Development and Evaluation of Bacteriophage Cocktail to Eradicate Biofilms Formed by an Extensively Drug-Resistant (XDR) Pseudomonas aeruginosa.

Extensive and multiple drug resistance in P. aeruginosa combined with the formation of biofilms is responsible for its high persistence in nosocomial infections. A sequential method to devise a suitable phage cocktail with a broad host range and high lytic efficiency against a biofilm forming XDR P. aeruginosa strain is presented here. Out of a total thirteen phages isolated against P. aeruginosa, five were selected on the basis of their high lytic spectra assessed using spot assay and productivity by efficiency of plating assay. Phages, after selection, were tested individually and in combinations of two-, three-, four-, and five-phage cocktails using liquid infection model. Out of total 22 combinations tested, the cocktail comprising four phages viz. φPA170, φPA172, φPA177, and φPA180 significantly inhibited the bacterial growth in liquid infection model (p < 0.0001). The minimal inhibitory dose of each phage in a cocktail was effectively reduced to >10 times than the individual dose in the inhibition of XDR P. aeruginosa host. Field emission-scanning electron microscopy was used to visualize phage cocktail mediated eradication of 4-day-old multi-layers of XDR P. aeruginosa biofilms from urinary catheters and glass cover slips, and was confirmed by absence of any viable cells. Differential bacterial inhibition was observed with different phage combinations where multiple phages were found to enhance the cocktail's lytic range, but the addition of too many phages reduced the overall inhibition. This study elaborates an effective and sequential method for the preparation of a phage cocktail and evaluates its antimicrobial potential against biofilm forming XDR strains of P. aeruginosa.

Tracking the phage trends: A comprehensive review of applications in therapy and food production.

In the present scenario, the challenge of emerging antimicrobial resistance is affecting human health globally. The increasing incidences of multidrug-resistant infections have become harder to treat, causing high morbidity, and mortality, and are posing extensive financial loss. Limited discovery of new antibiotic molecules has further complicated the situation and has forced researchers to think and explore alternatives to antibiotics. This has led to the resurgence of the bacteriophages as an effective alternative as they have a proven history in the Eastern world where lytic bacteriophages have been used since their first implementation over a century ago. To help researchers and clinicians towards strengthening bacteriophages as a more effective, safe, and economical therapeutic alternative, the present review provides an elaborate narrative about the important aspects of bacteriophages. It abridges the prerequisite essential requirements of phage therapy, the role of phage biobank, and the details of immune responses reported while using bacteriophages in the clinical trials/compassionate grounds by examining the up-to-date case reports and their effects on the human gut microbiome. This review also discusses the potential of bacteriophages as a biocontrol agent against food-borne diseases in the food industry and aquaculture, in addition to clinical therapy. It finishes with a discussion of the major challenges, as well as phage therapy and phage-mediated biocontrols future prospects.

Synergy of a virulent phage (φAB182) with antibiotics leading to successful elimination of biofilms formed by MDR Acinetobacter baumannii.

Emergence of multiple drug resistant (MDR) strains of Acinetobacter baumannii and a withering drug discovery pipeline necessitates the search for effective alternatives to replace or synergize with currently used antibiotics. In this report, we have described the synergy assessment of a virulent Acinetobacter baumannii phage φAB182 with a wide range of antibiotics. Myophage φAB182 was isolated from sewage against MDR A. baumannii and exhibited maximum stability at 25 °C and pH 7. It also had a short latent period of 9 min with a large burst size of 287. The phylogenetic analysis of its major capsid protein gene indicated an 84.15% similarity to the lytic A. baumannii phage Acj9. In the presence of antibiotics, phage φAB182 showed the highest synergy (p < 0.0001) with colistin, followed by polymixin B, ceftazidime and cefotaxime and this synergistic effect was further validated by time kill kinetics. The combined action of phage φAB182 with colistin, polymixin B, ceftazidime and cefotaxime was also synergistic for the eradication of biofilms formed by A. baumannii as measured by MBECcombination/MBECantibiotic values (<0.25). We thus propose bacteriophage φAB182 as a potential antibacterial candidate in combination therapy. The findings from this study strongly support the use of phage antibiotic synergy for the successful treatment of biofilm forming MDR A. baumannii infections.

Antibiotics targeting bacterial protein synthesis reduce the lytic activity of bacteriophages.

Combination therapy of bacteriophages and antibiotics requires careful selection of specific antibiotics as it is crucial towards determining the success of phage therapy to treat multiple drug-resistant bacterial infections. So, we examined how different antibiotics can affect phage lytic activity when used in combination against targeted bacteria. Various antibiotics targeting bacterial protein synthesis pathways were tested for their bactericidal action in combination with bacteriophages of Acinetobacter baumannii (φAB145, φAB182), Staphylococcus aureus (φSA115, φSA116) and Salmonella Typhimurium (φST143, φST188). The phages displayed highly significant antagonism with most of the protein/ribosomal machinery targeting antibiotics: φSA115 (13/13); φSA116 (13/13); φST143 (11/13); φAB145 (11/13); φST188 (9/13); φAB182 (7/13). To validate this antagonistic effect, synergy assessment of these phages with gentamicin (GEN) and tetracycline (TE) was performed using time kill curve assays and counting the remaining viable bacterial cells at the end of the experiment. An increase in bacterial turbidity in phage-antibiotic combination groups was observed as compared to the treatment with phages individually. Also, GEN exhibited 4.22, 5.90, 2.02, 3.15, 2.68, and 2.60 log proliferation in viable cell count, respectively, for φSA115, φSA116, φST145, φAB182, φST143 and φAB188 in combination group in comparison to their individual actions. TE supplementation also led to 2.40, 4.90, 1.61, 2.73, 3.93, and 1.81 log increments in viable bacterial count when combined with φSA115, φSA116, φST145, φAB182, φST143 and φAB188, respectively. This study concludes that antibiotics targeting the bacterial protein biosynthetic machinery may lead to a reduction in the lytic activity of bacteriophages, thus lowering their therapeutic potential. Hence, such compounds must be carefully screened before their employment in combination treatment regimens.

Phage Display Technique as a Tool for Diagnosis and Antibody Selection for Coronaviruses.

Phage display is one of the important and effective molecular biology techniques and has remained indispensable for research community since its discovery in the year 1985. As a large number of nucleotide fragments may be cloned into the phage genome, a phage library may harbour millions or sometimes billions of unique and distinctive displayed peptide ligands. The ligand-receptor interactions forming the basis of phage display have been well utilized in epitope mapping and antigen presentation on the surface of bacteriophages for screening novel vaccine candidates by using affinity selection-based strategy called biopanning. This versatile technique has been modified tremendously over last three decades, leading to generation of different platforms for combinatorial peptide display. The translation of new diagnostic tools thus developed has been used in situations arising due to pathogenic microbes, including bacteria and deadly viruses, such as Zika, Ebola, Hendra, Nipah, Hanta, MERS and SARS. In the current situation of pandemic of Coronavirus disease (COVID-19), a search for neutralizing antibodies is motivating the researchers to find therapeutic candidates against novel SARS-CoV-2. As phage display is an important technique for antibody selection, this review presents a concise summary of the very recent applications of phage display technique with a special reference to progress in diagnostics and therapeutics for coronavirus diseases. Hopefully, this technique can complement studies on host-pathogen interactions and assist novel strategies of drug discovery for coronaviruses.

Isolation and characterization of a novel, T7-like phage against Aeromonas veronii.

A virulent Aeromonas veronii biovar sobria and the corresponding novel, lytic bacteriophage (VTCCBPA5) were isolated from village pond water. The phage was found to belong to family Podoviridae. PCR analysis of major capsid protein gene confirmed its classification to T7-like genus. The protein profiling by SDS-PAGE indicated the major structural protein to be ~ 45 kDa. The phage (VTCCBPA5) is host specific and is stable over a range of pH (6-10) and temperatures (4-45 °C). On the basis of restriction endonuclease analysis combined with prediction mapping, it was observed to vary significantly from previously reported podophages of Aeromonas sp., viz. phiAS7 and Ahp1. The phylogenetic analysis on the basis of PCR-amplified segment of DNA polymerase gene of phage revealed it being an outgroup from podophages of Klebsiella sp. and Pseudomonas sp. though a small internal fragment (359 bp) showed the highest identity (77%) with Vibrio sp. phages. Thus, this is the first report of a novel Podoviridae phage against A. veronii. It expands the assemblage of podophages against Aeromonas sp. and BPA5 could be potentially useful in biocontrol of environmentally acquired Aeromonas veronii infections.