum	AMP, AMC, AMK, CRO, CXM, CHL, SXT, CN, MEM
PA 1139	Wound abscess	AMP, AMC, AMK, CXM, CHL, SXT, CRO, CTX, MEM
PA 1280	Pleural fluid	AMP, AMC, CFZ, CXM, CHL, SXT, TET
PA 1321	Wound abscess	AMP, AMC, CFZ, CAZ, CTX, CXM, CHL, SXT,
PA 1329	Wound abscess	AMP, AMC, CAZ, CRO, CTX, CXM, CHL, SXT, CN, TET, TOB
PA 1668	Urine	AMP, AMK, CRO, CXM, CIP, CN, NIT, NOR

Abbreviations: PA, Pseudomonas aeruginosa, AMC, amoxicillin-clavulanic acid; AMK, amikacin; AMP, ampicillin; CAZ, ceftazidime; CFZ, cefazolin; CHL, chloramphenicol; CIP, ciprofloxacin; CN, gentamicin; CRO, ceftriaxone; CTX, cefotaxime; CXM, cefuroxime; MEM, meropenem; NIT, nitrofurantoin; NOR, norfloxacin; SXT, sulfamethoxazole-trimethoprim; TET, tetracycline; TOB, tobramycin; TZP, piperacillin-tazobactam.

Table 2 Lytic Activity of Phages ΦJHS and ΦSMK Against Tested Bacterial Strains

Bacterial Strain	Phage Sensitivity
	ΦJHS	ΦSMK
Pseudomonas aeruginosa 1095	+	+
Pseudomonas aeruginosa 1098	++	++
Pseudomonas aeruginosa 1139*	++	++
Pseudomonas aeruginosa 1280	++	+
Pseudomonas aeruginosa 1321	+	+
Pseudomonas aeruginosa 1329	++	++
Pseudomonas aeruginosa 1668	++	++
Pseudomonas aeruginosa ATCC 27853	++	+
Escherichia coli ATCC 25922	–	–
Salmonella typhimurium ATCC 13311	–	–
Proteus mirabilis ATCC 35659	–	–
Klebsiella pneumoniae ATCC 700603	–	–
Enterobacter cloacae ATCC 13047	–	–
Acinetobacter baumannii ATCC 19606	–	–
Shigella dysenteriae ATCC 13313	–	–
Staphylococcus aureus ATCC 25923	–	–
Staphylococcus saprophyticus ATCC 15305	–	–

Notes: (++), clear plaques or complete lysis; (+), turbid plaque or partial lysis; (–) no plaque or no lysis; (*), host.

Figure 1 Medical implants used for biofilm experiment. (A) Foley balloon catheter. (B) Endotracheal tube. (C) 15-mm long catheter tube cut in half. (D) 12-mm long endotracheal tube cut in half. (E) Catheter segment and (F) endotracheal tube segment placed in (G) sterile 24-well culture plate.

Figure 2 Plaques formed by bacteriophages (A) ΦJHS and (B) ΦSMK on the lawn of PA 1139 strain using the double agar overlay method. The bar corresponds to 1 mm.

Table 3 Stability of Phages ΦJHS and ΦSMK to External Physical and Chemical Factors

External Factors	Time and Conditions of Incubation	Percentage Viability of Phages ± SD
		ΦJHS	ΦSMK
Temperature
4°C*	1 h	100 ± 0.0	100 ± 0.0
15°C	1 h	100 ± 7.5	100 ± 4.4
RT	1 h	100 ± 6.5	100 ± 3.2
37°C	1 h	100 ± 6.1	100 ± 6.5
40°C	1 h	100 ± 3.1	100 ± 5.5
50°C	1 h	76.9 ± 0.6	55.4 ± 2.5
90°C	1 h	0.0 ± 0.0	0.0 ± 0.0
pH
pH 2.0	1 h; 37°C	0.0 ± 0.0	0.0 ± 0.0
pH 4.0	1 h; 37°C	38.0 ± 2.0	29.8 ± 0.6
pH 7.0*	1 h; 37°C	100.0 ± 0.0	100.0 ± 0.0
pH 10.0	1 h; 37°C	78.9 ± 3.1	68.1 ± 1.1
pH 12.0	1 h; 37°C	0.0 ± 0.0	0.0 ± 0.0
Organic solvent
SM buffer*	1.5 h; RT	100 ± 0.0	100 ± 0.0
Chloroform	1.5 h; RT	82.0 ± 2.5	70.8 ± 1.5
Acetone	1.5 h; RT	0.0 ± 0.0	0.0 ± 0.0
96% ethanol	1.5 h; RT	0.0 ± 0.0	0.0 ± 0.0
48% ethanol	1.5 h; RT	90.0 ± 4.9	57.0 ± 2.1

Note: (*), controls.

Figure 3 Biofilms formed by MDR PA clinical isolates cultured for 24 h at static non-renewal condition, as analyzed by crystal violet staining procedure and shown as O.D values measured at a wavelength of 630 nm. All assays were performed in triplicates. The values presented are mean ± SD from two readings of triplicate experiments (n = 6).

Figure 4 Lytic activity of bacteriophages on biofilms formed by MDR PA clinical isolates after 6 h treatment with phages ΦJHS-PA1139 and ΦSMK-PA1139, as analyzed by crystal violet staining procedure and shown as O.D values measured at a wavelength of 630 nm. The values presented are mean ± SD from two readings of triplicate experiments (n= 6). Statistically significant differences between control and analyzed samples are marked with asterisks (p < 0.001 (⁎⁎⁎) in the multiple unpaired t-tests).

Figure 5 Microbial load inhibition of MDR PA 1098 (A) and 1668 (B) biofilm formation on non-coated and phage-coated catheter and ET segments, quantified as log₁₀CFU/mL. The values presented are mean ± SD from two counts of triplicate experiments (n= 6). Statistically significant differences between control and analyzed samples are marked with asterisks (p < 0.01 (⁎⁎), p < 0.001 (⁎⁎⁎) in the multiple unpaired t-tests).

Figure 6 Microbial load reduction with 6 h phage treatment of MDR PA 1098 (A) and 1668 (B) biofilms formed on catheter and ET segments, quantified as log₁₀CFU/mL. The values presented are mean ± SD from two counts of triplicate experiments (n = 6). Statistically significant differences between control and analyzed samples are marked with asterisks (p < 0.05 (⁎), p < 0.01 (⁎⁎), p < 0.001 (⁎⁎⁎) in the multiple unpaired t-tests).