Isolation and Characterization of Three Lytic Bacteriophages to Overcome Multidrug-, Extensive Drug-, and Pandrug-Resistant Pseudomonas aeruginosa

Abstract

Background:

The worrisome spread of multidrug-resistant (MDR) pathogens necessitates research on nonantibiotic therapeutics. Among these therapeutics, phage treatment uses bacteriophages (phages) as alternative antimicrobial agents.

Objectives:

This project evaluates the lytic efficiency of phage cocktails in vitro versus MDR, extensive drug-resistant (XDR), and pandrug-resistant (PDR) P. aeruginosa isolates.

Methods:

We utilized host range and genetic information to generate a three-phage cocktail capable of killing multiple clinical strains of P. aeruginosa and examined the effectiveness of the cocktail in this study. The isolates (114) had variable resistance to 13 antibiotics. A phage-enrichment approach was used to purify the bacteriophage cocktail; a phage lysate with a high titer (5 × 10⁹ PFU/mL) was prepared and tested against 114 P. aeruginosa isolates.

Findings:

The results showed that a cocktail of three phages (MMS1, MMS2, and MMS3) could lyse P. aeruginosa in both planktonic liquid and dish cultures. The MMS cocktail phages were shown to be viable between 4 and 50°C at pH 4–9. A one-step growth curve showed that the MMS phages had a latent period of 15 min and a burst period of approximately 18 min based on the size of approximately 265 offspring phages per host cell. The MMS3 phage was sequenced and shown to lack genes associated with bacterial pathogenicity or antibiotic resistance.

Conclusions:

Notably, XDR and PDR isolates were sensitive to the phage cocktail, a prospective substitute for antibiotics that does not contribute to the growth of antibiotic resistance, suggesting that the phage cocktail might be useful for generating personalized phage therapeutics.

Highlights

• In this study, we assembled a 3-phage cocktail considering the host range and genomic information for phages and assessed its in vitro efficacy.

• MMS3 is the first sequenced dsDNA phage in our country and serves as an excellent model for studying the biology of dsDNA phages.

• The cocktail lysed most multidrug-resistant (MDR) P. aeruginosa isolates and extensive drug-resistant (XDR) isolates, and one pandrug-resistant (PDR) isolate in our collection, implying that phage infection was independent of cells harboring an antibiotic resistance mechanism.

Introduction

These Gram-negative bacterial strains are naturally resistant to several drugs and have developed resistance to other drugs.¹ Previously, antibiotics, including fluoroquinolones, beta-lactam/beta-lactamase inhibitor mixtures, carbapenems, and/or aminoglycosides, among others, were used as the first line of defense against resistant P. aeruginosa strains that cause disease. However, the reputation of these antibiotics as trusted drugs has been damaged by the rise of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) pathogens.² The World Health Organization recently warned that the antimicrobial pathway for Gram-negative infections is not a positive outlook and ineffective, escalating this dilemma.³ The absence of meaningful techniques for treating P. aeruginosa strains and the poor promise of the combination of new drugs have produced a tremendous need to develop innovative antimicrobial therapeutics, such as nonantibiotic medications with new mechanisms of action.⁴ Bacteriophages (phages) are viruses capable of infecting and reproducing within bacteria, leading to host cell death in the case of lytic phages. Phages have been used for more than 100 years to treat bacterial diseases; however, phage therapy has mostly exceeded antibiotics, in part, because of medicine administration, restricted phage potency, and phage therapeutic unfamiliarity.⁴ Phages have recently regained attention as a therapeutic alternative in the antimicrobial resistance phase because they can escape classical antibiotic resistance processes to avoid damage to normal microbiota because of their specificity and biofilm decomposition mechanisms.⁵ Today, phage therapy occurs in Eastern and Western European regions, with the Eliava Institute in Tbilisi, Georgia, a research center dedicated to therapeutic phages.^6,7 In various case reports and in small clinical and preclinical trials, the efficacy of P. aeruginosa phage treatment has been assessed.^8–14 For the previous decade, the Centers for Disease Control and Prevention classified MDR P. aeruginosa as a severe danger, with approximately 32,600 incidences and 2700 related fatalities in 2017.^15,16

Thus, the aim of this research was to define the antibiotic and single-phage resistance of P. aeruginosa, perform phage retraining with the use of phages capable of breaking these resistant phenotypes, and develop a phage cocktail capable of limiting the growth of phage-resistant P. aeruginosa during treatment.

Materials and Methods

Bacterial isolates and media for growth

Clinical isolates (114) of P. aeruginosa were isolated from 365 clinical samples between December 2019 and April 2021 from Al-Ramadi Teaching Hospital, Anbar, Iraq. The bacteria were preserved in 60% glycerol at −20°C and maintained on 4°C slants of nutrient agar when necessary, with Vitek 2 and all the isolates identified as 114 P. aeruginosa clinical isolates (such as 59 burn isolates, 21 ear isolates, 14 Urinary Tract Infection (UTI) isolates, 11 sputum isolates, 6 wound isolates, 1 vaginal isolate, 1 blood isolate, and 1 Cerebrospinal Fluid (CSF) isolate).

Antibiotic sensitivity

Thirteen antibiotics were evaluated by the disc diffusion method to determine the antibiotic susceptibility of P. aeruginosa strains (17). The inhibition zone was measured (in millimeters) and the findings were interpreted according to Clinical and Laboratory Standards Institute documents.

Phage isolation

Twenty-three specimens from an individual with severe UTI and 33 hospital sewage samples before treatment and from different regions were separately centrifuged at 6000 rpm to exclude solids, and the supernatant was then filtered through sterile nitrocellulose filters with pore sizes of 0.45 m and 0.22 m (EMD Millipore Corporation (supplier Company). In 3 mL of melting Luria-Bertani (LB) soft agar, 50 μL of filtrate and 100 μL of P. aeruginosa in the early exponential phase were combined and incubated overnight at 37°C.

Isolation, separation, and purification of lytic phages

To isolate phages from natural specimens, including wastewater, in a 14-mL sterile tube, 1 mL of 5× concentrating LB broth (Becton Dickinson), 1 mL of a solution of the ‘host bacterium’ at 10⁸ CFU in LB broth, and 9 mL of wastewater were combined. The culture was incubated for 15–24 h at 37°C. The lysate was extracted using a sterile 5 mL syringe and filtered through 0.45- and 0.22-μm membrane filters (Minisart, Sartorius, Vilvoorde, Belgium). To titrate the phages, the agar overlay method was utilized. All plaques, regardless of their appearance, were inoculated in 2 mL of sterile LB broth in 14 mL sterile tubes and incubated for 2 h at 37°C. Following the addition of 50 μL of chloroform, the tube(s) were incubated at 4°C for 1 h.

Phage purification and quantification

As mentioned previously, phage purification was accomplished using a single sequential plaque propagation approach.¹⁷ By utilizing a sterile blade, a single plaque was removed from a plate and placed in a mid-log-phase P. aeruginosa culture (10⁸ CFU/mL) supplemented with 0.1M CaCl₂. At 37°C overnight, 10 μL of culture combination and phage combination were incubated. The lysate was filtered through a sterile filter with a pore size of 0.45 μm. Serial dilutions of the phage-containing filtrates were generated for quantification and titration, and plaques were allowed to form on a lawn of the same host bacterial culture. Three subsequent rounds of plating were used to purify single plaques, which were subsequently performed three additional times to obtain purified phages. All lysates were stored at 4°C.

Determination of phage–bacteria interactions

A. Plaque assay using the double-layer agar (DLA) technique: Bacterial cultures of 100 μL of the early exponential phase and 50 μL of the lysates were combined into 3 mL of the melting soft agar tube LB with CaCl₂ and MgSO₄ (0.1 M final concentration). Subsequently, the mixture was poured over the LB agar platform and incubated overnight at 37°C. The negative control was LB agar without lysates.^18,19

B. Spot assay using the DLA technique: A total of 100 μL of bacterial culture from the early exponential phase was mixed with 3 mL of LB soft agar, which was subsequently placed on an LB agar plate. Following solidification, 10 μL of phage lysate was added to the bacterial lawn and incubated overnight at 37°C.^20,21

Optimization of the phage lytic characteristics

The strains from the transient stocks of wild lytic phages were cultivated using the plate technique in conjunction with the relevant host bacterial strains and the representative.²² Plaque morphology and growth properties were reported based on the following criteria: (1) time required for plaque to be visible, (2) plaque clarity or turbidity, (3) plaque diameter, (4) plaque shape, (5) plaque depth, and (6) plaque edge. The best plaques according to the above parameters were selected.

Phage cocktail preparation

According to the host range spectrum, three phages were selected from high-titer phage lysate ∼10¹⁰ plaque-forming units (PFU)/mL; the selected phages were amplified to produce 100 mL of each phage at ∼10⁹ PFU/mL in phosphate-buffered saline (PBS). The phage cocktail was prepared by mixing equal aliquots of ∼10⁹ PFU/mL of each phage type.²³

Characterization of phages

Characterization was performed for the three phages individually and then for the entire cocktail.

Phage adsorption assays

A variety of bacteria taken from clinical specimens were cultivated overnight in LB at 37°C. Approximately 10⁷ PFU/mL isolated and filtered phage was combined with 500 μL of bacterial samples containing approximately 10⁸ CFU/mL. The suspension was incubated at 37°C, and the medium was centrifuged at 8000×g for 3 min after 0, 2, 4, 6, 8, 10, and 12 min of incubation. The DLA technique was used to measure the phage titer remaining in the supernatant. The isolated bacteria grown in LB media were used entirely as nonabsorbing controls.

Specificity

MMS and its component phages were tested in vitro versus clinical isolates of bacteria other than P. aeruginosa (Staphylococcus epidermidis, Staphylococcus aureus, Burkholderia cepacia, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae) using DLA and spot tests.

Effect of calcium and magnesium ions on the phage adsorption rate

Three flasks, each comprising 25 mL of a selected bacterial culture, were used. In the first flask, the phage suspension (250 μL ≈3.5 × 10⁸ PFU/mL) was introduced alone, whereas in the other flasks, CaCl₂ or MgSO₄ (250 μL, concentration of 10 mM) was administered to the phage filtrate (250 μL). After the desired incubation time, specimens were taken at various intervals (0, 10, 20, and 30 min). The titer of unattached phage was determined in control and calcium- or magnesium-enriched phage suspensions/specimens. We assessed the variety of existing free phages in each flask utilizing the DLA “soft agar overlay” technique. The number of free phages was determined using the equation N/N0 × 100, where N0 represents the PFU per mL at time zero and N represents the PFU per mL at 10, 20, and 30 min.²⁴

pH and thermal stability of phages

The heat tolerance of the phages was determined utilizing a conventional approach.²⁵ For 24 h, phage suspensions were incubated at various temperatures (30, 40, 50, 60, 70, and 80°C) in several microcentrifuge tubes (1.5 mL). The concentration (PFU/mL) was determined using the double agar overlay approach at 6-h periods. According to previous protocols,^26,27 the stability of the phages at different pH values (i.e., 3, 4, 5, 6, 7, 8, 9, 10, and 11) was measured. One mLof phage filtrate was added to 9 mL of SM buffer (at the prescribed pH) and incubated for 15 h at the stated temperature. Each phage suspension specimen was grown for 15 h and then compared to the host bacteria using a plaque assay.

One-step growth curve

To determine the phage’s latent period and burst size, a one-step growth experiment was performed utilizing the technique reported previously by Jiang et al.²⁸ The phage was introduced to a host bacterium in the mid-exponential phase and kept at 37°C for 15 min to allow the adsorption. Subsequently, the mixture was centrifuged (13,000 rpm for 30 s) to remove the residual phage and then incubated at 37°C with agitation. During a 40-minute incubation, samples were obtained every three minutes, and the phage titration was also calculated using the double-layer agar technique.

Purification of phage lysates by centrifugation through a glycerol gradient

A protocol was used to yield pure phage lysate with no artifact debris and appeared to be suitable for subsequent electron microscopy and molecular analysis.²⁹

Determination of host range

The host range of pure phages was determined using a spot assay.³⁰ Various bacteria obtained from clinical samples were cultured in LB broth until they reached the early log phase (OD = 0.4). Subsequently, 300 μL of bacteria were combined with 0.5% LB agar and injected onto 1% LB agar underneath to solidify. Next, 10 μL of purified phage at various concentrations (10⁷, 10⁵, 10³, and 10¹ PFU/mL) was placed on LB agar. The plates were incubated overnight at 37°C. The lysis zones and plaque generation were evaluated.

Transmission electron microscopy

Transmission electron microscopy was used to analyze bacteriophage particles with and without target bacteria.³¹ The suspensions were transferred to pioloform-coated grids (Agar Scientific, Stansted, UK), rinsed with water, and stained negatively with 2% uranyl acetate (Agar Scientific) in water before being examined using a Tecnai Spirit transmission electron microscope at 100 kV. Micrographs were taken with a digital camera positioned on the bottom (Eagle, 4X4K, FEI). The ImageJ program was used for analysis.³² The scale bar (nm) for each image was established, and then the head dimensions (width and length) and tail dimensions (width and length) were obtained. Each phage was measured independently five times, and the mean value was recorded.

Bacteriophage genomic DNA isolation

Phage genomic DNA was extracted according to the manufacturer’s instructions (Sacace Biotechnologies). Numerous phases of buffer treatment and centrifugation were carried out according to the manufacturer’s phage DNA separation guidelines. Subsequently, 0.5 μL of DNA was placed onto a 1% agarose gel and run at 160 V for 30 min. Under an ultraviolet transilluminator, nucleic acid bands were observed.

Genome sequencing

Genomic DNA was submitted to the core facility for next-generation sequencing and sequenced using Illumina technology (Illumina, Inc.) at Macrogen, Inc., Seoul Capital Area, Republic of Korea. The sequencing library was generated according to the standard Illumina DNA shotgun library preparation technique using the TruSeq DNA Sample Prep kit. Ultrasonication was used to fragment the DNA, followed by adaptor ligation and PCR amplification. The sequencing by synthesis (SBS) method was used to yield paired-end reads of 410 bp. FastQC v0.11.5 was used to examine the sequence files before and after trimming. Trimmomatic v0.36 was used to trim reads from the NGS data (including adapter removal) to eliminate sequences with a per-base sequence quality score of less than 30. Following selection, sequences with a total of 139,516 pairs of reads and an average coverage of >100× were retained (approx. 72 kb). The raw sequence data were de novo assembled using SPAdes 3.13.0. SPAdes automatically selected the appropriate k-mer length based on the read length and then improved mismatches and short indels in the resulting contigs and scaffolds. The genome was sequenced and submitted as a raw sequence in the NCBI Sequence Read Archive (SRA) database. BLAST analysis of the whole-genome sequences against GenBank sequences revealed high degrees of matching and congruence with previously sequenced genomes, enabling bacteriophage taxonomic identification.

Whole-genome sequencing and prophage prediction of host P. aeruginosa

The chromosomal DNA of the isolate was extracted from 20 mL of overnight Luria broth cultures with a DNA extraction kit (Promega, Korea). Genomic DNA was submitted to the core facility for next-generation sequencing and sequenced using Illumina technology (Illumina, Inc.) at Macrogen, Inc., Seoul Capital Area, Republic of Korea. Prophet software was used for prophage prediction.

Results

Antibiotic susceptibility test results

Figure 1 summarizes the antibiotic susceptibility patterns of 144P. aeruginosa isolates to 13 drugs from seven various antimicrobial classes. Among 144 clinical P. aeruginosa isolates, 53% (60) were classified as MDR, 5% (6) as XDR, and approximately 3% (3) as possibly PDR. Figure 1 shows the proportions of P. aeruginosa susceptible to antibiotics.

FIG. 1.

(A) Percentage of isolates according to clinical case. (B) Percentage susceptibility of P. aeruginosa to antibiotics.

Imipenem was shown to be the most potent antimicrobial drug, with a susceptibility rate of 90%, followed by gentamicin (CN) (86%), Ofloxacin (OFX) (82%), colistin (77%), and Azithromycin (ATM) (70%) (Fig. 2).

FIG. 2.

Antibiotic susceptibility results.

Bacteriophage isolation and host range

Three phages were isolated in total from 33 distinct wastewater and 23 urine specimens at various times (note that the phages in the cocktail were all from sewage samples). Clinical isolates were employed as host strains for bacteriophage isolation. Purified phage lysates were combined with the appropriate host and placed onto agar plates to determine their infectivity. Nine of the thirty-three wastewater samples contained phages with unique morphologies regarding plaque size, plaque edge, and turbidity. Phages were isolated using the double-layer plaque technique depending on their plaque shape (Fig. 3). Host range experiments with 114 strains were conducted on each purified phage to determine its wide host activity. Forty-five percent (n = 51) of 114 MDR P. aeruginosa strains were infected with at least one of the phages examined. Five MDR P. aeruginosa isolates were more sensitive to phage infection, with sensitivities ranging from 45% to 71%.

FIG. 3.

illustrates the soft agar overlay method for phage extraction and validation (A). The plate displays well-defined plaques of phage varying in size from 1.5 to 3.5 mm, whereas (B) depicts the phage isolation procedure using the spot agar overlay approach. The plate depicts a spot test for the isolation of bacteriophages against P. aeruginosa. On the bacterial lawn, clear patches emerged, which were visible.

Individual phage activity rates were determined depending on their wide host range virulence against all 114 MDR strains. The percentages of the activity of individual phages MMS1, MMS2, and MMS3 against 114 MDR P. aeruginosa determined using DLA and spot tests are shown in Figure 4 and Figure 5. Among the three phages examined, two showed high infectivity rates of 48% (MMS2) and 57% (MMS3) against MDR strains. MMS1 phages exhibited a 45% infectivity rate.

FIG. 4.

Percentage of infectivity for phages.

FIG. 5.

Host range of bacteriophages against different clinical isolates of P. aeruginosa (green = susceptible and red = resistant). It showed high infectivity rates of 45% (MMS1), 48% (MMS2), and 57% (MMS3) against P. aeruginosa isolates.

In vitro characterization of the phage cocktail

The efficiency of plating (EOP) of the cocktail was contrasted with the EOP of each particular phage on 114 P. aeruginosa strains, and the results are shown in Figure 5. As predicted, the phage cocktail lysed a broader range of bacterial species than any particular phage. A single phage with the broadest host range dissolved 65 of the 114 strains in the whole strain collection (57%), while merging the phages in a cocktail boosted the host range lysis by 14% (81 out of 114 strains). Notably, the host range of the phage cocktail in vitro was smaller than what would be expected theoretically if the host ranges of each individual phage in the cocktail were combined. This phenomenon could be explained by phages competing for host bacterial cells.

Specificity

Table 1 summarizes the in vitro potency of MMS and its component phages against bacteria other than P. aeruginosa.

Table 1.

MMS and Its Constituent Phages Exhibit in Vitro Action against Bacteria Other than P. aeruginosa

Bacteria	MMS1	MMS2	MMS3	Cocktail
Staphylococcus epidermidis	0	0	0	0
Staphylococcus aurous	0	0	0	0
Burkholderia cepacia	1	0	0	0
Acinetobacter baumannii	0	0	0	0
Escherichia coli	0	0	0	0
Klebsiella pneumoniae	0	0	0	0

0: not effective, 1: effective.

Thermal and pH stability

Thermal stability tests were conducted to determine the thermal stability of the phage cocktail MMS. The phages were kept at 4°C for a long period of time during the experiments. The phages were completely stable at room temperature. Based on these findings, phage MMS shows vigorous activity following a 6-h incubation at 30–60°C. The weakest activity was detected at 70°C, even though it was completely inert at 80°C and above (Fig. 6A). In addition, the phage was stable at 37°C for 15 h at pH 6, 7, 8, or 9, and the maximum stability was observed at pH 7, whereas at pH 4, the phage was less active. No plaque growth was observed at pH 3; however, substantial plaque activity was detected at pH 7 (Fig. 6B).

FIG. 6.

(A) Thermal stability evaluation at different temperatures. The graphic illustrates the thermal stability of the phages. The phages exhibited high stability at 37°C, but deteriorated at 70°C. (B) shows the results of a pH stability test. The phages were incubated at 37°C for 15 h at various pH values to ensure pH stability. The figure shows that the phages are stable at pH 6, 7, 8, and 9, resulting in a decrease in stability at pH 4.

Impact on the phage adsorption rate of Ca++ and Mg++ ions

The influence of Mg++ and Ca++ ions on bacteriophage adsorption to the host was investigated. CaCl₂ or MgSO₄ (10 mM) was added to the bacterium and phage combination. The plaque assay was utilized to identify the number of free pages in combinations at intervals of 0, 10, 20, and 30 min. The observation revealed a distinction between the treated and control samples (Fig. 7A). Compared with those in the control (samples without Mg⁺⁺/Ca⁺⁺), the number of free phages was dramatically lower in the Ca⁺⁺ and Mg⁺⁺ ion-treated specimens.

FIG. 7.

The bacteriophage adsorption rate was determined in (A). For the measurement of free bacteriophage components, supernatants were taken at various time intervals. By introducing 10 mM MgSO4 and/or CaCl2 to phages and P. aeruginosa, the capability of divalent metal ions was demonstrated. (B) One-step growth curves (B1, B2, and B3 for MMS1, MMS2, and MMS3, respectively). The burst size and latency time of the bacteriophage are depicted in the figure (triphasic pattern).

Latent time and burst size

A one-step growth method was used to assess the latency and burst size of the phages. This experiment yielded a triphasic curve (latent, log, and stationary phases). For MMS3, the latent period and burst size were 15 min and 265 virions per cell, respectively (Fig. 7D). The latency periods were 21 and 18 min, and the burst sizes were 240 and 267 virions per cell for MMS1 and MMS2, respectively (Fig. 7B, 7C).

Transmission electron microscopy (TEM) analysis

Following the host range identification research, phages with an extensive host range were chosen for characterization examination, including transmission electron microscopy. MMS1 has a 63 nm head and a 136 nm long noncontractile tail, resembling members of the family Siphoviridae. Phage MMS2 has a 65 nm diameter head and a 150 nm tail. The presence of a long contractile tail indicates that the phage belongs to the Myoviridae family of the order Caudovirales. Phage MMS3 features a 65 nm head, a short neck, and a 15 nm contractile tail. The morphological traits of the phage were similar to those of a Podoviridae species (Fig. 8).

FIG. 8.

Transmission electron microscopy images of MMS1, MMS2, and MMS3 (from left to right). In MMS1, the siphovirus contains a hexagonal head measuring 60 nm in diameter and a tail measuring 280 nm in length. In MMS2, the myovirus has a hexagonal head measuring 65 nm in diameter and a tail measuring 150 nm in length. In MMS3, the podovirus has a hexagonal head with a diameter of 65 nm and a tail of 15 nm.

Genome sequence and bioinformatics analysis of the selected phages

MMS3 phages were sequenced, and their genomic features are listed in Table 2. The linear genome of the MMS3 phage was 72,747 bp in length. Alignments revealed that the sequences of MMS3 are associated with the Podoviridae Litunavirus group and share a high degree of conservation with the sequences of Pseudomonas phage vB PaeP DEV (Fig. 9), which are publicly accessible (accession no. MF490238.1). To compare all suspected viral open reading frames (ORFs) to a custom-built library of genes encoding bacterial virulence factors, integrases/excisionases/recombinases, and antibiotic resistance genes, all putative viral ORFs were compared to a proprietary database encoding bacterial virulence factors, integrases/excisionases/recombinases, and antibiotic resistance genes. The results showed that the MMS3 genome does not encode proteins with undesired functionalities, which led us to designate them virulent.

Table 2.

Genomic Features of MMS3 Phages

Contig	Contig length	Subject description	Subject length	E-Value	ANI (%)	Aln. Cov.
contig1	72,747	MF490238.1 Pseudomonas phage vB_PaeP_DEV, complete genome	72,697	0.0	93.09	92.28

ANI: % of relatedness at the whole-genome level.

Aln. Cov.: Alignment coverage. % of coverage by assembly sequence alignments against.

FIG. 9.

(A) Gel image of a 1% agarose gel run at 160 V for 30 min. (B) Alignment coverage heatmap resulting from ANI analysis.

Prediction revealed three sequences of prophages in the isolate. These prophages were aligned with the isolated Podovirus phage (MMS3) sequence and public sequences in the NCBI database of the other two phages, and no matching was observed.

Nucleotide sequence accession numbers

Genome sequences of the phage MMS3 have been deposited in the Sequence Read Archive (SRA) under accession number PRJNA748094.

The genome sequences of the P. aeruginosa isolates were deposited in the Sequence Read Archive (SRA) under accession number SAMN21212333.

Discussion

P. aeruginosa is an infectious agent that poses a significant health risk, especially to individuals with traumatic burns or cystic fibrosis and immunocompromised individuals. P. aeruginosa is an antibiotic resistance expert that exhibits resistance to a wide variety of drugs through reduced membrane permeability and efflux pump expression, as well as a remarkable capacity for mutation and horizontal transfer of additional characteristics; as a result, MDR and XDR strains are generated.³³ Following the identification of these characteristics, which considerably constrain existing therapeutic options, phage therapy has been reassessed as a prospective alternative medication. However, phage therapy is not commonly employed due to legislative limitations and few adequate clinical trials and performance research. One of the primary impediments to effective phage therapy is the rapid generation of phage-resistant mutants. Modifications to the LPS molecules accessible on the cell surface are a primary phage resistance mechanism in P. aeruginosa. As mutant phages with an enlarged or changed host range can be isolated and used as complementing components of a therapeutic phage cocktail to avoid the creation of phage-resistant mutants, bacterial resistance to phages can be gained by a variety of mechanisms, including modifications to abortive infection systems or cell surface receptors and CRISPR and restriction-modification.^34,35

Accumulating evidence suggests the efficacy of phages in treating experimental bacterial infections, arguing for their use as first-line therapy, particularly for infections associated with MDR pathogens.^28,36–39 Researchers initially drove the selection of phages with in vitro efficacy against the pathogen of the individual. A complementary approach would be to create a solution with a broad host range, along with such a tailored approach. We constructed a three-phage cocktail and evaluated its efficacy in vitro, taking into account the host range and genetic material for phages. The three phages have distinct host ranges in vitro, which contributes to the effectiveness of the cocktail. For instance, isolate 50 is lysed by phage MMS1, but not by phage MMS2. In vitro, our cocktail had greater host breadth for clinically isolated P. aeruginosa than the individual phages. In addition, the cocktail destroyed the majority of MDR and XDR strains and one PDR strain in our collection, indicating that phage infection occurred independent of the presence of an antibiotic resistance pathway in the cells. Also, the phage cocktail was capable of infecting and killing superbug isolates isolated from various cases of infection. The results showed the effectiveness of MMS1 against Burkholderia cepacia, which could be explained by the fact that B. cepacia is very closely related to P. aeruginosa. Moreover, analyses of the diversity of P. aeruginosa revealed the presence of genome islands that contain genes highly homologous to ones identified in strains of Burkholderia sp. This finding suggests that there is frequent exchange of genetic material between the two organisms.

Treatment of a P. aeruginosa strain with the phage cocktail indicated that phages could penetrate the biofilm, damaging the biomass and eliminating the bacteria trapped therein. In this regard, the phage cocktail significantly enhanced the impact of single-phage infections. In addition, phage cocktails composed of podovirus, myovirus, and siphovirus (vB_PaeP_PYO2, vB_PaeP_DEV, vB_PaeM_E215, and vB_PaeM_E217) decreased biofilms generated by various strains to varying degrees, which might be explained by the reported disparities in biofilm production and composition among clinical P. aeruginosa isolates.^35,41 This shows a synergistic effect when many phages are used. Additional research should be undertaken to elucidate the processes underlying this synergy. However, in light of prior results about the function of the immune system during monophage therapy, we can assume that the cocktail decreases the likelihood of phage-resistant bacteria growth.⁴²

In summary, our technique, which involves information on (i) host range, (ii) genomic material, and (iii) in vitro efficacy and safety, resulted in the construction of a three-phage cocktail (MMS cocktail) that requires validation using in vitro assays. Notably, the in vitro potency in liquid cultures and plates was positive. Thus, in vitro testing under conditions unrelated to the treatment of human bacterial infections can be challenging when developing phage cocktails, despite their reasoning being at the heart of phage therapy.²³

The MMS cocktail, which is prescribed for the treatment of P. aeruginosa infections, is composed of three component phages that are obligatory lytic (not temperate), kill a broad range of clinical P. aeruginosa strains, are deficient in specialized transduction, are likely insufficient for generalized transduction, and do not contain any bacterial virulence factor or drug resistance gene. Given that phage therapy can be targeted to a specific pathogen and thus reduce disruption of the patient’s commensal flora than a wide range of antibiotics can, it is significant that the MMS cocktail component phages are P. aeruginosa specific, exhibiting no in vitro cross-genus activity. dsDNA phages are a distinct subfamily of phages categorized within the Podoviridae family and belong to Duplodnaviria.⁴³ There is currently no sequenced dsDNA phage in our country, and MMS3 is the only phage sequenced in this work, which infects a human pathogen. MMS3 is the first sequenced dsDNA phage and is an effective model for studying dsDNA phage biology.

Along with the properties of individual phages, there is a reason for the particular combination of phages that comprise the MMS cocktail. Within MMS, the phages MMS1, MMS2, and MMS3 each contribute a unique anti-P. aeruginosa activity; there is occasional synergy to kill otherwise resistant P. aeruginosa isolates, the intrinsic frequency of resistance within populations of sensitive bacteria is low, and complementation is possible when resistance does develop. The therapeutic value of antibacterial agents is highly dependent on their spectrum of activity against both target and nontarget bacteria. The MMS cocktail exhibits strong in vitro activity, with a roughly comparable percentage of sensitive isolates among the MDR and non-MDR P. aeruginosa isolates. This finding is consistent with the findings of an independent investigation that examined one of the three MMS component phages and discovered no significant correlation between phage susceptibility and antibiotic resistance in 20 clinical P. aeruginosa isolates.⁴⁴ Unlike static small molecules, phages can evolve in situ, adapting to local bacterial populations and experiencing hostile coevolution to circumvent newly established resistance.⁴⁶ The most frequently hypothesized processes center on the interaction of unrelated or distantly related phages, in which two phages, for instance, use distinct receptors.⁴⁷ Perhaps one phage contains a tail spike protein with depolymerase activity, which destroys the bacterial capsule and enhances the permeability of a cell surface receptor to a second phage with such enzymatic activity.⁴⁸

Randomized, controlled preclinical and clinical studies are required to demonstrate that single-patient clinical observational data acquired in a research setting translate into comprehensive clinical effectiveness.

Conclusion

This study revealed that hospital wastewater is an excellent source of phages against MDR bacteria. The isolated phage cocktail MMS exhibited excellent heat and pH stability. Furthermore, there was a burst size of 240, 267, and 265 virions per cell for MMS1, MMS2, and MMS3, respectively, which is considered an extremely promising metric for employing it as a phage therapy candidate. Finally, the host range can be increased by using a phage cocktail, resulting in the isolation of more phages. Nonetheless, a cocktail (combination of three or more phages) will be an appropriate alternative for bacterial infection and contamination management for eradication and to prevent the development of bacterial resistance to phages.

Authors’ Contributions

M.M.S.: conception and design, experiments, data analysis, writing, and review and editing; Z.K.Z.: review and editing; and M.A.S.: conception and design and review and editing.

Author Disclosure Statement

All authors have approved the final article and declare no conflict of any financial or other interest.

Funding Information

No funding was received for this article.