Prasanth Manohar

Small lytic phages of the ssRNA Leviviricetes and ssDNA Microviricetes achieve host lysis by expression of a single protein, designated as an Sgl protein. Four ssRNA Sgls have been shown to inhibit steps in peptidoglycan biosynthesis: three target MurJ, the Lipid II flippase, and one targets MurA, the committed step of the pathway. A fifth Sgl, the E protein of the canonical ssDNA microvirus φX174, inhibits MraY, which catalyzes the synthesis of the first lipid-linked intermediate. This inhibition requires SlyD, a cytoplasmic FKBP-type peptidyl-prolyl cis-trans isomerase. The Sgl of the Caulobacter ssRNA phage φCb5 (Cebevirus halophobicum) has not been characterized. Here, we present physiological evidence that SglφCb5 targets MraY. Moreover, we show that, despite the absence of detectable sequence similarity, SglφCb5 shares several features with the φX174 E protein, including the distribution of hydrophobic and charged residues, a proposed N-out, C-in membrane topology, and retention of lytic activity in an SglφCb5 -LacZ fusion. Furthermore, expression of the Caulobacter cytoplasmic FKBP-type peptidyl-prolyl cis-trans isomerase CC2799 enhances SglφCb5 -mediated lysis in E. coli. These parallels between Sgl function in a DNA phage and an RNA phage either represent remarkably consonant convergent evolution or some evolutionary cross-over event for which there is currently no precedent.

Background: Salmonella infecting phages are one of the attractive alternatives to treat antibiotic-resistant infections. Here, we aimed to isolate Salmonella phages and study their lysis genes. Materials and Methods: The water samples were collected from the Texas A&M University (TAMU) wastewater treatment plant, Texas and Salmonella Typhi LT2 was used as a host strain. Host range, life cycle, growth kinetics, and thermal stability were studied. The predicted lysis genes were subsequently evaluated with the λ complementation method. Results: Two Salmonella myoviruses, Manna and Mansal, belonging to Tequatrovirus and Kuttervirus genera, respectively, were isolated. The lysis genes of Manna are all closely related to the T4 counterparts. The Mansal gp21 was shown to be a class I holin by complementation tests. Conclusion: Mansal was found to exhibit canonical lysis inhibition (LIN) but has no homologs of the T4 LIN genes rI and rIII , thus representing the first experimentally confirmed LIN system among class I holins.

Pseudomonas jumbophage phiKZ is prominent because of the proteinaceous nuclear structure that is formed during the infection cycle, protecting the replicating phage DNA from host nucleases. Progress has been made in deciphering the molecular basis of nucleus formation and other unique aspects of phiKZ biology, but the understanding of how it causes host lysis remains minimal. Here we present bioinformatic, physiological and molecular evidence for a “lysis cassette”, the expression of which is necessary and sufficient for the temporally regulated disruption of the host envelope. This cluster contains genes for the usual components of an MGL (multigene lysis) system, including the holin, endolysin, i-spanin and o-spanin. In addition, a fifth gene in the cluster, encoding a cytoplasmic protein, was found to accelerate the timing of holin-mediated lethality when expressed in trans. Evidence is provided that suggests this lysis regulator protein interacts with the cytoplasmic domain of the phiKZ class III holin. In support of this notion, alpha-fold analysis generated a high-confidence structure of a conserved holin-lysis regulator heterodimer complex. Infections at high multiplicity resulted in slower bulk culture lysis profiles than at low multiplicity, suggesting that phiKZ might have a lysis-inhibition system.

Pseudomonas jumbophage phiKZ is prominent because of the proteinaceous nuclear structure that is formed during the infection cycle, conferring resistance of the replicating phage DNA to anti-phage resistance systems. Progress has been made in deciphering the molecular basis of nucleus formation and other unique aspects of phiKZ biology, but the understanding of how it causes host lysis remains minimal. Here we present bioinformatic, physiological and molecular evidence for a “lysis cassette”, the expression of which is necessary and sufficient for the temporally regulated disruption of the host envelope. This cluster contains genes for the usual components of an MGL (multigene lysis) system, including the holin, endolysin, i-spanin and o-spanin. In addition, a fifth gene in the cluster, encoding a cytoplasmic protein, was found to accelerate the timing of holin-mediated lethality when expressed in trans. Evidence is provided that suggests this lysis regulator protein interacts with the cytoplasmic domain of the phiKZ class III holin. In support of this notion, alpha-fold analysis generated a high-confidence structure of a conserved holin-lysis regulator heterodimer complex. Infections at high multiplicity resulted in slower bulk culture lysis profiles than at low multiplicity, suggesting that phiKZ might have a lysis-inhibition system.

The emergence of multi-drug resistance in Salmonella, causing food-borne infections, is a significant issue. With over 2,600 serovars in in Salmonella sp., it is crucial to identify specific solutions for each serovar. Phage therapy serves as an alternate treatment option. In this study, vB_SalP_792 phage was obtained from sewage, forming plaques in eight out of 13 tested clinical S. enterica isolates. Transmission electron microscopy (TEM) examination revealed a T7-like morphotype. The phage was characterized by its stability, life cycle, antibiofilm, and lytic ability in food sources. The phage remains stable throughout a range of temperatures (−20 to 70°C), pH levels (3–11), and in chloroform and ether. It also exhibited lytic activity within a range of MOIs from 0.0001 to 100. The life cycle revealed that 95% of the phages attached to their host within 3 min, followed by a 5-min latent period, resulting in a 50 PFU/cell burst size. The vB_SalP_792 phage genome has a dsDNA with a length of 37,281 bp and a GC content of 51%. There are 42 coding sequences (CDS), with 24 having putative functions and no resistance or virulence-related genes. The vB_SalP_792 phage significantly reduced the bacterial load in the established biofilms and also in egg whites. Thus, vB_SalP_792 phage can serve as an effective biocontrol agent for preventing Salmonella infections in food, and its potent lytic activity against the clinical isolates of S. enterica, sets out vB_SalP_792 phage as a successful candidate for future in vivo studies and therapeutical application against drug-resistant Salmonella infections.

Phage-antibiotic combinations to treat bacterial infections are gaining increased attention due to the synergistic effects often observed when applying both components together. Most studies however focus on a single pathogen, although in many clinical cases multiple species are present at the site of infection. The aim of this study was to investigate the anti-biofilm activity of phage-antibiotic/antifungal combinations on single- and dual-species biofilms formed by P. aeruginosa and the fungal pathogen Candida albicans. The Pseudomonas phage Motto in combination with ciprofloxacin had significant anti-biofilm activity. We then compared biofilms formed by P. aeruginosa alone with the dual-species biofilms formed by bacteria and C. albicans. Here, we found that the phage together with the antifungal fluconazole was active against 6-h-old dual-species biofilms but showed only negligible activity against 24-h-old biofilms. This study lays the first foundation for potential therapeutic approaches to treat co-infections caused by bacteria and fungi using phage-antibiotic combinations.

Introduction: The emergence of antibiotic resistance is a significant challenge in the treatment of bacterial infections, particularly in patients in the intensive care unit (ICU). Phage-antibiotic combination therapy is now being utilized as a preferred therapeutic option for infections that are multi-drug resistant in nature.Methods: In this study, we examined the combined impact of the staph phage vB_Sau_S90 and four antibiotics on methicillin-resistant Staphylococcus aureus (MRSA). We conducted experiments on three different treatment sequences: a) administering phages before antibiotics, b) administering phages and antibiotics simultaneously, and c) administering antibiotics before phages.Results: When the media was supplemented with sub-inhibitory concentrations of 0.25 μg/mL and 1 μg/mL, the size of the plaque increased from 0.5 ± 0.1 mm (in the control group with only the phage) to 4 ± 0.2 mm, 1.6 ± 0.1 mm, and 1.6 ± 0.4 mm when fosfomycin, ciprofloxacin, and oxacillin were added, respectively. The checkerboard analysis revealed a synergistic effect between the phages and antibiotics investigated, as indicated by a FIC value of less than 0.5. The combination treatment of phages and antibiotics demonstrated universal efficacy across all treatments. Nevertheless, the optimal effectiveness was demonstrated when the antibiotics were delivered subsequent to the phages. Utilizing the Galleria mellonella model, in vivo experiments showed that the combination of phage-oxacillin effectively eliminated biofilm-infected larvae, resulting in a survival rate of up to 80% in the treated groups.Discussion: Our findings highlight the advantages of using a combination of phage and antibiotic over using phages alone in the treatment of MRSA infections.

Infections caused by Pseudomonas aeruginosa are becoming increasingly difficult to treat due to the emergence of strains that have acquired multidrug resistance. Therefore, phage therapy has gained attention as an alternative to the treatment of pseudomonal infections. Phages are not only bactericidal but occasionally show activity against biofilm as well. In this study, we describe the Pseudomonas phage Motto, a T1-like phage that can clear P. aeruginosa infections in an animal model and also exhibits biofilm-degrading properties. The phage has a substantial anti-biofilm activity against strong biofilm-producing isolates (n = 10), with at least a twofold reduction within 24 h. To demonstrate the safety of using phage Motto, cytotoxicity studies were conducted with human cell lines (HEK 293 and RAW 264.7 macrophages). Using a previously established in vivo model, we demonstrated the efficacy of Motto in Caenorhabditis elegans, with a 90% survival rate when treated with the phage at a multiplicity of infection of 10.

Escherichia fergusonii is a rarely isolated opportunistic pathogen in animals and humans. Here, we present the annotated genome sequence of Escherichia phage Mangalyan, a T4-like bacteriophage infecting E. fergusonii isolated from chickens. Phage Mangalyan has a genome length of 140,513 bp and belongs to the Vequintavirinae family.

Background: Galleria mellonella , an advantageous model organism, to investigate the therapeutic potential of bacteriophages. Employing the Galleria mellonella (waxworm) model, this study involved infecting the larvae with pathogenic Staphylococcus aureus , followed by combination treatment with staph phages; vB_Sau_Saa90 and vB_Sau_Saa165 at the concentrations of 10 ⁷ and 10 ⁹ PFU/mL and oxacillin, at 100 mg/kg. The treatment modalities were executed in three distinct orders: pre-phage treatment, simultaneous treatment, and post-phage treatment. Each treatment was administered at 90 min intervals. The health state index of the waxworms was monitored throughout the study. Results: Among the three treatment orders, both pre-phage treatment and post-antibiotic treatment exhibited the significant outcomes. The survival percentages ranged from 40% to 50%, 30% to 50%, and 50% to 70% across strong, moderate, and weak biofilm conditions. Notably, the phages vB_Sau_Saa90 and vB_Sau_Saa165 displayed remarkable effectiveness in combination with antibiotics. This successful synergy showcased the potential of combinatorial treatment in effectively addressing biofilm-related infections. Conclusions: This study holds considerable significance as it underscores the suitability of G. mellonella as a model organism for exploring bacteriophage-based therapeutic approaches. The study’s findings shed light on the efficacy of combining bacteriophages and antibiotics to target S. aureus biofilm cells. The observed results in the waxworm model highlight a promising avenue for managing biofilm infections. However, the need for further validation through larger animal models is emphasized, as this could potentially pave the way for novel treatment strategies with broader clinical implications.