Interlaboratory comparison of Pseudomonas aeruginosa phage susceptibility testing.

Standardized approaches to phage susceptibility testing (PST) are essential to inform selection of phages for study in patients with bacterial infections. There is no reference standard for assessing bacterial susceptibility to phage. We compared agreement between PST performed at three centers: two centers using a liquid assay standardized between the sites with the third, a plaque assay. Four Pseudomonas aeruginosa phages: PaWRA01ø11 (EPa11), PaWRA01ø39 (EPa39), PaWRA02ø83 (EPa83), PaWRA02ø87 (EPa87), and a cocktail of all four phages were tested against 145 P. aeruginosa isolates. Comparisons were made within measurements at the two sites performing the liquid assay and between these two sites. Agreement was assessed based on coverage probability (CP8), total deviation index, concordance correlation coefficient (CCC), measurement accuracy, and precision. For the liquid assay, there was satisfactory agreement among triplicate measurements made on different days at site 1, and high agreement based on accuracy and precision between duplicate measurements made on the same run at site 2. There was fair accuracy between measurements of the two sites performing the liquid assay, with CCCs below 0.6 for all phages tested. When compared to the plaque assay (performed once at site 3), there was less agreement between results of the liquid and plaque assays than between the two sites performing the liquid assay. Similar findings to the larger group were noted in the subset of 46 P. aeruginosa isolates from cystic fibrosis. Results of this study suggest that reproducibility of PST methods needs further development.

Genomic characterization of key bacteriophages to formulate the potential biocontrol agent to combat enteric pathogenic bacteria.

Combating bacterial pathogens has become a global concern especially when the antibiotics and chemical agents are failing to control the spread due to its resistance. Bacteriophages act as a safe biocontrol agent by selectively lysing the bacterial pathogens without affecting the natural beneficial microflora. The present study describes the screening of prominent enteric pathogens NDK1, NDK2, NDK3, and NDK4 (Escherichia, Klebsiella, Enterobacter, and Serratia) mostly observed in domestic wastewater; against which KNP1, KNP2, KNP3, and KNP4 phages were isolated. To analyze their potential role in eradicating enteric pathogens and toxicity issue, these bacteriophages were sequenced using next-generation sequencing and characterized based on its genomic content. The isolated bacteriophages were homologous to Escherichia phage (KNP1), Klebsiella phage (KNP2), Enterobacter phage (KNP3), Serratia phage (KNP4), and belonged to Myoviridae family of Caudovirales except for the unclassified KNP4 phage. Draft genome analysis revealed the presence of lytic enzymes such as holing and lysozyme in KNP1 phage, endolysin in KNP2 phage, and endopeptidase with holin in KNP3 phage. The absence of any lysogenic and virulent genes makes this bacteriophage suitable candidate for preparation of phage cocktail to combat the pathogens present in wastewater. However, KNP4 contained a virulent gene rendering it unsuitable to be used as a biocontrol agent. These findings make the phages (KNP1-KNP3) as a promising alternative for the biocontrol of pathogens in wastewater which is the main culprit to spread these dominated pathogens in different natural water bodies. This study also necessitates for genomic screening of bacteriophages for lysogenic and virulence genes prior to its use as a biocontrol agent.

An Insight into Phage Diversity at Environmental Habitats using Comparative Metagenomics Approach.

Bacteriophages play significant role in driving microbial diversity; however, little is known about the diversity of phages in different ecosystems. A dynamic predator-prey mechanism called "kill the winner" suggests the elimination of most active bacterial populations through phages. Thus, interaction between phage and host has an effect on the composition of microbial communities in ecosystems. In this study, secondary phage metagenome data from aquatic habitats: wastewater treatment plant (WWTP), fresh, marine, and hot water spring habitat were analyzed using MG-RAST and STAMP tools to explore the diversity of the viruses. Differential relative abundance of phage families-Siphoviridae (34%) and Myoviridae (26%) in WWTP, Myoviridae (30%) and Podoviridae (23%) in fresh water, and Myoviridae (41%) and Podoviridae (8%) in marine-was found to be a discriminating factor among four habitats while Rudiviridae (9%), Globuloviridae (8%), and Lipothrixviridae (1%) were exclusively observed in hot water spring. Subsequently, at genera level, Bpp-1-like virus, Chlorovirus, and T4-like virus were found abundant in WWTP, fresh, and marine habitat, respectively. PCA analysis revealed completely disparate composition of phage in hot water spring from other three ecosystems. Similar analysis of relative abundance of functional features corroborated observations from taxa analysis. Functional features corresponding to phage packaging machinery, replication, integration and excision, and gene transfer discriminated among four habitats. The comparative metagenomics approach exhibited genetically distinct phage communities among four habitats. Results revealed that selective distribution of phage communities would help in understanding the role of phages in food chains, nutrient cycling, and microbial ecology. Study of specific phages would also help in controlling environmental pathogens including MDR bacterial populations using phage therapy approach by selective mining and isolation of phages against specific pathogens persisting in a given environment.

A comparison of phage susceptibility testing with two liquid high-throughput methods.

Phage therapy is a promising antibacterial strategy, especially given that drug-resistant bacterial infections are escalating worldwide. Because phages are not active against all strains of a given species, phages being considered for therapeutic use would ideally be tested against bacterial isolates from individual patients prior to administration. Standardized, clinically validated phage susceptibility testing (PST) methods are needed for assessing in vitro phage activity. This study compared two high-throughput liquid-based PST assays. The first, using the Biolog Omnilog™, assessed changes in microbial respiration leading to color changes based on a tetrazolium dye. The second, Agilent BioTek Cytation 7, assessed changes in optical density. Both used 96-well microtiter plate formats. A total of 55 diverse phages with activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, or Enterococcus faecalis were studied against their respective susceptible bacterial hosts and non-susceptible controls, with susceptibility defined based on plaque assay. PST was performed by both assays in replicates, with results compared in terms of hold times (time through which bacterial growth is inhibited by phage compared to controls). Coefficients of variance and interclass correlation coefficients were used to assess inter- and intra-assay reproducibility. Based on a ≤50% coefficient of variance cutpoint, 87% of Biolog and 84% of Agilent assays were considered valid for susceptible bacteria, with 100% considered valid for non-susceptible bacteria by both systems. Using a 8 h hold time cutpoint, 100% of the results matched between the two assays. The interclass correlation coefficient showed 26% excellent agreement, 35% good agreement, and 17% moderate agreement between the two assays for susceptible isolates and 100% excellent agreement for non-susceptible isolates. Overall, the assays compared provided good/fair statistical reproducibility for the assessment of phage susceptibility.

Engineered Bacteriophage-Polymer Nanoassemblies for Treatment of Wound Biofilm Infections.

The antibacterial efficacy and specificity of lytic bacteriophages (phages) make them promising therapeutics for treatment of multidrug-resistant bacterial infections. Restricted penetration of phages through the protective matrix of biofilms, however, may limit their efficacy against biofilm infections. Here, engineered polymers were used to generate noncovalent phage-polymer nanoassemblies (PPNs) that penetrate bacterial biofilms and kill resident bacteria. Phage K, active against multiple strains of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), was assembled with cationic poly(oxanorbornene) polymers into PPNs. The PPNs retained phage infectivity, while demonstrating enhanced biofilm penetration and killing relative to free phages. PPNs achieved 3-log10 bacterial reduction (∼99.9%) against MRSA biofilms in vitro. PPNs were then incorporated into Poloxamer 407 (P407) hydrogels and applied onto in vivo wound biofilms, demonstrating controlled and sustained release. Hydrogel-incorporated PPNs were effective in a murine MRSA wound biofilm model, showing a 1.5-log10 reduction in bacterial load compared to a 0.5 log reduction with phage K in P407 hydrogel. Overall, this work showcases the therapeutic potential of phage K engineered with cationic polymers for treating wound biofilm infections.

Phylogenetic diversity and plant growth-promoting activities of rhizobia nodulating fenugreek (Trigonella foenum-graecum Linn.) cultivated in different agroclimatic regions of India.

Fenugreek (Trigonella foenum-graecum Linn.), is an extensively cultivated legume crop used as a herb, spice, and traditional medicine in India. The symbiotic efficiency and plant growth-promoting potential of fenugreek rhizobia depend on the symbiont strain and environmental factors. We isolated 176 root-nodulating bacteria from fenugreek cultivated in different agroclimatic regions of India. MALDI-TOF MS-based identification and phylogenetic analyses based on 16S rRNA and five housekeeping genes classified the fenugreek-rhizobia as Ensifer (Sinorhizobium) meliloti. However, the strains represent separate sub-lineages of E. meliloti, distinct from all reported sub-lineages across the globe. We also observed the spatial distribution of fenugreek rhizobia, as the three sub-lineages of E. meliloti recorded during this study were specific to their respective agroclimatic regions. According to the symbiotic gene (nodC and nifH) phylogenies, all three sub-lineages of E. meliloti harboured symbiotic genes similar to symbiovar meliloti; as with the housekeeping genes, these also revealed a spatial distribution for different clades of sv. meliloti. The strains could nodulate fenugreek plants and they showed plant growth-promoting potential. Significant differences were found in the plant growth parameters in response to inoculation with the various strains, suggesting strain-level differences. This study demonstrates that fenugreek rhizobia in India are diverse and spatially distributed in different agro-climatic regions.

Control of Multidrug-Resistant Gene Flow in the Environment Through Bacteriophage Intervention.

The spread of multidrug-resistant (MDR) bacteria is an emerging threat to the environment and public wellness. Inappropriate use and indiscriminate release of antibiotics in the environment through un-metabolized form create a scenario for the emergence of virulent pathogens and MDR bugs in the surroundings. Mechanisms underlying the spread of resistance include horizontal and vertical gene transfers causing the transmittance of MDR genes packed in different host, which pass across different food webs. Several controlling agents have been used for combating pathogens; however, the use of lytic bacteriophages proves to be one of the most eco-friendly due to their specificity, killing only target bacteria without damaging the indigenous beneficial flora of the habitat. Phages are part of the natural microflora present in different environmental niches and are remarkably stable in the environment. Diverse range of phage products, such as phage enzymes, phage peptides having antimicrobial properties, and phage cocktails also have been used to eradicate pathogens along with whole phages. Recently, the ability of phages to control pathogens has extended from the different areas of medicine, agriculture, aquaculture, food industry, and into the environment. To avoid the arrival of pre-antibiotic epoch, phage intervention proves to be a potential option to eradicate harmful pathogens generated by the MDR gene flow which are uneasy to cure by conventional treatments.

Intriguing Interaction of Bacteriophage-Host Association: An Understanding in the Era of Omics.

Innovations in next-generation sequencing technology have introduced new avenues in microbial studies through "omics" approaches. This technology has considerably augmented the knowledge of the microbial world without isolation prior to their identification. With an enormous volume of bacterial "omics" data, considerable attempts have been recently invested to improve an insight into virosphere. The interplay between bacteriophages and their host has created a significant influence on the biogeochemical cycles, microbial diversity, and bacterial population regulation. This review highlights various concepts such as genomics, transcriptomics, proteomics, and metabolomics to infer the phylogenetic affiliation and function of bacteriophages and their impact on diverse microbial communities. Omics technologies illuminate the role of bacteriophage in an environment, the influences of phage proteins on the bacterial host and provide information about the genes important for interaction with bacteria. These investigations will reveal some of bio-molecules and biomarkers of the novel phage which demand to be unveiled.