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Original Article
ARTICLE IN PRESS
doi:
10.25259/JHS-2024-9-16-R1-(1508)

Phage Mediated Degradation of Pseudomonas aeruginosa Exopolysaccharides

, , ,
1Department of Infectious Diseases and Microbial Genomics, Nitte University Centre for Science Education and Research, NITTE (Deemed to be University), Mangaluru, Karnataka, India
2Department of Bio and Nano Technology, Nitte University Centre for Science Education and Research, NITTE (Deemed to be University), Mangaluru, Karnataka, India

* Corresponding author: Dr. Juliet Roshini Mohan Raj, Department of Infectious Diseases and Microbial Genomics, Nitte University Centre for Science Education and Research, NITTE (Deemed to be University), Mangaluru, Karnataka, India. julietm@nitte.edu.in

Received: , Accepted: ,
© 2025 Published by Scientific Scholar on behalf of Journal of Health and Allied Sciences NU
Licence
This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

How to cite this article: Prabell S, Suresh S, Premanath R, Mohan Raj JR. Phage Mediated Degradation of Pseudomonas aeruginosa Exopolysaccharides. J Health Allied Sci NU. doi: 10.25259/JHS-2024-9-16-R1-(1508)

Abstract

Objectives

Pseudomonas aeruginosa is a highly adaptive, nosocomial, and multi-drug-resistant opportunistic bacterium known for the production of extracellular polymeric substances (EPS). The presence of EPS hinders the penetration of antibiotics and other antimicrobial agents and thus poses a significant challenge in healthcare. The interaction between phages and the EPS of P. aeruginosa in terms of carbohydrate hydrolysis and structural changes were studied to assess the potential of phages to overcome the barrier of the EPS.

Material and Methods

Twenty-six P. aeruginosa isolates, including 18 multidrug-resistant clinical isolates and 8 drug-sensitive environmental isolates, were tested for sensitivity to a panel of 18 non-redundant phage lysates. EPS was extracted from each bacterial culture, and the total carbohydrate content was assayed before and after phage treatment using the phenol-sulfuric acid method. The effect of candidate phage activity on the structure of the EPS was further analysed using Fourier transform infrared spectroscopy (FTIR), comparing EPS that were treated with phages for different time intervals with untreated controls. The combinatorial effect of phages that degraded EPS but could not lyse the host cell, with cell wall and cell membrane targeting antibiotics, was determined.

Results

Of the 468 phage-host combinations tested, 380 (80%) exhibited host cell lysis, indicating phage sensitivity. However, only 30% of these phage-sensitive instances showed phage-mediated hydrolysis of the extracted EPS. However, it was interesting to note that phage-mediated EPS hydrolysis was observed in 40% of instances where the cultures were resistant to lysis due to phage infection. Phages that do not lyse the bacteria but degrade the EPS are effective in facilitating cell envelope-targeting antibiotics to lyse the bacterial pathogen.

Conclusion

This study highlights the effectiveness of bacteriophage in overcoming the hurdle of the exopolysaccharides in Pseudomonas aeruginosa in antimicrobial therapy.

Keywords

Bacteriophage
Combinatorial effect
Extracellular polymeric substance
Multidrug resistance
Pseudomonas aeruginosa

INTRODUCTION

Extracellular polymeric substances (EPS) are high-molecular-weight organic polymers secreted by microorganisms into their environment, playing a crucial role in biofilm formation and maintenance. Pseudomonas aeruginosa, a multidrug-resistant pathogen, is well-known for its ability to form biofilms, which significantly contributes to its tolerance and resistance to antibiotics and antimicrobial therapies. This organism is an opportunistic pathogen, frequently implicated in nosocomial infections, particularly in immunocompromised patients.[1] The EPS matrix is essential for the structural and functional integrity of biofilms, governing key physicochemical properties such as adhesion, mechanical stability, and protection from external threats.[2] The composition of the P. aeruginosa biofilm matrix can vary depending on the strain, biofilm age, and environmental conditions, including pH, oxygen levels, and nutrient availability.[3] Notably, the presence of the pslA gene in 90% of multidrug-resistant (MDR) P. aeruginosa isolates is strongly associated with enhanced biofilm formation and increased antibiotic resistance.[4]

Given the rising challenge of antibiotic resistance in P. aeruginosa, alternative therapeutic strategies are urgently required. Bacteriophage (phage) therapy has emerged as a promising approach. Phages, viruses that specifically infect bacteria, have been successfully used in various industries, including food production and wastewater treatment, to target and eradicate bacterial biofilms.[5-7] Phage therapy has demonstrated efficacy in lysing MDR P. aeruginosa strains and disrupting biofilms.[8] In this study, we investigated the depolymerisation of the EPS matrix in both P. aeruginosa PAO1 and drug-resistant strains by previously characterised lytic bacteriophages with known biofilm-degrading capabilities.

MATERIAL AND METHODS

Bacterial cultures

Pseudomonas aeruginosa PAO1 strain, along with multidrug-resistant clinical isolates from sputum (n=10) and wound (n=8) samples,[9] and drug-sensitive environmental isolates (n=8),[9] were obtained from glycerol stocks (10%) stored at -80°C at Nitte University Centre for Science Education and Research, Mangaluru, India. The cultures were revived in Asparagine broth (HiMedia, India) and subsequently maintained on Luria-Bertani (LB) agar for routine use. All bacterial cultures were incubated at 37°C.

Pseudomonas phages

A total of 18 Pseudomonas bacteriophages, previously designated Pa1 to Pa189, along with 4 newly characterised phages (Pa19 to Pa22), were propagated using P. aeruginosa PAO1 as the host strain. The bacteriophage lysates were concentrated by ultracentrifugation at 26,300 g for 4 hours (Optima XPN-100 Ultracentrifuge, Beckman Coulter), following filtration through a 0.22 μm membrane. The resulting phage pellet was suspended in SM buffer, and the phage titre was determined using the routine test dilution method. For phage susceptibility testing, P. aeruginosa PAO1 and clinical isolates were lawned on LB agar plates. Each phage (Pa1–Pa18) (5 µL) was spotted on the bacterial lawns, which were incubated at 37°C for 6 hours to observe for lysis.

Extraction of exopolysaccharide

EPS was extracted from P. aeruginosa cultures as per the method described by Chug et al.[10] Briefly, bacterial cultures were grown in 50 mL LB broth for 96 hours at 37°C. Cells were harvested by centrifugation at 10,621 g for 30 minutes (Eppendorf® Centrifuge 5804R). The EPS in the supernatant was precipitated using 95% ethanol in a 1:3 v/v ratio at 4°C overnight. The EPS was pelleted by centrifugation at 15,295 g for 20 minutes, resuspended in 30 mL Milli-Q water, and passed through a 0.45 µm membrane filter to remove residual cells. The extracted EPS was stored at 4°C till further analyses.

Colorimetric estimation of EPS depolymerization

The phenol-sulfuric acid method was used to quantify the carbohydrate concentration in the extracted EPS before and after phage treatment. Briefly, 500 µL of extracted EPS was mixed with an equal volume of bacteriophage lysate (∼5 *108 PFU/mL) and incubated for 1 hour and 18-hour time points. At each time point, 40 µL of 80% phenol prepared from 99.5% phenol (LOBAChemie,05168) and 2.5 mL of 98% extra pure sulfuric acid (LOBAChemie,00289) were added, and the mixture was incubated at room temperature for 20 minutes in the dark. The absorbance was measured at 490 nm using a photoelectric colorimeter (SYSTRONICS Type: 113). A standard curve was generated using a sodium alginate concentration gradient (HiMedia MB114) to extrapolate EPS concentrations.[11] Control reactions included EPS alone, phage alone, and SM buffer.

Statistical analysis: Analysis of variance (ANOVA) was employed to determine if time affected phage-mediated EPS degradation. A significance level of p ≤ 0.05 was considered statistically significant.

Fourier transform infrared spectroscopy of the EPS on phage treatment

The structural changes in EPS after phage treatment were analysed using FTIR spectroscopy. EPS samples treated with bacteriophages were treated with ZnCl2 (HiMedia MB046) in a 1:50 ratio to remove the phages[9,12]). The samples were centrifuged at 4300 g (Eppendorf Minispin®/Minispin Plus® centrifuge), and the supernatant was collected in a fresh 1.5 mL tube. The FTIR spectra were obtained using an ALPHA II FTIR spectrometer (Bruker Optics GmbH & Co) at the Nitte Gulabi Shetty Memorial Institute of Pharmaceutical Sciences, Mangaluru, India. Controls of phage suspensions and phage suspensions treated with ZnCl₂ in the absence of EPS were also analysed via FTIR to monitor for any noise /peaks due to either ZnCl2 or the phages.

Combinatorial effect of antibiotics and phages

The minimal inhibitory concentration (MIC) for the combinational therapy of phage with antibiotics was assessed in bacterial isolates that were resistant to lysis by the bacteriophages but whose EPS was degraded by the specific phage. The antibiotics utilised included Cefotaxime (Himedia MB134-1G, India), Colistin (Himedia PCT1142-1G, India), and Penicillin G (Himedia TC187-1MU, India). MIC was determined in a 96-well plate as per CLSI guidelines using antibiotic concentrations ranging from 64 mg/L to 0.125 mg/L with bacteriophages at a concentration of 109 PFU/mL. Appropriate controls, including antibiotic-only controls, media-only controls, phage only and culture controls, were included to ensure the reliability of the results. 96-well plates were incubated at 37°C for 18 hours. The absorbance at 620 nm was used to record culture turbidity. Wells with the least concentration of antibiotic showing > 90% reduction in absorbance compared to cell control was considered the MIC80.

RESULTS

Host range of the phages

The host range, reflecting the susceptibility or resistance of clinical P. aeruginosa isolates to the tested bacteriophages, has been summarised in Table 1. Notably, isolate ADS-34 was resistant to lysis by all phages tested, while isolates ADS-03 and ADS-13 exhibited resistance to more than 80% of the phages.

Table 1: Host range of phages on the isolates used
Isolates/phages
 Bacteriophages
Pa1 Pa3 Pa5 Pa6 Pa7 Pa9 Pa10 Pa11 Pa12 Pa13 Pa14 Pa15 Pa16 Pa17 Pa18 Pa19 Pa20 Pa22
Sputum isolates ADS 03 + - + - - + - - - - - - - - - - - -
ADS 04 + + + + + + + + + + + + + + + + + +
ADS 13 + + + - - - - - - - - - - - - - - +
ADS 14 + + + + + + + + + + + + + + + + + +
ADS 26 + + + + + + + + + + + + + + + + + +
ADS 28 + + + + + + + + + + + + + + + + + +
ADS 34 - - - - - - - - - - - - - - - - - -
ADS 46 + + + + + + + + + + + + + + + + + +
ADS 47 + + + + + + + + + + + + + + + + + +
ADS 49 + + + + + + + + + + + + + + + + + +
Environmental isolates AMO1 + + + + + + + + + + + + + + + + + +
FTOA1 + + + - + - - + + + - + + + - + + +
FLO1 + - + - - - - + - - + + + + + + + +
JMO2 + + + + + + + + + + + + + + + + + +
FO1 + + + + - + - + - + - + + + + + + +
FTOA + + + + - + - + - + - + + + + + + +
NOA - + + - - - - + - + - + + + + + + +
FO2 + + + - + - + + + + + - + + - + + +
Wound isolates ADWS-11 + + + + + + + + + + + + + + + + + -
ADWS-44 + + + + + + + + + + + + + + + + + +
ADWS-36 + + + + + + + + + + + + + + + + + +
ADWS-25 + + + + + + + + + + + + + + - + - -
ADWS-62 + + + + - + - - - + + + + + - + + -
ADW-24 + + + + + + + + + + + + + + + + + +
ADW-37 + + + + + + + + + + + + + + + + + +
ADW-22 + + + + + + + + + + + + + + + + + +

ADS: Asparagine diabetic sputum isolate; Pa: phage, +: Isolate shows lysis on exposure to the phage, -: Isolate resistant to phage infection.

EPS degradation by phages

Phages demonstrated the ability to lyse P. aeruginosa biofilms, reducing biofilm formation by >50% in P. aeruginosa PAO1 and other isolates, as previously confirmed by the crystal violet biofilm assay. 9 In this study, phages were able to degrade between 20% to 75% of the carbohydrate content in extracted EPS, as shown in Figure 1. A statistically significant reduction in EPS carbohydrate content was observed after phage treatment compared to controls (p1 hour = 2.28481E-18 and p18 hours =5.32412 E-16). However, the difference in EPS degradation between 1 and 18 hours of phage treatment was not statistically significant (= 0.035603327).

The average ability of the 18 bacteriophages to degrade the exopolysaccharides of Pseudomonas aeruginosa. EPS: Extracellular polymeric substances.
Figure 1:
The average ability of the 18 bacteriophages to degrade the exopolysaccharides of Pseudomonas aeruginosa. EPS: Extracellular polymeric substances.

The composition of EPS can vary depending on factors such as strain type and environmental conditions. While phages were able to lyse 93% of isolates from wound specimens, EPS degradation was only observed in 56% of these cases. In sputum-derived isolates, although lysis sensitivity was lower (74%), phage-mediated EPS degradation occurred in 50% of phage-resistant isolates. Environmental isolates were generally less susceptible to both phage infection and EPS degradation [Figure 2].

Sensitivity of isolates to phage-mediated EPS degradation. (a) Sputum isolates: 74% phage-host combinations (133/180) showed lysis, but of these, only 11% (19/180) showed EPS degradation. Of the 26% phage-host combinations (49/180) that did not show lysis, EPS degradation was observed in 23/180 combinations. (b) Environmental isolates: 78% phage-host combinations (113/ 144) showed lysis, but 29% (42 /144) showed EPS degradation. Of the 22% phage-host combinations (31/144) that resisted phage infections, 8 /144 showed EPS degradation. (c) Wound Isolates: 93% combinations (134/144) were positive for lysis, of which 53/144, i.e., (37%), showed EPS degradation. Four of ten combinations that did not show lysis were positive for EPS degradation. Phage -host combinations were calculated as the number of phages multiplied by the number of isolates. The bigger pie chart represents phage sensitivity, wherein the area in complete grey represents cell lysis due to phage activity, but the phages did not degrade the EPS of these isolates. The stripped area indicates isolates that were resistant to phage infection, as indicated by the absence of lysis. The dotted areas represent isolates that lyses on phage infection and whose EPS was degraded by the phages. The smaller pie chart represents only the proportion of isolates that were resistant to phage infection. In this small pie chart, the striped area represents resistance to phage infection and degradation of EPS. The dotted areas represent isolates whose EPS was degraded by the phages, even though cell lysis was not observed in the plaque assay.
Figure 2:
Sensitivity of isolates to phage-mediated EPS degradation. (a) Sputum isolates: 74% phage-host combinations (133/180) showed lysis, but of these, only 11% (19/180) showed EPS degradation. Of the 26% phage-host combinations (49/180) that did not show lysis, EPS degradation was observed in 23/180 combinations. (b) Environmental isolates: 78% phage-host combinations (113/ 144) showed lysis, but 29% (42 /144) showed EPS degradation. Of the 22% phage-host combinations (31/144) that resisted phage infections, 8 /144 showed EPS degradation. (c) Wound Isolates: 93% combinations (134/144) were positive for lysis, of which 53/144, i.e., (37%), showed EPS degradation. Four of ten combinations that did not show lysis were positive for EPS degradation. Phage -host combinations were calculated as the number of phages multiplied by the number of isolates. The bigger pie chart represents phage sensitivity, wherein the area in complete grey represents cell lysis due to phage activity, but the phages did not degrade the EPS of these isolates. The stripped area indicates isolates that were resistant to phage infection, as indicated by the absence of lysis. The dotted areas represent isolates that lyses on phage infection and whose EPS was degraded by the phages. The smaller pie chart represents only the proportion of isolates that were resistant to phage infection. In this small pie chart, the striped area represents resistance to phage infection and degradation of EPS. The dotted areas represent isolates whose EPS was degraded by the phages, even though cell lysis was not observed in the plaque assay.

Phage Pa1 degraded the EPS of 37.5% of environmental isolates and 62.5% of wound isolates after 1 hour but failed to degrade the EPS of sputum isolates. After 18 hours, Pa1 exhibited EPS degradation in 62.5% of environmental, 62.5% of wound, and 10% of sputum isolates. Phage Pa3 degraded the EPS of 37.5% of environmental, 50% of wound, and 50% of sputum isolates after 1 hour, with this increasing to 50%, 62.5%, and 80%, respectively, after 18 hours. However, phages Pa14 to Pa20 were less effective, degrading less than 40% of the EPS content. For clinical isolates from wounds and sputum, ∼50% EPS degradation occurred among those isolates resistant to phage lysis.

Fourier transform infrared spectroscopy (FTIR/IR spectroscopy)

FTIR spectroscopy confirmed the structural digestion of specific molecules within the EPS [Figure 3 and Supplementary Tables S1 to S4]. The untreated EPS of P. aeruginosa PAO1 [Figure 3a] exhibited characteristic absorption peaks at 1260.84 cm-1 (CO-C-CO stretching), 1747.38 cm-1 (C=O stretching), 2359.42 cm-1 (CO2), and 2850.87 cm-1 (C-H stretching). After treatment with phage Pa1, these peaks disappeared, indicating phage-induced structural alterations in the EPS.

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4
Fourier-transform infrared (FTIR) spectra of P. aeruginosa PAO1 EPS: (a) With phage Pa1 treatment at hour one and hour eighteen, specific peak disappearance at 2850.87 cm-1, 2359.42 cm-1, 1747.38 cm-1, and 1260.84 cm-1 were observed. (b) With phage Pa10 treatment, peak disappearance at 1167.08 cm-1 and the appearance of new peaks in the range 3674 to 3566 cm-1 were observed. (c) FTIR spectra of P. aeruginosa clinical isolate ADS-28 EPS with phage Pa1 treatment at hours one and eighteen showed peak disappearance at 2357.50 cm-1. (d) FTIR spectra of P. aeruginosa clinical isolate ADS-46 EPS with and without phage treatment showed no significant difference as phage Pa1 was incapable of degrading the exopolysaccharide of this particular strain. EPS: Extracellular polymeric substances, PAO1: Pseudomonas aeruginosa strain.
Figure 3:
Fourier-transform infrared (FTIR) spectra of P. aeruginosa PAO1 EPS: (a) With phage Pa1 treatment at hour one and hour eighteen, specific peak disappearance at 2850.87 cm-1, 2359.42 cm-1, 1747.38 cm-1, and 1260.84 cm-1 were observed. (b) With phage Pa10 treatment, peak disappearance at 1167.08 cm-1 and the appearance of new peaks in the range 3674 to 3566 cm-1 were observed. (c) FTIR spectra of P. aeruginosa clinical isolate ADS-28 EPS with phage Pa1 treatment at hours one and eighteen showed peak disappearance at 2357.50 cm-1. (d) FTIR spectra of P. aeruginosa clinical isolate ADS-46 EPS with and without phage treatment showed no significant difference as phage Pa1 was incapable of degrading the exopolysaccharide of this particular strain. EPS: Extracellular polymeric substances, PAO1: Pseudomonas aeruginosa strain.

Phage Pa10 treatment at 1 hour resulted in the disappearance of the 1167.08 cm-1 (C-O stretching) peak, further indicating phage-mediated structural changes. Additionally, new peaks at 3673.88 cm-1, 3648.71 cm-1, 3629.21 cm-1, 3587.33 cm-1, and 3566.70 cm-1 emerged, indicating the presence of free alcohols, which suggests EPS digestion [Figure 3b].

FTIR spectra of the clinical isolate ADS-28 showed the disappearance of the 2357.50 cm-1 (O=C=O) peak and the appearance of a 3735.39 cm-1 peak, suggesting a distinct alteration in the EPS molecular structure due to phage treatment [Figure 3c]. In contrast, the EPS of isolate ADS-46, which did not exhibit any significant carbohydrate reduction after phage treatment, showed no significant changes in the FTIR spectra [Figure 3d], consistent with the phenol-sulfuric acid estimation results.

Combinatorial effect of antibiotics and phages

The MIC80 ​values for antibiotics alone and in combination with bacteriophages were determined for bacterial isolates resistant to bacteriophage-mediated lysis but whose EPS was effectively degraded by the phage. Of 12 such combinations tested, only 4 phage-antibiotic combinations showed a synergistic effect [Table 2]. MIC80 ​ for isolate FLO1 decreased from 64 mg/L to 32 mg/L for Penicillin in the presence of phage Pa10, and for ADWS-62, a significant reduction was observed with the MIC80 ​decreasing from 64 mg/L to < 0.125 mg/L in the presence of phage Pa10. The isolate ADS-34, which was MDR and showed complete resistance to phage lysis, displayed a reduction of MIC for colistin from 8 mg/L to 4 mg/L with a combinatorial effect with phage Pa14. ADWS-11 showed a significant reduction in MIC80 ​from 64 mg/L with cefotaxime alone to 2 mg/L when cefotaxime was used in combination with phage Pa22.

Table 2: Minimal inhibitory concentration (mic) in combination with phages
Antibiotic Isolate Bacteriophage degrading EPS but not capable of host lysis MIC80 for antibiotics only (mg/L) MIC80 breakpoint for antibiotics with bacteriophage (combinational therapy) (mg/L)
Penicillin FLO1 Pa10 > 64 32
ADWS-62 Pa10 > 64 < 0.125
Colistin ADS-03 Pa14 2 8
ADS-13 Pa14 2 4
ADWS-11 Pa22 8 16
ADS-34 Pa14 8 4
FLO1 Pa10 4 4
Cefotaxime ADS-03 Pa14 4 4
ADS-13 Pa14 4 8
ADS-34 Pa14 2 4
FLO1 Pa10 1 8
ADWS-11 Pa22 > 64 2
Penicillin PAO1 Pa22 < 0.125 < 0.125
Colistin Pa12 < 0.125 < 0.125
Cefotaxime Pa15 0.25 0.25

FLO1, ADWS, ADS, PAO1: Pseudomonas aeruginosa strain.

DISCUSSION

Extracellular polymeric compounds, including polysaccharides, proteins, lipids, and nucleic acids, are produced by bacteria in biofilms and contribute to their pathogenicity and resistance.[13-15] P. aeruginosa is known to colonise the lungs of cystic fibrosis patients, diabetic wounds, and urinary catheters, forming biofilms that hinder antimicrobial diffusion and lead to chronic infections.[16,17]

Although phages can lyse bacterial cells, the extracellular matrix can protect the host in response to antimicrobial agents, especially antibiotics.[18,19] For instance, P. aeruginosa shows increased biofilm formation in response to sub-inhibitory concentrations of aminoglycoside antibiotics like tobramycin.[20,21] Additionally, the alginate matrix in the biofilm protects P. aeruginosa from leukocyte destruction mediated by IFN-γ.[22] Several studies confirm that microbial EPS hinders sulfamethizole and sulfonamide antibiotics diffusion, hence alleviating the inhibitory effect.[23] Further, the EPS reduces the negative effects of certain antibiotics like fluoroquinolones and β-lactams that generate reactive oxygen species.[24] Phage cocktails have been shown to eradicate infections in in vivo studies.[25,26] Alemayehu et al.,[27] using a phage cocktail, demonstrated complete elimination of P. aeruginosa biofilm in a murine model. While some strains can resist phage lysis due to the presence of EPS, phage-mediated degradation of EPS can weaken the biofilm matrix, making the host more susceptible to antimicrobial agents.[28,29] Thus, for successful therapeutic application, phages must navigate the EPS to find suitable adhesion sites, which is a challenge due to their lack of intrinsic motility.[30] Combinational therapy remains an ongoing area of investigation.[31] This study evaluated the ability of bacteriophages to degrade the EPS produced in P. aeruginosa recovered from clinical and environmental isolates. FTIR spectroscopy confirmed bond breakages, indicating the digestion of exopolysaccharide molecules by the bacteriophages.

Bacteriophages were able to degrade EPS of the bacterial strain ADS-34, which was resistant to all tested antibiotics,[9] and phage-resistant isolates ADS-03 and ADS-13 (resistant to 80% of the phages). While the sensitivity of sputum isolates to phage lysis was 93%, the EPS degradation was observed in only 56% of the isolates. A substantial reduction in EPS content following phage treatment suggests that phage therapy could effectively diminish the biofilm matrix. However, the lack of a significant difference in the degradation between 1 and 18 hours of treatment implies that the initial phase of phage interaction is critical for EPS degradation. The following criteria must be accomplished for a phage to successfully degrade the biofilm and lyse the bacteria: Host susceptibility to the phage and the ability of the phage polysaccharide depolymerase to degrade the EPS. A substantial degree of biofilm degradation can occur even if only one of these criteria is met, and phage-free depolymerase enzyme activity alone results in rapid biofilm degradation.[32]

Phage Pa1 and Pa3 displayed different efficiencies in degrading EPS from various sources over time. Phage Pa1 showed a marked increase in degradation efficacy from 1 hour to 18 hours for environmental and wound isolates but was notably less effective against EPS from sputum isolates. Phage Pa3, on the other hand, demonstrated a consistent and increasing degradation capacity across all isolate types over time. This indicates that while some phages may act quickly, others may require longer exposure to achieve significant EPS degradation, and their effectiveness can vary based on the EPS composition of bacterial isolate and the EPS degradation mechanism that the phage possesses[33] or the amount of exopolysaccharide in the biofilm[34] or that the phages response to biofilm is strain-dependent.[35] Phage-derived depolymerases show significant anti-biofilm and bactericidal effects.[29] Studying phage enzymatic properties is crucial, as novel phage proteins could provide targeted strategies against biofilm degradation.[36]

FT-IR spectroscopy has been used to study structural attributes like macromolecular conformation and molecular interaction.[37,38,39] In this study, FTIR was used to study any structural alteration of the EPS upon phage treatment. The disappearance of characteristic bonds in the EPS of PAO1 after treatment with phage Pa1 and the appearance of peaks in Pa10-treated EPS indicates effective enzymatic activity and structural degradation. Distinct spectral changes observed in clinical isolate ADS-28 suggest that phage treatment can significantly alter EPS composition, supporting the potential for phage therapy to disrupt biofilms. Conversely, the lack of significant spectral changes in ADS-46 after phage treatment, though the host was susceptible to all phages tested, indicated that some EPS compositions resist phage-mediated degradation.

Biofilms present a major obstacle in treating infections as they impede antibiotic penetration and provide a protective barrier for bacteria. The EPS, which represents the “house of the biofilm cells,” is thus a primary barrier that needs to be overcome for effective antimicrobial action.[40] Antibiotic combinatorial therapies and non-antibiotic treatments are being actively researched as promising strategies to address the growing threat of antibiotic-resistant infections. Phage-antibiotic combinations in Staphylococcus aureus indicate an enhanced susceptibility of biofilms to antibiotics after subsequent phage treatment.[41,42] Burkholderia species pretreated with sublethal concentrations of antibiotics show higher susceptibility to phage.[43] A combination of phages with antibiotics targeting bacterial protein synthesis reduces the lytic activity of bacteriophages and thus does not function with synergy.[44] Hence, in this study, antibiotics targeting the cell envelop were tested for phage-antibiotic synergy. However, combinatorial treatment can show mixed outcomes, including synergy, no effect, and antagonism were observed in concordance with other reports.[45,46] The drastic decrease in the MIC80 of penicillin toward ADWS-62 and that of cefotaxime to ADWS-11 when used in combination with phages that could not lyse these isolates is a promising beacon in the application of phages in therapy. These findings emphasize the need for tailored phage-antibiotic combinations to overcome biofilm-associated resistance and improve therapeutic outcomes against MDR P. aeruginosa infections.

CONCLUSION

Certain bacteriophages demonstrated significant EPS degradation, effectively weakening biofilm structures and enhancing bacterial susceptibility to antimicrobial agents. FTIR analysis provided structural evidence of EPS digestion by specific phages, corroborating the colorimetric results and confirming phage-induced alterations in EPS composition.

The ability of phages to degrade EPS can play a more critical role in their therapeutic potential when used in combination with antibiotics compared to their lytic ability alone. A deeper understanding of the composition differences in the EPS and mechanisms underlying phage-mediated EPS degradation and bacterial lysis could facilitate the development of novel antimicrobial strategies. Further research in this area could enable the use of bacteriophages in targeted, combinatorial, and personalised therapies against biofilm-forming and antibiotic-resistant bacteria.

Ethical approval

The research/study approved by the Central Ethics Committee Review Board at NITTE (Deemed to be University), number NU/CEC/2021/159, dated 7th September 2021.

Declaration of patient consent

Patient’s consent not required as patients identity is not disclosed or compromised.

Financial support and sponsorship

N/RG/NUFR1/NUCSER/2020/02 funded by Nitte (Deemed to be University).

Conflicts of interest

There are no conflicts of interest.

Use of artificial intelligence (AI)-assisted technology for manuscript preparation

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

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