Phage Strains Research Articles

Inferring strain-level mutational drivers of phage-bacteria interaction phenotypes arising during coevolutionary dynamics.

The enormous diversity of bacteriophages and their bacterial hosts presents a significant challenge to predict which phages infect a focal set of bacteria. Infection is largely determined by complementary - and largely uncharacterized - genetics of adsorption, injection, cell take-over and lysis. Here we present a machine learning approach to predict phage-bacteria interactions trained on genome sequences of and phenotypic interactions amongst 51 Escherichia coli strains and 45 phage strains that coevolved in laboratory conditions for 37 days. Leveraging multiple inference strategies and without a priori knowledge of driver mutations, this framework predicts both who infects whom and the quantitative levels of infections across a suite of 2,295 potential interactions. We found that the most effective approach inferred interaction phenotypes from independent contributions from phage and bacteria mutations, accurately predicting of interactions while reducing the relative error in the estimated strength of the infection phenotype by . Feature selection revealed key phage and E. coli mutations that have a significant influence on the outcome of phage-bacteria interactions, corroborating sites previously known to affect phage infections, as well as identifying mutations in genes of unknown function not previously shown to influence bacterial resistance. The method's success in recapitulating strain-level infection outcomes arising during coevolutionary dynamics may also help inform generalized approaches for imputing genetic drivers of interaction phenotypes in complex communities of phage and bacteria.

Leveraging mathematical modeling framework to guide regimen strategy for phage therapy

Bacteriophage (phage) cocktail therapy has been relied upon more and more to treat antibiotic-resistant infections. Understanding of the complex kinetics between phages, target bacteria, and the emergence of phage resistance remain hurdles to successful clinical outcomes. Building upon previous mathematical concepts, we develop biologically-motivated nonlinear ordinary differential equation models to explore single, cocktail, and sequential phage treatment modalities. While the optimal pairwise phage treatment strategy was the double simultaneous administration of two highly potent and asymmetrically binding phage strains, it appears unable to prevent the evolution of resistance. This treatment regimen did have a greater lysis efficiency, promoted higher phage population sizes, reduced bacterial density the most, and suppressed the evolution of resistance the longest compared to all other treatments strategies tested. Conversely, the combination of phages with polar potencies allows the more efficiently replicating phages to monopolize susceptible host cells, thereby quickly negating the intended compounding effect of cocktails. Together, we demonstrate that a biologically-motivated modeling-based framework can be leveraged to quantify the effects of each phage’s properties to more precisely predict treatment responses.

Specific aspects of bacteriophage regulation: international practices and future developments

INTRODUCTION. The increasing prevalence of multidrug-resistant strains of pathogens determines the need for fundamentally new antibacterial agents, including bacteriophage preparations. The consistent implementation of phage therapy is hindered by the lack of generally accepted standardised regulatory documents governing the legal and methodological aspects of the production and preclinical and clinical studies of bacteriophage preparations.AIM. This study aimed to analyse the international experience with the production and lifecycle management of bacteriophage preparations, as well as the main regulatory requirements for the control of their quality, safety, and efficacy.DISCUSSION. It is difficult to develop virulent bacteriophage preparations in accordance with the existing requirements for other medicinal products because of the biological characteristics of bacteriophages, the wide variety of bacteriophage strains, and the potential for rapid changes both in the bacteriophage population and in the pathogen population. Therefore, it is reasonable to develop streamlined marketing authorisation routes for phage therapies and methods for the assessment of their safety and efficacy. As part of these efforts, it is necessary to assess the adverse events specific to this group of medicinal products, such as the risks of lysogeny, resistance to bacteriophages, and antibiotic resistance gene transfer between bacterial strains. The pharmaceutical development of bacteriophage preparations can be based on several approaches. Many countries worldwide, including the United States, are implementing the concept of Quality by Design, considering approaches based on the Biological Master File, and conducting Expanded Access programmes. The Active Substance Master File procedure allows the submission of a separate document package covering only part of the registration dossier for regulatory approval. Expanded Access programmes provide individual patients with access to innovative medicinal products without approved treatment protocols. In the Russian Federation, the commercial production of bacteriophage medicinal products is conducted in accordance with the quality standards specified in the State Pharmacopoeia of the Russian Federation.CONCLUSIONS. There are fundamental differences in the approaches to phage therapy and its regulation around the world and in the Russian Federation. It is reasonable to supplement the current national guidelines for the safety and efficacy evaluation of bacteriophage preparations, in particular, to specify the requirements for conducting preclinical studies.

Biological and genome characteristics of a novel broad host-range phage ΦEP1 infecting enteroinvasive Escherichia coli

We analyzed the biological and genome characteristics of a phage infecting enteroinvasive Escherichia coli (EIEC), aiming to provide resources and a reference for the prevention and treatment of EIEC. With the EIEC preserved in our laboratory as the host bacterium, one strain of phage was isolated from the effluent sample from a chicken farm in Huzhou, Zhejiang and named ΦEP1. The titer, optimal multiplicity of infection, one-step growth curve, temperature, pH value, chloroform and bile salt sensitivity of ΦEP1 were determined by the double-layer agar plate method. The morphology of the phage was observed by transmission electron microscopy. The biocontrol effects of ΦEP1 in different food matrixes and the protective effect of this phage on Caco-2 cells were tested. The phage ΦEP1 showed the optimal multiplicity of infection of 0.1, the titer of 1.3×1010 PFU/mL, strong tolerance to temperature, pH, chloroform, and bile salt, and a broad host spectrum. Furthermore, it expressed lysis activity against multiple strains of multiple antibiotic-resistant pathogenic E. coli and Shigella with different serotypes. Phage ΦEP1 had an incubation period of 10 min, an outbreak period of 80 min, and an outbreak volume of 48 PFU/cell. According to the morphology observed by transmission electron microscopy, phage ΦEP1 belonged to the order of Caudovirales, and it had a good protective effect on Caco-2 cells. Phage ΦEP1 had a genome of 87 182 bp with the GC content of 39.80%, 128 putative open reading frames, and no antibiotic resistance genes or virulence genes. ΦEP1 inhibited the growth of EIEC in artificially contaminated milk and beef and eliminated EIEC in cell protection experiments. It significantly increased the survival rate of Caco-2 cells and down-regulated the expression of interleukin (IL)-6 and IL-1β to reduce inflammation. We obtained an EIEC-targeting phage ΦEP1 with a high titer and strong tolerance to the environment, which provided a basis for the application of phages in food preservation and other fields.

Assessment of the Safety of Anti-Salmonella Disinfectant for Veterinary Use Based on a Cocktail of Bacteriophages.

The toxicity and safety of a veterinary anti-salmonella disinfectant based on three highly virulent bacteriophage strains (titers 1010 PFU/ml) were studied. Acute, chronic, and inhalation toxicity, as well as local irritancy of the disinfectant were evaluated on outbred white mice CD1 (n=65), Soviet chinchilla rabbits (n=20), and rats (n=20). No toxic effects of the disinfectant was observed after its intraperitoneal or intragastric administration to mice and intragastric administration to rats; in rabbits, application on the skin and eyes produced no local irritation effect. Inhalation of 10% of the disinfectant did not cause any pathologies in mice. Thus, the tests confirmed the high level of safety of the disinfectant based on a mixture of bacteriophages for use as an additional specific disinfection agent against Salmonella in veterinary and livestock facilities.

A New Casjensviridae Bacteriophage Isolated from Hospital Sewage for Inactivation of Biofilms of Carbapenem Resistant Klebsiella pneumoniae Clinical Isolates.

Klebsiella pneumoniae, a member of the ESKAPE pathogen group, is a prominent cause of hospital-acquired infections. The WHO has recognized carbapenem-resistant K. pneumoniae as a critical-one priority pathogen. These resilient superbugs have the ability to form biofilms and present a significant global threat. In the present study, we isolated and characterized a bacteriophage SAKp02, from hospital sewage, infectious to carbapenem-resistant K. pneumoniae patient isolates. SAKp02 could infect 43 of 72 clinical isolates, indicating a broad host spectrum. Whole genome analysis classified SAKp02 within the family Casjensviridae, with a 59,343 bp genome encoding 82 ORFs. Comparative genomic analysis revealed significant differences between SAKp02 and its closest viruses, indicating a distinct genetic makeup positioning it as a novel phage strain within the lineage. The SAKp02 genome comprises bacteriolytic enzymes, including holin, endolysin, and phage depolymerase, crucial for bacterial lysis and biofilm disruption. It reduced biofilm biomass by over threefold compared to the control and eradicated 99% of viable cells within a 4 h treatment period. Scanning electron microscopy corroborated the ability of the phage to dismantle biofilm matrices and lyse bacterial cells. Safe and effective treatments are warranted, and hence, the fully characterized lytic phages with therapeutic potential against drug-resistant clinical isolates of bacteria are needed. Our study is the first to report the antibacterial and antibiofilm activity of Casjensviridae phages, and our discovery of a novel K. pneumoniae phage broadens the arsenal against the bacteria.

Isolation and characterisation of Pseudomonas aeruginosa bacteriophage isolated from Batu Pahat, Johor, Malaysia

The World Health Organization has classified Pseudomonas aeruginosa as a 'Priority One and Critical Pathogen' for which research and design of new antibiotics are urgently needed due to its high rate of antimicrobial resistance. Phage therapy, which uses bacteriophages (phages), has been proposed as an antibacterial agent and shows potential for combating this issue. This study aimed to isolate and characterise bacteriophages from different environmental samples that act specifically against P. aeruginosa. The phages were tested to determine their ability to lyse P. aeruginosa using a spot test. Transmission electron microscopy (TEM) was employed to determine the structure, size and phage family, while specificity and sensitivity tests were conducted using six different bacterial species and 20 clinical multi-drug resistant P. aeruginosa isolates, respectively. Phage PA1 was isolated from Batu Pahat, Johor and using a spot test, PA1 could form clear plaques against P. aeruginosa. PA1 was present in a high titer of 1.06 (± 32.2) x 1010 PFUs/ml. Based on TEM analysis, PA1 was classified as a member of the Myoviridae family. Host-range analysis displayed that PA1 had 100% specificity towards P. aeruginosa and only 45% sensitivity towards different P. aeruginosa clinical isolates. Phage PA1 demonstrated lysis of P. aeruginosa but exhibited a narrow host range, presenting a challenge for phage therapy. A promising approach to overcome this limitation involves using phage cocktails containing multiple strains of phages to broaden the host range and enhance the overall efficacy of phage therapy.

Bacteriophage-cocktail hydrogel dressing to prevent multiple bacterial infections and heal diabetic ulcers in mice.

Bacteriophage (phage) has been reported to reduce the bacterial infection in delayed-healing wounds and, as a result, aiding in the healing of said wounds. In this study we investigated whether the presence of phage itself could help repair delayed-healing wounds in diabetic mice. Three strains of phage that target Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa were used. To prevent the phage liquid from running off the wound, the mixture of phage (phage-cocktail) was encapsulated in a porous hydrogel dressing made with three-dimensional printing. The phage-cocktail dressing was tested for its phage preservation and release efficacy, bacterial reduction, cytotoxicity with 3T3 fibroblast, and performance in repairing a sterile full-thickness skin wound in diabetic mice. The phage-cocktail dressing released 1.7%-5.7% of the phages embedded in 24 h, and reduced between 37%-79% of the surface bacteria compared with the blank dressing (p <.05). The phage-cocktail dressing exhibited no sign of cytotoxicity after 3 days (p <.05). In vivo studies showed that 14 days after incision, the full-thickness wound treated with a phage-cocktail dressing had a higher wound healing ratio compared with the blank dressing and control (p <.01). Histological analysis showed that the structure of the skin layers in the group treated with phage-cocktail dressing was restored in an orderly fashion. Compared with the blank dressing and control, the repaired tissue in the phage-cocktail dressing group had new capillary vessels and no sign of inflammation in its dermis, and its epidermis had a higher degree of re-epithelialization (p <.05). The slow-released phage has demonstrated positive effects in repairing diabetic skin wounds.

Isolation and complete genome sequence of Aeromonas bacteriophage Gekk3-15.

Bacteria of the genus Aeromonas, especially A. hydrophila and A. veronii are recognized as important fish pathogens that cause significant economic losses in aquaculture. Environmentally friendly bacteriophage-based solutions for the treatment of fish and for the reduction of colonization by pathogenic bacteria in production facilities are currently in high demand. The bacteriophage Gekk3-15 was isolated during a search for novel phage strains potentially suitable for Aeromonas biocontrol applications. Genome sequencing revealed that this virus is a relatively small myovirus with a 64847 bp long dsDNA genome, which is consistent with virion electron microscopy data. Bacteriophage Gekk3-15 is distinct in its nucleotide and encoded aa sequences from all other sequenced bacteriophage genomes, and may represent a new viral taxon at the genus or subfamily level.

VB_EcoM-P896 coliphage isolated from duck sewage can lyse both intestinal pathogenic Escherichia coli and extraintestinal pathogenic E. coli.

Pathogenic Escherichia coli strains cause diseases in both humans and animals. The limiting factors to prevent as well as control infections from pathogenic E. coli strains are their pathotypes, serotypes, and drug resistance. Herein, a bacteriophage (vB_EcoM-P896) has been isolated from duck sewage. Furthermore, aside from targeting intestinal pathogenic E. coli strains like enteropathogenic E. coli, Shiga toxin-producing E. coli, entero-invasive E. coli, and enteroaggregative E. coli, vB_EcoM-P896 can cause lysis in extraintestinal pathogenic E. coli strains such as avian pathogenic E. coli. Stability analysis revealed that vB_EcoM-P896 was stable under the following conditions: temperature, 4℃-50℃; pH, 3-11. The sequencing of the vB_EcoM-P896 genome was conducted utilizing an HiSeq system (Illumina, San Diego, CA) and subjected to de novo assembling with the aid of Spades 3.11.1. The characteristics of the DNA genome were as follows: size, 170,656bp; GC content, 40.4%; the number of putative coding regions, 294. Transmission electron microscopy analysis of morphology and genome analysis revealed that the phage vB_EcoM-P896 belonged to the order Caudovirales and the family Myoviridae. The pan-genome analysis of vB_EcoM-P896 was divided into two levels. The first level involved the analysis of 91 strains of muscle tail phages, which were mainly divided into 5 groups. The second level involved the analysis of 24 strains of myophage with high homology. Of the 1480 gene clusters, 23 were shared core genes. Neighbor-joining phylogenetic trees were constructed using the Poisson model with MEGA6.0 based on the conserved sequences of phage proteins, the amino acid sequence of the terminase large subunit, and tail fibrin. Further analysis revealed that vB_EcoM-P896 was a typical T4-like potent phage with potential clinical applications.

Isolation and grouping of RNA phages by Itaru Watanabe et al. (1967).

I. Watanabe et al. isolated approximately 30 strains of RNA phages from various parts of Japan. To isolate RNA phages, they assessed the infection specificity of male Escherichia coli and RNase sensitivity. They found that the isolated strains of RNA phages could be serologically separated into three groups. Furthermore, most of them were serologically related, and the antiphage rabbit serum prepared by one of these phages neutralized most of the other phages. The only serologically unrelated phage was the RNA phage Qβ, which was isolated at the Institute for Virus Research, Kyoto University, in 1961.

Bacteriophage adhering to mucus provide protection against Aeromonas veronii infection in scaleless fish

Yellow catfish (Pelteobagrus fulvidraco) is an economically important freshwater cultured fish in China. As a typical scaleless bony fish, the skin mucus plays a critical role in protecting against water-borne microbes. The major challenges in yellow catfish farming are motile Aeromonas septicemia (MAS) and skin ulcerative syndrome, resulting in significant financial losses. In this study, we isolated and identified Aeromonas veronii ZYAH128 as the pathogen responsible for the mass mortality of yellow catfish. To control this pathogen, we explored the use of phages as an alternative approach. Thirty-three phage strains were identified and evaluated for their ability to lyse A. veronii ZYAH128 in vitro. Thereafter, 11 phages were assessed in controlling the infection of A. veronii in yellow catfish via immersion. The two phages, ZPAV-18 and ZPAV-25 provided outstanding prophylactic rates of 85.7% and 71.4%, therapeutic rates of 85.7% and 85.7%, respectively. Phage was able to sustain in yellow catfish mucus for 7 d in flowing water, and the presence of fish mucus increased the susceptibility of A. veronii to phage infection. Our findings supported the symbiotic relationship where phages inhabit mucosal surfaces and provide external immunity against bacterial infection. Overall, this study provides valuable insights into the potential of phage therapy for combating bacterial infections in scaleless fish.

Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice

The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we show that bacteriophage strains D29 and DS6A can efficiently lyse Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently kills H37Rv in liquid culture and in Mtb-infected human primary macrophages. We further show in subsequent experiments that, after the humanized mice were infected with aerosolized H37Rv, then treated with DS6A intravenously, the DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduces Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrate the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.

Isolation of novel &lt;i&gt;Actinomyces oris&lt;/i&gt; and &lt;i&gt;Aggregatibacter actinomycetemcomitans&lt;/i&gt; bacteriophages and study of their biological characteristics &lt;i&gt;in vitro&lt;/i&gt;

SCIENTIFIC RELEVANCE. The incidence of periodontal infections with antibiotic-resistant strains of Actinomyces oris and Aggregatibacter actinomycetemcomitans capable of forming biofilms in the oral cavity is increasing, and the effectiveness of antimicrobials against biofilms is insufficient. Therefore, the isolation of bacteriophages active against A. oris and Ag. actinomycetemcomitans is an urgent task.AIM. This study aimed to isolate bacteriophages active against A. oris and Ag. actinomycetemcomitans, study their biological properties, and select optimum culture conditions providing pure lines and high titres of bacteriophages.MATERIALS AND METHODS. Bacteriophages were isolated from biosamples of saliva, dental plaque, and periodontal pocket contents. The study determined bacteriophage characteristics, including the optimum multiplicity of infection, phage–cell system cultivation time, resistance to various temperatures and pH, and storage stability. Bacteriophage morphology was studied using transmission electron microscopy. Negative colony morphology, lytic activity, host range, and specificity were assessed using spot testing and the Gratia method. Bacterial strains were obtained from the strain collection of the Micromir research and production centre.RESULTS. The authors isolated and studied 3 novel bacteriophages active against A. oris (vB_AorP_1/G-12, vB_AorP_2/Ch-28, and vB_AorP_3/Bl-35) and 1 bacteriophage active against Ag. actinomycetemcomitans (vB_AacS_1/Dc-1). The A. oris bacteriophages were classified as podoviruses, and the vB_AacS_1/Dc-1 bacteriophage was classified as a siphovirus. The phages formed completely transparent round plaques without a halo, with a diameter ranging from 0.8±0.1 to 4.0±0.2 mm. The optimum parameters established to obtain pure phages with maximum titres included a multiplicity of infection of 0,1–10 and phage–cell system cultivation time of 8–12 hours. The study demonstrated the ability of A. oris bacteriophages to lyse Actinomyces naeslundii strains. Of the 15 A. oris bacteriophage strains studied, vB_AorP_1/G-12, vB_AorP_2/Ch-28, and vB_AorP_3/Bl-35 lysed 10, 8, and 12 bacterial strains, respectively. The vB_AacS_1/Dc-1 phage isolate exhibited lytic activity against both tested strains of Ag. actinomycetemcomitans. The studied phages demonstrated stability under abiotic stress and long-term storage conditions.CONCLUSIONS. The authors isolated 3 novel bacteriophages active against A. oris and 1 bacteriophage active against Ag. actinomycetemcomitans and studied their biological properties. The isolated bacteriophages are promising as candidates for further research using clinical strains and whole-genome sequencing.

Two novel bacteriophages isolated from the environment that can help control activated sludge foaming.

Nocardia spp., which belongs to one of the Nocardio-form filamentous bacteria, is usually surface hydrophobic and when overproduced attaches to the surface of bubbles under the action of surfactants, allowing the stable presence of foam on the surface of aeration tanks, leading to the occurrence of sludge-foaming events. Two novel phages, P69 and KYD2, were isolated from the environment, and their hosts were Nocardia transvalensis and Nocardia carnea, respectively. These two phages are Siphophages-like with long tails. An aeration tank pilot plant was constructed in the laboratory to simulate sludge foaming, and these two strains of phage were applied. Compared with the reactor not dosed with phage, the application of phage could reduce the host level in the reactor, resulting in the highest decrease in turbidity by more than 68% and sludge volume index by more than 25%. The time for surface foam disappearance was 9h earlier than that of the control group (the group with the same concentration of Nocardia carnea but no bacteriophage applied), significantly improving water quality. The phage can effectively inhibit the propagation of Nocardia in the actual sludge-foaming event, control the sludge foaming, and improve the effluent quality. It provides a novel and relatively economical solution for controlling sludge foaming in sewage treatment plants in the future, shows that the phages have potential application value in the prevention and control of Nocardia, and provides another way to control the sludge-foaming event caused by the excessive reproduction of Nocardia in the future.

System analysis of the ecological distribution of bacteriophages in hospital wastewater

Phage therapy is one of the most important tools for the treatment of infections with multi-drug resistant bacteria. Such phages are usually isolated from hospital effluents, however, no systematic study on the distribution of phages in hospital effluents has been conducted so far. The aim of this study was to isolate the corresponding phages of common pathogenic bacteria isolated in the clinic as hosts, so as to assess the ecological distribution of phages in hospital wastewater and to provide a reference for the isolation and application of phages of drug-resistant bacteria in the clinic. A cross-sectional study design was used in this study. The 125 pathogenic bacteria (belonging to 16 different strains) isolated from the clinical microbiology laboratory of Qilu Hospital of Shandong University from May to June 2023 were selected as the target strains, and the phages corresponding to these strains were isolated and purified from the hospital wastewater by using the double-layer plate sandwich method. At the same time, the distribution of pathogenic bacteria in the same batch of wastewater was analyzed with the help of mNGS sequencing technology, so as to preliminarily investigate the abundance correspondence between pathogenic bacteria and phages in wastewater. The results showed that a total of 56 phage strains were isolated from 125 clinical pathogens as hosts, corresponding to six pathogens, including Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. All six pathogenic bacteria contained strains with different degrees of drug resistance, with a higher percentage of multi-drug resistant strains in A. baumannii, Escherichia coli and P. aeruginosa. The phage acquisition rates of these six pathogens were, in descending order, Escherichia coli (80%), Stenotrophomonas maltophilia (75%), Pseudomonas aeruginosa (70%), Klebsiella pneumoniae (66.67%), Acinetobacter baumannii (36.36%) and Staphylococcus aureus (12.5%). Preliminary mNGS sequencing results showed that the pathogenic bacteria with higher abundance in the batch of effluent were Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Acinetobacter baumannii, Klebsiella aerogenes, Klebsiella michiganensis and Pseudomonas aeruginosa. In conclusion, phages of most common clinical Gram-negative pathogens were isolated from hospital wastewater with high isolation rates; however, phages of Gram-positive pathogens were isolated at lower rates, and only phages corresponding to Staphylococcus aureus were isolated in this study. The corresponding mNGS sequencing results showed that the distribution of Gram-negative pathogens in sewage may had a positive correlation with the ecological distribution of phages.

Thermal Inactivation of Escherichia Phage OSYSP and Host Strain Escherichia coli O157:H7 EDL933: A Comparative Kinetic Analysis

Lytic bacteriophages are promising biocontrol agents against pathogenic bacteria for food and therapeutic applications. Investigating the feasibility of combining phage and physical lethal agents, such as heat, as an effective hurdle combination could lead to beneficial applications. The current research was initiated to compare the thermal inactivation kinetics of a lytic phage (Escherichia phage OSYSP) and its host (Shiga toxin-producing Escherichia coli O157:H7 EDL933), considering they have different critical thermal targets in their structures. To provide a basis for comparison, thermal inactivation kinetics were determined on suspensions of these agents in buffered peptone water using a thermally controlled circulating water bath. Results showed that the bacteriophage virions have a remarkable heat resistance (p < 0.05) compared to their host cells. The D-values of the populations of phage (PFU/mL) and EDL933 strain (CFU/mL) were 166.7 and 7.3 min at 55°C, compared to 44.4 and 0.3 min at 60°C, respectively. Additionally, D-values were significantly (p < 0.05) more influenced by temperature changes in the case of E. coli O157:H7 EDL933 (z-value 3.7°C) compared to that for phage OSYSP (z-value 7.7°C). When the phage suspension was heat-treated in a thermal cycler instead of a water bath, no significant differences between the two treatment procedures (p > 0.05) in estimating virus D- and z-values were observed. Based on these findings, it may be feasible to combine phage OSYSP with mild heat during processing of food to selectively inactivate E. coli O157:H7 EDL933 and subsequently maintain product safety during storage by the surviving phage population; however, the feasibility of this application needs to be investigated. Additionally, the relatively heat-resistant phage OSYSP could qualify as a biological indicator to validate thermal treatments of minimally processed foods in which E. coli O157:H7 EDL933 is the pathogen-of-concern.

Infectious characteristics of some Vibrio spp. phages isolated in shrimp farming of the Mekong delta

Vibrio spp. Were a cause of serious disease in aquaculture. This study aimed to develop a Vibrio spp. inhibition method that is safe and friendly for the environment, namely bacteriophage (phage). Vibrio spp. and their phages were isolated from shrimp, water, and mud from farms in Mekong Delta provinces (Kien Giang, Soc Trang, and Bac Lieu provinces). Then, the phage influence on the bacterial concentration and the toxR gene was investigated by comparing the gene sequences pre- and post-infection. The polymerase chain reaction identified 31 strains of Vibrio spp., including 9 of Vibrio parahaemolyticus. Then, using several isolated Vibrio spp. as hosts, 32 phage strains were isolated. Results found that each bacteriophage affected the Vibrio toxR gene in specific ways. Phage ST9 and phage ST10 did not change bacterial concentration, but they could change nucleotides at 4 positions compared to the V. parahaemolyticus sequence. Nevertheless, phage KG6 could reduce bacterial concentration but did not affect gene sequences. The bacteriophages can affect bacteria through several mechanisms, including reducing the bacterial concentration via the lytic process or affecting genes encoding bacterial virulence. These findings open a new direction in phage therapy research if such an effect can reduce or neutralize bacterial virulence.

Machine Learning Mediated Advanced Phage and Antimicrobial Therapy- A Futuristic Approach

The emergence of antimicrobial resistance (AMR) has overwhelmed the contemporary curatives and have turned into one of the major challenges in the biomedical sector. With increasing deaths being associated with AMR every year; early detection of pathogens and development of novel drugs and alternative therapies, have all become ad hoc in diagnosis, prognosis and patient survival. Bacteriophage therapy remains a viable strategy to counteract AMR, yet unduly restrained by phage resistance. Phage infection is a natural phenomenon and can be widely manipulated in vitro using advanced techniques including the CRISPR/Cas systems which renders phage therapy an upper hand in comparison to conventional drugs. Phage identification, host range detection, determination of phage-receptor binding efficiency, adsorption rate, phage genome analysis are crucial stages in phage selection and phage cocktail preparation and moreover pivotal in flourishing phage therapy. The ascent of translational research and omics has allowed the development of quick, reliable and precise strategies for phage-based diagnosis and treatment techniques. However, in vitro evaluation of AMR and phage factors as well as storing, processing and analyzing large laboratory data outputs are expensive, time-consuming and labor-intensive. Machine learning (ML) is a utilitarian strategy to organize, store, analyze data sets and more importantly allows prediction of certain features by recognizing patterns in the data sets. With the huge number of research been carried out around the globe and enormous data sets being published and stored in databases, ML can utilize the available data to perform and guide in developing alternative therapeutics. Several ML based tools have been developed to predict resistance in host, phage grouping for cocktail preparation, resistance and lysogenic genes detection, phage genomic evaluation and to understand phage-host interactions. ML also allows the in silico analysis of large samples (drug/phage) and reduces sample size for in vitro evaluation thereby reducing overall costs, time and labor. The present review summarizes the available ML algorithms and corresponding databases used in AMR and phage research. It also emphasizes the status quo of antimicrobial and phage resistance in the healthcare sector and analyses the role of ML in analyzing biological databases in order to predict possible phage/drug-host interaction patterns, phage susceptibility, suitability of phage strains for therapy and recommends the most efficient drug combinations and treatment strategies.

Rapid Bench to Bedside Therapeutic Bacteriophage Production.

It has been over 100years since bacteriophages (phages) were used as a human therapeutic. Since then, phage production has dramatically evolved. Current phage preparations have fewer adverse effects due to their low bacterial toxin content. As a result, therapeutic phages have become a predominant class of new antimicrobials and are being widely used for compassionate treatment of multidrug-resistant (MDR) infections. We describe herein a protocol for the production and ultrapurification of phages. By this technique, it is possible for a lab experienced with the process to produce >109 plaque-forming units (PFU) per mL of Gram-negative phages that meet FDA endotoxins limits for intravenous infusions in as little as 48hours. We provide illustrations of the process and tips on how to safely remove bacterial toxins from phage lysates. Although dependent on the phage strain, the approach described can rapidly generate and purify phages for a variety of applications.