Biological Therapy on Infected Traumatic Wounds: A Case-Control Study

Background:Traumatic wound is a great challenging issue to surgeons, because of large in size, heavily contaminated, infected and unscenic. Infection proceeded to progressive tissue necrosis, septicemia, organ failure or even death. Majority has polymicrobial infections. Bacteriohage therapy will have revolutionized in the treatment of wound. The present study was planned to evaluate the efficacy of topical bacteriophage therapy on large traumatic wounds in comparison with conventional therapy. Methods:The Study conducted from Sept. 2018 to July 2020. Samples between 12- 60 years was taken into study. Customized bacteriophage applied over the wound after serial debridement in case and conventional dressing in control. Fifty four wounded person met the clinical inclusion criteria; 27 in each group. Wound swab and tissue biopsy was taken for bacterial isolation. Isolated specific phage was applied over the wound on alternate day till the wound become sterile and fit for further definitive management. Results: A significant and rapid improvement was observed in wound healing in cases then control group. Average number of day required for complete granulation of wound and attaining sterility was half in cases then control. The hospital stay of the patients on BT was half (20days) than those on CT (40 days). The financial analysis also favours the BT over CT as only 1/third expenditure incurred in BT group as compared to CT Conclusion:Topical Bacteriophage therapy is efficient, effective to clearing the infection in shorter length of time and cost effective for infected traumatic wounds as compared to conventional dressing.

Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay

Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage’s ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics. Other techniques, such as classic adsorption assays, are required to define a phage’s “adsorptive host range.” The issue shared amongst all adsorption assays, however, is that they rely on the use of a complete bacteriophage and thus inherently describe when all adsorption-specific machinery is working together to facilitate bacterial surface adsorption. These techniques cannot be used to examine individual interactions between a singular set of a phage’s adsorptive machinery (like long tail fibers, short tail fibers, tail spikes, etc.) and that protein’s targeted bacterial surface receptor. To address this gap in knowledge we have developed a high-throughput, filtration-based, bacterial binding assay that can evaluate the adsorptive capability of an individual set of a phage’s adsorption machinery. In this manuscript, we used a fusion protein comprised of an N-terminal bioluminescent tag translationally fused to T4’s long tail fiber binding tip (gp37) to evaluate and quantify gp37’s relative adsorptive strength against the Escherichia coli reference collection (ECOR) panel of 72 Escherichia coli isolates. Gp37 could adsorb to 61 of the 72 ECOR strains (85%) but coliphage T4 only formed plaques on 8 of the 72 strains (11%). Overlaying these two datasets, we were able to identify ECOR strains incompatible with T4 due to failed adsorption, and strains T4 can adsorb to but is thwarted in replication at a step post-adsorption. While this manuscript only demonstrates our assay’s ability to characterize adsorptive capabilities of phage tail fibers, our assay could feasibly be modified to evaluate other adsorption-specific phage proteins.

Molecular Modeling the Proteins from the exo-xis Region of Lambda and Shigatoxigenic Bacteriophages

Despite decades of intensive research on bacteriophage lambda, a relatively uncharacterized region remains between the exo and xis genes. Collectively, exo-xis region genes are expressed during the earliest stages of the lytic developmental cycle and are capable of affecting the molecular events associated with the lysogenic-lytic developmental decision. In Shiga toxin-producing E. coli (STEC) and enterohemorragic E. coli (EHEC) that are responsible for food- and water-borne outbreaks throughout the world, there are distinct differences of exo-xis region genes from their counterparts in lambda phage. Together, these differences may help EHEC-specific phage and their bacterial hosts adapt to the complex environment within the human intestine. Only one exo-xis region protein, Ea8.5, has been solved to date. Here, I have used the AlphaFold and RoseTTAFold machine learning algorithms to predict the structures of six exo-xis region proteins from lambda and STEC/EHEC phages. Together, the models suggest possible roles for exo-xis region proteins in transcription and the regulation of RNA polymerase.

The Isolation of specifically lytic phages along with their extracted endolysins as antibacterial agents to MDR Enterococcus faecalis

Background: Multidrug-resistant (MDR) enterococci have become a major problem in recent times and have been reported increasingly around the world. Lytic phages infect bacteria leading to rapid host death with limited risk of phage transduction, underlining the increasing interest in potential phage therapy in the future. Objective (s): The aim of this study is to use phage therapy as alternative approach for treatment of Enterococcus faecalis infections that recorded as MDR in Iraq to tackle this problem. Materials and Methods: Thirty E. faecalis isolates were collected from patients with different infectious diseases such as urinary tract infection (UTI), diabetic foot, septicemia, and wound infections. The isolation of specific lytic phages was from different environmental sources such as (sewage, and wastewater). The biokinetic assays were carried out to measure the characteristics of the isolated phage. The study of the bacteriophage and the formed phage cocktail infectivity against isolates E.faecalis was tested by the top layer assay. The phage endolysin was extracted from the best bacteriophage that gave best results. Results: All the isolated E.faecalis was reported as MDR in this study. About 75 E.faecalis specific phages were isolated and purified. All the isolated bacteria were 100% sensitive to the lytic phages. The formed phage cocktail was capable to create inhibition zones on the most bacterial isolates' lawns. The molecular weight and the concentration of the extracted endolysin was evaluated in this study and found to be as (48 kDa) and (0.5mg/ml), respectively. The antibacterial activity of the extracted endolysin was evaluated by the turbidity reduction assay. A clear decline in the bacterial growth was manifested (5x107 CFU/ml) to (1x104 CFU/ml), in which the bacterial growth was reduced by (3.63 log). The endolysin found to be effective against 90% of E.faeclais isolates. Conclusion: The activity of the isolated specific phage together with the activity of the formed phage cocktail, were efficient as successful and inexpensive method of therapy against MDR E.faecalis. The potential of the extracted endolysin over the phage therapy was verified in this study. The coverage rate and the absence of resistant E.faecalis to the phage and its endolysin had emphasized on the importance of this alternative therapy to commonly used antibiotics. List of abbreviation: MDR = multiple drug-resistant, E. faecalis = Enterococcus faecalis, BT = burst time, BS = burst size, IP = infective percentage, Phage =bacteriophage, CFU = Colony forming unit, kDa = Kilodalton, OD = Optical Densities.

Topically Applied Bacteriophage to Control Multi-Drug Resistant Klebsiella pneumoniae Infected Wound in a Rat Model

(Background): Multi-drug-resistant Klebsiella pneumoniae (MDR-KP) has steadily grown beyond antibiotic control. Wound infection kills many patients each year, due to the entry of multi-drug resistant (MDR) bacterial pathogens into the skin gaps. However, a bacteriophage (phage) is considered to be a potential antibiotic alternative for treating bacterial infections. This research aims at isolating and characterizing a specific phage and evaluate its topical activity against MDR-KP isolated from infected wounds. (Methods): A lytic phage ZCKP8 was isolated by using a clinical isolate KP/15 as a host strain then characterized. Additionally, phage was assessed for its in vitro host range, temperature, ultraviolet (UV), and pH sensitivity. The therapeutic efficiency of phage suspension and a phage-impeded gel vehicle were assessed in vivo against a K. pneumoniae infected wound on a rat model. (Result): The phage produced a clear plaque and was classified as Siphoviridae. The phage inhibited KP/15 growth in vitro in a dose-dependent pattern and it was found to resist high temperature (˂70 °C) and was primarily active at pH 5; moreover, it showed UV stability for 45 min. Phage-treated K. pneumoniae inoculated wounds showed the highest healing efficiency by lowering the infection. The quality of the regenerated skin was evidenced via histological examination compared to the untreated control group. (Conclusions): This research represents the evidence of effective phage therapy against MDR-KP.

Diagnostic application of sensitive and specific phage-exposed epitopes for visceral leishmaniasis and human immunodeficiency virus coinfection

The diagnosis of visceral leishmaniasis (VL) has improved with the search of novel antigens; however, their performance is limited when samples from VL/human immunodeficiency virus (HIV)-coinfected patients are tested. In this context, studies conducted to identify more suitable antigens to detect both VL and VL/HIC coinfection cases should be performed. In the current study, phage display was performed using serum samples from healthy subjects and VL, HIV-infected and VL/HIV-coinfected patients; aiming to identify novel phage-exposed epitopes to be evaluated with this diagnostic purpose. Nine non-repetitive and valid sequences were identified, synthetized and tested as peptides in enzyme-linked immunosorbent assay experiments. Results showed that three (Pep2, Pep3 and Pep4) peptides showed excellent performance to diagnose VL and VL/HIV coinfection, with 100% sensitivity and specificity values. The other peptides showed sensitivity varying from 50.9 to 80.0%, as well as specificity ranging from 60.0 to 95.6%. Pep2, Pep3 and Pep4 also showed a potential prognostic effect, since specific serological reactivity was significantly decreased after patient treatment. Bioinformatics assays indicated that Leishmania trypanothione reductase protein was predicted to contain these three conformational epitopes. In conclusion, data suggest that Pep2, Pep3 and Pep4 could be tested for the diagnosis of VL and VL/HIV coinfection.

Novel Host Recognition Mechanism of the K1 Capsule-Specific Phage of Escherichia coli : Capsular Polysaccharide as the First Receptor and Lipopolysaccharide as the Secondary Receptor

K1 capsule-specific phages of Escherichia coli have been reported in recent years, but the molecular mechanism involved in host recognition of these phages remains unknown. In this study, the interactions between PNJ1809-36, a new K1-specific phage and its host bacteria E. coli DE058, were investigated. A transposon mutation library was used to screen for receptor-related genes. Gene deletion, lysis curve determination, plaque formation test, adsorption assay and inhibition assay of phage by lipopolysaccharide (LPS) showed that capsular polysaccharide (CPS) was the first receptor for the initial adsorption of PNJ1809-36 to E. coli DE058 and LPS was a secondary receptor for the irreversible binding of the phage. The penultimate galactose in the outer core was identified as the specific binding region on LPS. Through antibody blocking assay, fluorescence labeling and high-performance gel permeation chromatography (HPGPC), the tail protein ORF261 of phage PNJ1809-36 was identified as the receptor binding protein on CPS. Given these findings, we propose a model for the recognition process of phage PNJ1809-36 on E. coli DE058: The phage PNJ1809-36 tail protein ORF261 recognizes and adsorbs to the K1 capsule; then the K1 capsule is partially degraded, exposing the active site of LPS which is recognized by phage PNJ1809-36. This model provides insight into the molecular mechanisms between K1-specific phages and their host bacteria. IMPORTANCE It has been speculated that CPS is the main receptor of K1-specific phages belonging to Siphoviridae . In recent years, a new type of K1-specific phage belonging to Myoviridae has been reported, but its host recognition mechanisms remain unknown. Here, we studied the interactions between PNJ1809-36, a new type of K1 phage, and its host bacteria E. coli DE058. Our research showed that the phage initially adsorbed to the K1 capsule mediated by ORF261 and then bound to the penultimate galactose of LPS to begin the infection process.

Investigation of Salmonella Phage–Bacteria Infection Profiles: Network Structure Reveals a Gradient of Target-Range from Generalist to Specialist Phage Clones in Nested Subsets

Bacteriophages that lyse Salmonella enterica are potential tools to target and control Salmonella infections. Investigating the host range of Salmonella phages is a key to understand their impact on bacterial ecology, coevolution and inform their use in intervention strategies. Virus–host infection networks have been used to characterize the “predator–prey” interactions between phages and bacteria and provide insights into host range and specificity. Here, we characterize the target-range and infection profiles of 13 Salmonella phage clones against a diverse set of 141 Salmonella strains. The environmental source and taxonomy contributed to the observed infection profiles, and genetically proximal phages shared similar infection profiles. Using in vitro infection data, we analyzed the structure of the Salmonella phage–bacteria infection network. The network has a non-random nested organization and weak modularity suggesting a gradient of target-range from generalist to specialist species with nested subsets, which are also observed within and across the different phage infection profile groups. Our results have implications for our understanding of the coevolutionary mechanisms shaping the ecological interactions between Salmonella phages and their bacterial hosts and can inform strategies for targeting Salmonella enterica with specific phage preparations.

Use of a Specific Phage Cocktail for Soft Rot Control on Ware Potatoes: A Case Study

Using bacteriophages (bacterial viruses) to control pathogenic bacteria is a promising approach in horticulture. However, the application of this strategy in real conditions requires compliance with particular technological and environmental restraints. The presented paper concerns the process of phage selection to create a cocktail that is efficient against the circulating causal agents of potato soft rot. The resulting phage cocktail causes a complete lysis of a mixture of circulating pectobacterial strains in vitro. In the context of being used to treat ware potatoes during off-season storage, the protocol of phage application via the humidity maintenance system was designed. The phage cocktail was shown to reduce the population of Pectobacterium spp. 10–12-fold, achieving a population that was below a symptomatic threshold.