Lytic Efficacy Research Articles

Discovery of Mycobacterium avium subsp. paratuberculosis Lytic Phages with Extensive Host Range Across Rapid- and Slow-Growing Pathogenic Mycobacterial Species

Background/Objectives: Developing interventions for Johne’s disease, which focuses on controlling Mycobacterium avium subsp. paratuberculosis (MAP) in contaminated environments by treating infected cows and preventing transmission from diseased animals, is a critical priority. Bacteriophage (phage) therapy, an emerging biological intervention, offers a promising alternative for the treatment and management of MAP infections. Methods: In this study, we generated an MAP-specific lytic phage library aimed at characterizing the therapeutic potential of phages under environmental and biological conditions that mimic those encountered in infected cattle such as ruminal fluid, milk, colostrum, and the bovine intestinal epithelium, a key site of MAP colonization and, later, transmission. Results: Our library contains a diverse collection of phages that have demonstrated robust lytic activity against MAP. The host range of these phages was thoroughly assessed, revealing that several isolates produce clear plaques on a range of MAP strains, as well as other pathogenic non-tuberculous mycobacterial (NTM) species and M. tuberculosis strains. This broad host range expands the therapeutic potential of the phage collection, positioning it as a potential cross-species antimicrobial tool. In vitro tests under conditions replicating the rumen, milk, and colostrum environments show that selected phages maintain stability and lytic efficacy, even in the presence of complex biological fluids. Furthermore, a subset of these phages was capable of preventing MAP colonization and invasion in cultured bovine epithelial cells, suggesting their potential for direct prophylactic application in cattle. Conclusions. Our collection of MAP phages represents a valuable source that can be developed into probiotic-like preparations, offering a cost-effective solution for prophylaxis and control of Johne’s disease.

Bacteriophages Mediating Effective Elimination of Multidrug-Resistant Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis and septicemia; in certain cases, mortality leads to economic losses and elicits potential foodborne zoonotic risk. The study aimed to determine the prevalence of APEC pathotypes and serotypes in poultry, followed by characterization for virulence markers and antibiotic sensitivity and analysis of lytic efficacy of bacteriophages in the eradication of APEC. We successfully isolated and characterized 34 E. coli isolates from poultry farms. The lytic efficacy of seven bacteriophages, as well as a phage cocktail, was evaluated for biological control of multiple drug resistance (MDR) APEC. A total of 67.65% of isolated E. coli were APEC. A total of 94.11% of the isolates were multidrug-resistant bacteria harboring virulence genes. The lytic ability of seven bacteriophages ranged from 0.98% to 36.76%, with a cocktail of EscoΦA-06 and ΦA-07 exhibiting lysis of 48.04% isolates. As serological variability in APEC limits the application and development of vaccines, the findings support the employment of bacteriophages against elimination of MDR APEC in poultry settings.

Development and characterization of a bacteriophage cocktail with high lytic efficacy against field-isolated Salmonella enterica

Salmonella spp. is a prevalent pathogen that causes great public health concern worldwide. Bacteriophage-based cocktails have arisen as an alternative to antibiotics to inhibit the growth of Salmonella. However, the bactericidal effect of bacteriophage cocktails in vivo largely differs from their observed effect in vitro. This is partly because in vitro developments of cocktails do not always consider the bacterial diversity nor the environmental conditions where bacteriophages will have to replicate. Here, we isolated and sequenced 47 bacteriophages that showed variable degrees of lytic activity against 258 Salmonella isolates from a commercial broiler company in Brazil. Three of these bacteriophages were characterized and selected to assemble a cocktail. In vitro quantitative assays determined the cocktail to be highly effective against multiple serovars of Salmonella, including Minnesota and Heidelberg. Remarkably, the in vitro lytic activity of the cocktail was retained or improved in conditions that more closely resembled the chicken gut, such as anaerobiosis, 42°C, and Salmonella mono-strain biofilms. Analysis of bacterial cross-resistance between the 3 bacteriophages composing the cocktail revealed limited or no generation of cross-resistance. Our results highlight the relevance of an optimized flux of work to develop bacteriophage cocktails against Salmonella with high lytic efficacy and strong potential to be applied in vivo in commercial broiler farms.

Inhibition of Tumor Metastasis by Liquid-Nitrogen-Shocked Tumor Cells with Oncolytic Viruses Infection.

Despite the superior tumor lytic efficacy of oncolytic viruses (OVs), their systemic delivery still faces the challenges of limited circulating periods, poor tumor tropism, and spontaneous antiviral immune responses. Herein, a virus-concealed tumor-targeting strategy enabling OVs' delivery toward lung metastasis via systemic administration is described. The OVs can actively infect, be internalized, and cloak into tumor cells. Then the tumor cells are subsequently treated with liquid-nitrogen-shocking to eliminate the pathogenicity. Such a Trojan Horse-like vehicle avoids virus neutralization and clearance in the bloodstream and facilitates tumor-targeted delivery for more than 110-fold virus enrichment in the tumor metastasis. In addition, this strategy can serve as a tumor vaccine and initiate endogenous adaptive antitumor effects through increasing the memory T cells and modulating the tumor immune microenvironment, including reducing the M2 macrophage, downregulating Treg cells, and priming T cells.

In planta interactions of a novel bacteriophage against Pseudomonas syringae pv. tomato

The biology and biotechnology of bacteriophages have been extensively studied in recent years to explore new and environmentally friendly methods of controlling phytopathogenic bacteria. Pseudomonas syringae pv. tomato (Pst) is responsible for bacterial speck disease in tomato plants, leading to decreased yield. Disease management strategies rely on the use of copper-based pesticides. The biological control of Pst with the use of bacteriophages could be an alternative environmentally friendly approach to diminish the detrimental effects of Pst in tomato cultivations. The lytic efficacy of bacteriophages can be used in biocontrol-based disease management strategies. Here, we report the isolation and complete characterization of a bacteriophage, named Medea1, which was also tested in planta against Pst, under greenhouse conditions. The application of Medea1 as a root drenching inoculum or foliar spraying reduced 2.5- and fourfold on average, respectively, Pst symptoms in tomato plants, compared to a control group. In addition, it was observed that defense-related genes PR1b and Pin2 were upregulated in the phage-treated plants. Our research explores a new genus of Pseudomonas phages and explores its biocontrol potential against Pst, by utilizing its lytic nature and ability to trigger the immune response of plants.Key points• Medea1 is a newly reported bacteriophage against Pseudomonas syringae pv. tomato having genomic similarities with the phiPSA1 bacteriophage• Two application strategies were reported, one by root drenching the plants with a phage-based solution and one by foliar spraying, showing up to 60- and 6-fold reduction of Pst population and disease severity in some cases, respectively, compared to control• Bacteriophage Medea1 induced the expression of the plant defense-related genes Pin2 and PR1b

Lytic efficiency of immunosuppressive drug-resistant armoured T cells against circulating HBV-related HCC in whole blood.

Recurrence of hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) after liver transplant (LT) is mediated by circulating tumour cells (CTCs) and exacerbated by the immunosuppressants required to prevent graft rejection. To circumvent the effects of immunosuppressants, we developed immunosuppressive drug-resistant armoured HBV-specific T-cell receptor-redirected T cells (IDRA HBV-TCR). However, their ability to eliminate HBV-HCC circulating in the whole blood has never been tested, and whether their lytic efficacy is compatible with the number of adoptively transferred T cells in vivo has never been measured. Hence, we developed a microscopy-based assay to quantify CTCs in whole blood. The assay was then used to quantify the efficacy of IDRA HBV-TCRs to lyse free-floating HBV-HCC cells in the presence of Tacrolimus and Mycophenolate Mofetil (MMF). We demonstrated that a panel of antibodies (AFP, GPC3, Vimentin, pan-Cytokeratin, and CD45) specific for HCC tumour antigens and immune cells can effectively differentiate HCC-CTCs in whole blood. Through dose-titration experiments, we observed that in the presence of immunosuppressive drugs, a minimum of 20 000 IDRA HBV-TCR T cells/ml of whole blood is necessary to lyse ~63.5% of free-floating HBV-HCC cells within 16 hours. In conclusion, IDRA HBV-TCR T cells can lyse free-floating HBV-HCC cells in whole blood in the presence of Tacrolimus and MMF. The quantity of IDRA-HBV TCR T cells required can be achieved by the adoptive transfer of 5 × 106 IDRA-HBV TCR-T cells/kg, supporting the utilisation of IDRA HBV-TCR T cells to eliminate CTCs as prophylaxis against recurrence after LT.

Dose-Dependent Von Willebrand Factor Inhibition by Aptamer BB-031 Correlates with Thrombolysis in a Microfluidic Model of Arterial Occlusion.

Von Willebrand Factor (VWF) plays a critical role in thrombus formation, stabilization, and propagation. Previous studies have demonstrated that targeted inhibition of VWF induces thrombolysis when administered in vivo in animal models of ischemic stroke. The study objective was to quantify dose-dependent inhibition of VWF-platelet function and its relationship with thrombolysis using BB-031, an aptamer that binds VWF and inhibits its function. VWF:Ac, VWF:RCo, T-TAS, and ristocetin-induced impedance aggregometry were used to assess BB-031-mediated inhibition of VWF. Reductions in original thrombus surface area and new deposition during administration of treatment were measured in a microfluidic model of arterial thrombolysis. Rotational thromboelastometry was used to assess changes in hemostasis. BB-031 induced maximal inhibition at the highest dose (3384 nM) in VWF:Ac, and demonstrated dose-dependent responses in all other assays. BB-031, but not vehicle, induced recanalization in the microfluidic model. Maximal lytic efficacy in the microfluidic model was seen at 1692 nM and not 3384 nM BB-031 when assessed by surface area. Minor changes in ROTEM parameters were seen at 3384 nM BB-031. Targeted VWF inhibition by BB-031 results in clinically measurable impairment of VWF function, and specifically VWF-GPIb function as measured by VWF:Ac. BB-031 also induced thrombolysis as measured in a microfluidic model of occlusion and reperfusion. Moderate correlation between inhibition and lysis was observed. Additional studies are required to further examine off-target effects of BB-031 at high doses, however, these are expected to be above the range of clinical targeted dosing.

Isolation and therapeutic efficacy of enteropathogenic E. coli phage cocktail against colibacillosis in neonatal goats

Phage based therapeutics have shown promising results against the infections caused by the drug resistant bacteria. To combat the problem of antibiotic resistance posed by diarrhoeagenic E. coli, here, we identified and characterized 38 E. coli phages which were isolated from 70 solid sources (goat-faeces and soil). The in vitro lytic range of phage isolates (n=38) against 439 isolates of E. coli was found between 16 and 53%. Three phage isolates with highest host range showed lytic efficacy against 53, 48 and 46% of E. coli isolates, respectively. A preparation with above three phages was developed, and the phages of the preparation were found stable at wide range of temperature, pH and chloroform treatment. Endotoxin content of the preparation was found below the threshold level and it also passed safety and sterility tests. a total of 40 diarrheic goat kids were administered orally with the therapeutic phage preparation for two days twice daily. Total 21 diarrheic goat-kids were successfully treated using the therapeutic phage preparation, whereas 19 kids could not be treated (success rate: 52.5%; 21/40). The results of the current study provide insight for using lytic bacteriophages for therapeutic interventions against drug resistant E. coli responsible for colibacillosis in neonatal goat kids

Feasibility of Microbubble-Accelerated Low-Dose Thrombolysis of Peripheral Arterial Occlusions Using an Ultrasound Catheter.

Intra-arterial administration of microbubbles (MBs) through an ultrasound (US) catheter increases the local concentration of MBs into the thrombus and may further enhance outcomes of contrast-enhanced sonothrombolysis (CEST). The objective of this study was to evaluate the feasibility and lytic efficacy of intra-arterial infusion of MBs during US-enhanced thrombolysis in both in vitro and in vivo peripheral arterial occluded models. SonoVue and Luminity MBs were infused at a flow rate of 20 mL/h through either the drug delivery lumen of the US catheter (DDC, n=20) or through the tube lumen of the vascular phantom (systematic infusion, n=20) during thrombolysis with a low-dose urokinase (UK) protocol (50 000 IU/h) with(out) US application to assess MB survivability and size by pre-treatment and post-treatment measurements. A human thrombus was placed into a vascular phantom of the flow system to examine the lytic effects of CEST by post-treatment D-dimer concentrations measurements of 5 treatment conditions (saline, UK, UK+US, UK+US+SonoVue, and UK+US+Luminity). Thrombolytic efficacy of localized MBs and US delivery was then investigated in vivo in 5 porcine models by arterial blood flow, microcirculation, and postmortem determined thrombus weight and remaining length. US exposure significantly decreased SonoVue (p=0.000) and Luminity (p=0.000) survivability by 37% and 62%, respectively. In vitro CEST treatment resulted in higher median D-dimer concentrations for the SonoVue (0.94 [0.07-7.59] mg/mL, p=0.025) and Luminity (0.83 [0.09-2.53] mg/mL, p=0.048) subgroups when compared with thrombolysis alone (0.36 [0.02-1.00] mg/mL). The lytic efficacy of CEST examined in the porcine model showed an improved median arterial blood flow of 21% (7%-79%), and a median thrombus weight and length of 1.02 (0.96-1.43) g and 2.25 (1.5-4.0) cm, respectively. One allergic reaction and 2 arrhythmias were observed due to the known allergic reaction on lipids in the porcine model. SonoVue and Luminity can be combined with an US catheter and could potentially accelerate thrombolytic treatment of peripheral arterial occlusions. Catheter-directed thrombolysis showed to be an effective alternative to surgery for acute peripheral arterial occlusions, but this technique is still associated with several limb and life-threatening complications. The effects of thrombolysis on clot dissolution may be further enhanced by intra-arterial administration of microbubbles through an ultrasound catheter. This study demonstrates the feasibility and lytic efficacy of intra-arterial infusion of microbubbles during US-enhanced thrombolysis in both in vitro and in vivo peripheral arterial occluded models.

Improving treatment for acute ischemic stroke-Clot busting innovation in the pipeline.

Acute ischemic stroke is a consequence of disrupted blood flow to the brain, caused by thrombosis—the pathological formation of occlusive clots within blood vessels, which can embolize distally to downstream tissues and microvasculature. The highest priority of stroke treatment is the rapid removal of occlusive clots and restoration of tissue perfusion. Intravenous thrombolysis is the pharmacological standard-of-care for the dissolution of blood clots, wherein thrombolytic drugs are administered to restore vessel patency. While the introduction of recombinant tissue-plasminogen activator (rtPA) in 1996 demonstrated the benefit of acute thrombolysis for clot removal, this was countered by severe limitations in terms of patient eligibility, lytic efficacy, rethrombosis and safety implications. Development of safer and efficacious treatment strategies to improve clot lysis has not significantly progressed over many decades, due to the challenge of maintaining the necessary efficacy-safety balance for these therapies. As such, rtPA has remained the sole approved acute therapeutic for ischemic stroke for over 25 years. Attempts to improve thrombolysis with coadministration of adjunct antithrombotics has demonstrated benefit in coronary vessels, but remain contraindicated for stroke, given all currently approved antithrombotics adversely impact hemostasis, causing bleeding. This Perspective provides a brief history of stroke drug development, as well as an overview of several groups of emerging drugs which have the potential to improve thrombolytic strategies in the future. These include inhibitors of the platelet receptor glycoprotein VI and the signaling enzyme PI3-Kinase, novel anticoagulants derived from hematophagous creatures, and proteolysis-targeting chimeras.

In vitro lytic efficacy of bacteriophages against multidrug-resistant pathogenic bacterial species isolated from pyogenic skin infections

Bacterial multidrug resistance has worsened the situation by adding to economic burden but also poses a greater risk of patient death. The aim of the study was to characterize the multidrug resistance (MDR) properties of the bacterial isolates from skin infections and then to isolate and evaluate lytic efficacy of bacteriophages against the pathogenic bacteria. Antimicrobial susceptibilities of the isolates (n=84) from pyogenic skin infections against 14 antibiotics was studied using CLSI guidelines. Phylogenetic analyses of the MDR strains from each species was performed. Lytic efficacy of the sewage-derived phages was assessed by spot test. was the most predominant (57, 68%) of the total of 84 isolates. The number of Gram-negative isolates that were resistant to all antibiotics (except amikacin) were significantly higher (P<0.05). On the other hand, significant number of strains were susceptible only to clindamycin and erythromycin (P<0.05). Phylogenetic analysis based on the 16S rRNA gene revealed close relatedness of the strains with MDR strains previously reported. analysis of select MDR strains (n=20) showed that the bacteriophages ΦDMSA-2, ΦDMEC-1 and ΦDMPA-1 against , , and , respectively, showed lytic efficacy against 4 of 5 MDR strains tested from each species. These preliminary, but still important results emphasize the potential of phages as an effective alternative therapy against MDR bacteria. Further, the lytic efficacy of phages underscores the importance of developing need-based and locally isolated bacteriophages as potential antimicrobial therapy alternative to antibiotics.

Isolation and Characterization of Group III Campylobacter jejuni-Specific Bacteriophages From Germany and Their Suitability for Use in Food Production.

Campylobacter spp. are a major cause of bacterial foodborne diarrhea worldwide. While thermophilic Campylobacter species asymptomatically colonize the intestines of chickens, most human infections in industrial countries have been attributed to consumption of chicken meat or cross-contaminated products. Bacteriophages (phages) are natural predators of bacteria and their use at different stages of the food production chain has been shown to reduce the public health burden of human campylobacteriosis. However, regarding regulatory issues, the use of lytic phages in food is still under discussion and evaluation. This study aims to identify lytic phages suitable for reducing Campylobacter bacteria along the food production chain. Therefore, four of 19 recently recovered phages were further characterized in detail for their lytic efficacy against different Campylobacter field strains and their suitability under food production settings at different temperatures and pH values. Based on the results of this study, the phages vB_CjM-LmqsCP1-4 and vB_CjM-LmqsCP1-5 appear to be promising candidates for the reduction of Campylobacter jejuni in food production settings.

Characterization of staphylococcal endolysin LysSAP33 possessing untypical domain composition.

Endolysin, a peptidoglycan hydrolase derived from bacteriophage, has been suggested as an alternative antimicrobial agent. Many endolysins on staphylococcal phages have been identified and applied extensively against Staphylococcus spp. Among them, LysK-like endolysin, a well-studied staphylococcal endolysin, accounts for most of the identified endolysins. However, relatively little interest has been paid to LysKunlike endolysin and a few of them has been characterized. An endolysin LysSAP33 encoded on bacteriophage SAP33 shared low homology with LysK-like endolysin in sequence by 41% and domain composition (CHAP-unknown CBD). A green fluorescence assay using a fusion protein for LysSAP33_CBD indicated that the CBD domain (157-251 aa) was bound to the peptidoglycan of S. aureus. The deletion of LysSAP33_CBD at the C-terminal region resulted in a significant decrease in lytic activity and efficacy. Compared to LysK-like endolysin, LysSAP33 retained its lytic activity in a broader range of temperature, pH, and NaCl concentrations. In addition, it showed a higher activity against biofilms than LysK-like endolysin. This study could be a helpful tool to develop our understanding of staphylococcal endolysins not belonging to LysK-like endolysins and a potential biocontrol agent against biofilms.

A new Streptomyces scabies-infecting bacteriophage from Egypt with promising biocontrol traits.

Potato common scab caused by Streptomyces scabies is one of the most economically important diseases infecting potato. It reduces the quality of potato tubers, which subsequently decreases the tuber prices and causes significant economic losses for potato growers. Biological control using bacteriophages is a promising strategy for controlling this disease. In this study, a novel bacteriophage with high lytic efficacy against S. scabies was isolated from a potato field at El-Minya, Egypt, and was designated SscP1EGY. The phage has an icosahedral head of 55nm and a short tail of 7.5nm, typical of a podovirus. Its infection cycle was 90min, including 50min of latent time and 40min of rise period with a burst size of approximately 200 PFU per infected cell. The genome of SscP1EGY consists 51,751 nucleotides with 76 predicted genes. SscP1EGY infected and completely lysed seven tested S. scabies strains but showed no lytic activity against three beneficial Streptomyces species, other beneficial bacterial species, and non-target plant pathogenic bacteria. In greenhouse experiments, treatment of S. scabies-inoculated potato tubers with phage SscP1EGY resulted in reductions of (1) the severity of scab, (2) the number of lesions, and (3) the percentage of lesion surface, as compared to the inoculated tubers without phage treatment. Also, scab lesions appeared superficial in phage-treated tubers but pitted in non-phage-treated tubers. Our results suggest that SscP1EGY has a potential as a biological control agent for S. scabies. Based on our knowledge, SscP1EGY is the first sequenced S. scabies-infecting phage in Egypt.

Isolation and Characterization of Streptococcusmutans Phage as a Possible Treatment Agent for Caries.

Streptococcus mutans is a key bacterium in dental caries, one of the most prevalent chronic infectious diseases. Conventional treatment fails to specifically target the pathogenic bacteria, while tending to eradicate commensal bacteria. Thus, caries remains one of the most common and challenging diseases. Phage therapy, which involves the use of bacterial viruses as anti-bacterial agents, has been gaining interest worldwide. Nevertheless, to date, only a few phages have been isolated against S. mutans. In this study, we describe the isolation and characterization of a new S. mutans phage, termed SMHBZ8, from hundreds of human saliva samples that were collected, filtered, and screened. The SMHBZ8 genome was sequenced and analyzed, visualized by TEM, and its antibacterial properties were evaluated in various states. In addition, we tested the lytic efficacy of SMHBZ8 against S. mutans in a human cariogenic dentin model. The isolation and characterization of SMHBZ8 may be the first step towards developing a potential phage therapy for dental caries.

Lytic Capacity Survey of Commercial Listeria Phage Against Listeria spp. with Varied Genotypic and Phenotypic Characteristics.

Listeria monocytogenes is regularly isolated from food processing environments and is endemic in some facilities. Bacteriophage have potential as biocontrol strategies for L. monocytogenes. In this study, the lytic capacity of a commercial Listeria phage cocktail was evaluated against a library of 475 Listeria spp. isolates (426 L. monocytogenes and 49 other Listeria spp.) with varied genotypic and phenotypic characteristics. The lytic capacity of the Listeria phages was measured by spot assays where lysis was scored on a scale of 0-3 (0 = no lysis; 1 = slight lysis; 2 = moderate lysis; 3 = confluent lysis). Only 5% of all tested Listeria spp. isolates, including L. monocytogenes, were either moderately or highly susceptible (score 2 or 3) to lysis by Listeria phage when scores were averaged across temperature and phage concentration; 155 of 5700 treatment (multiplicity of infection [MOI] and temperature) and characteristic (genotype, sanitizer tolerance, and attachment capacity) combinations resulted in confluent lysis (score = 3). Odds ratios for susceptibility to lysis were calculated using multinomial logistic regression. The odds of susceptibility to lysis by phage decreased (p < 0.05) if the L. monocytogenes isolate was previously found to persist or if the phage-bacteria culture was incubated at 30°C; neither isolate persistence or temperature was significant (p ≥ 0.05) when all factors were considered. In addition, lytic efficacy varied (p < 0.05) among pulse field gel electrophoresis (PFGE) pulsotypes and may be affected by host MOI (p < 0.05). There was no effect (p > 0.05) of attachment capacity or sanitizer tolerance on phage susceptibility. This study underscores the complexity of using Listeria phage as a biocontrol for Listeria spp. in food processing facilities and highlights that phage susceptibility is most greatly impacted by genotype. Further studies are needed to evaluate these findings within a processing environment.

A potent enzybiotic against methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is one of the most dreadful infectious agents, responsible for high mortality and morbidity in both humans and animals. The increased prevalence of multidrug-resistant (MDR) Staphylococcus aureus strains has limited the number of available treatment options, which calls for the development of alternative and effective modalities against MDR S. aureus. Endolysins are bacteriophage-derived antibacterials, which attack essential conserved elements of peptidoglycan that are vital for bacterial survival, making them promising alternatives or complements to existing antibiotics for tackling such infections. For developing endolysin lysin-methicillin-resistant-5 (LysMR-5) as an effective antimicrobial agent, we evaluated its physical and chemical characteristics, and its intrinsic antibacterial activity against staphylococcal strains, including methicillin-resistant Staphylococcus aureus (MRSA). In this study, we cloned, expressed, and purified LysMR-5 from S. aureus phage MR-5. In silico analysis revealed that LysMR-5 harbors two catalytic and one cell wall-binding domain. Biochemical characterization and LC-MS analysis showed that both catalytic domains were active and had no dependence on divalent ions for their action, Zn2+ exerted a negative effect. The optimal lytic activity of the endolysin was at 37°C/pH 7.0 and in the presence of ≥ 300mM concentration of NaCl. Circular dichroism (CD) demonstrated a loss in secondary structure with an increase in temperature confirming the thermosensitive nature of endolysin. Antibacterial assays revealed that LysMR-5 was active against diverse clinical isolates of staphylococci. It showed high lytic efficacy against S. aureus ATCC 43300, as an endolysin concentration as low as 15µg/ml was sufficient to achieve maximum lytic activity within 30min and it was further confirmed by scanning electron microscopy. Our results indicate that rapid and strong bactericidal activity of LysMR-5 makes it a valuable candidate for eradicating multidrug-resistant S. aureus.

Synthesis, Characterization and Estimation the Biological Activity of New Mesomorphic Heterocyclic Compounds

Methicillin-resistant Staphylococcus aureus (MRSA) is a S. aureus that resistant to β-lactam antibiotics (e.g., Cefoxitin and Oxacillin). MRSA has a tremendous capacity to develop resistance to other classes of antibiotics and forming a real threat to patients. The process of exploring a new tactic of non-antibiotic treatments has become an urgent need. A bacteriophage is one of the possible treatments that strongly suggested. Bacteriophages are viruses that infect bacteria as a natural host with a bactericidal capability against multidrug-resistant bacteria that do not respond to conventional antibiotics. The current study investigates the lytic efficacy of phage-cocktail in vitro, specifically against S. aureus isolated from skin infections and find out the possible association of phage-antibiotic resistance. A total of 43 isolates of Methicillin-resistant staphylococcus aureus were isolated from skin infections. The isolates are distributed as (10 isolates of burn, 4 isolates of diabetic foot ulcer, 7 isolates of surgical wounds, 3 isolates of pressure ulcer, and 19 of skin and soft tissue infection). The isolates exhibited variant antibiotic susceptibility against 12 antibiotics (Cefoxitin FOX, Vancomycin VAN, Oxacillin OX, Rifampin RA, Chloramphenicol C, Nitrofurantoin F, Clindamycin DA, Azithromycin AZM, Amikacin AK, Trimethoprim-sulfamethoxazole SXT, Ciprofloxacin CIP, and Gentamicin CN). A bacteriophage cocktail was isolated using a phage-enrichment technique, high titer phage lysate (5*109 pfu/ml) was obtained and investigated against 43 MRSA isolates. The phage-cocktail showed high specificity to S. aureus but variable susceptibility to 43 MRSA isolates. It was observed that there was no association (p greater than 0.05) between phage and antibiotic resistance of (FOX, OX, VAN, RA, C, F, and DA) where the significant association was observed (p less than 0.05) with (AZM, AK, SXT, CIP, and CN). Significantly, the more antibiotic-resistant isolates exhibited more sensitivity to phage-cocktail, which represents a promising alternative to antibiotics that do not affect with increasing antibiotic resistance.

Isolation and Genomic Characterization of phiVibrioH1 a Myoviridae Phage for Controlling Pathogenic Vibrio parahaemolyticus from Seafood and Human

Vibrio parahaemolyticus is a zoonotic pathogen causing vibriosis in marine fish and associated with food poisoning outbreaks in humans. This study aimed to isolate and characterize bacteriophage against multidrug resistant (MDR) Vibrio parahaemolyticus from seafood and humans, and also to assess the lytic efficacy of phage on growth. Vibrio Parahaemolyticus was isolated from flesh of 80 white shrimp, 70 blue crabs and 50 mullets, and from 50 hand swabs of humans (fish handlers) on Thiosulphate Citrate bile salts sucrose agar media. The suspected colonies were biochemically identified. Pathogenic V. parahaemolyticus carrying thermostable direct hemolysin (tdh+) gene was molecularly detected . The pathogenic tdh+V. parahaemolyticus (n=25) isolates were explored for antimicrobial vulnerability resistant to 12 antimicrobials employing the disc diffusion method. Fifteen isolates of tdh+V. parahaemolyticus were 100% resistant to five antimicrobials. Bacteriophage was isolated from sewage water using spot test and double over layer agar assay. The phiVibrioH1 was belonged to family Myoviridae according to transmission electron microscopy. This study revealed a polyvalent phage infecting wide host range of MDR V. parahaemolyticus, V. vulnificus, V. fluvialis, V. alginolyticus, Pseudomonas aeruginosa, E. coli O26 and Proteus vulgaris. The genome of phage was sensitive to digestion with BamH1 and ECoR1 restriction enzymes indicating double stranded DNA. From 24 to 42 hours post-treatment of pathogenic V. parahaemolyticus with the PhiVibrioH1 phage showed complete lysis of bacterial cells. This study confirmed that the phiVibrioH1 is a lytic phage and has a high potential to control pathogenic V. parahaemolyticus strains recovered from seafood and humans.

Efficacy of Bacteriophages Against Staphylococcus aureus Isolates from Bovine Mastitis.

The lytic efficacy of bacteriophages against Staphylococcus aureus isolates from bovine milk was investigated in vitro, regarding possible applications in the therapy of udder inflammation caused by bacterial infections (mastitis). The host range of sequenced, lytic bacteriophages was determined against a collection of 92 Staphylococcus (S.) aureus isolates. The isolates originated from quarter foremilk samples of clinical and subclinical mastitis cases. A spot test and a subsequent plaque assay were used to determine the phage host range. According to their host range, propagation and storage properties, three phages, STA1.ST29, EB1.ST11, and EB1.ST27, were selected for preparing a bacteriophage mixture (1:1:1), which was examined for its lytic activity against S. aureus in pasteurized and raw milk. It was found that almost two thirds of the isolates could be lysed by at least one of the tested phages. The bacteriophage mixture was able to reduce the S. aureus germ density in pasteurized milk and its reduction ability was maintained in raw milk, with only a moderate decrease compared to the results in pasteurized milk. The significant reduction ability of the phage mixture in raw milk promotes further in vivo investigation.