Use Of Lytic Bacteriophages Research Articles

Genomic Analysis and Characterization of Lytic Bacteriophages that Target Antimicrobial Resistant Escherichia coli in Addis Ababa, Ethiopia

The emergence of antibiotic resistance in E. coli strains has sparked a fervent investigation of alternative therapies, such as the use of lytic bacteriophages. Phage genome sequence analysis is a novel method for learning more about proteins and other biomolecules encoded by phages, particularly phage-lytic enzymes that are crucial to the lysis of bacterial cells. Seven potential lytic E. coli phages—EH-B-A (A1), EP-M-A, EP-B-K (E2), EI-SP-GF, ET-SD-TH, and ST-TK—isolated from activated dairy farm sludges, rivers, and hospital liquid waste genomes were described in this study. The Illumina NextSeq 550 sequencer was used for sequencing phage isolates. The virus nucleotide collection (nr/nt) (taxid:10239) was used to evaluate whole-genome sequences. Phylogenetic analysis was performed using the MEGA11 software. Genome sequencing revealed that each bacteriophage contained a linear double-stranded DNA genome. Phage isolates were taxonomically identified as the Caudoviricetes class with four genera of phages, including Kagunavirus, Vequintavirus, Dhillonvirus, and Jilinvirus. Phage genome lengths varied from 24,264 to 136,204 bp, and GC contents ranged from 44% to 55%. In total, 33-218 CDSs (coding sequences) were predicted, with 19–77% of CDSs encoding functional proteins. All phages lacked tRNA genes in their genomes, except for EI-SP-GF, which possessed five tRNA genes. Based on the phylogenetic tree analysis, the phage isolates were related to Enterobacteria and E. coli phage sequences in the database. Screening did not show any genes encoding for a CRISPR-like system, virulence, antibiotic resistance, or lysogeny. Because of their stringent lytic nature, these phage isolates might be used in the future to treat E. coli infections. This study might also provide some primary data for developing phage control techniques and advance our understanding of the genetic composition of E. coli phages.

Bioconservation of hard rent cheese using bacteriophages

Several technological approaches are used to increase the safety of perishable food products during their production and realization. In particular, it is the use of temperature, high pressure, ultraviolet radiation and added preservatives. One of the generally recognized and common disadvantages of all these methods is that they have an influence on the microorganisms indiscriminately, that is, they inhibit both pathogenic and potentially beneficial autochthonous lactic acid bacteria of the "normal" microflora of fermented products. The purpose of this investigation was to monitor hard rennet cheeses for the content of Staphylococcus aureus and to develop a technology for its preservation using a lytic staphylococcal bacteriophage. Contamination of milk-raw material and Dutch cheese with Staphylococcus aureus was determined by inoculating the selected samples and carrying out tenfold dilutions on a selective hemoagar medium with 5% sodium chloride, followed by thermostating for +37-48 hours. Isolation of bacteriophages from milk raw materials was carried out by filtering through bacterial filters with pores of 0.45 μm. During the improvement of the technology of Dutch cheese with staphylococcal bacteriophage, two samples of cheese were produced - one experimental with bacteriophage, and the second control - without bacteriophage. It was set up that in milk-raw material, which is processed for the production of hard cheeses, Staphylococcus aureus was not detected in 25 to 40% of the samples, on average 45% of the milk samples were contaminated with this pathogen up to 5×102 CFU/ml and from 18 to 30% samples had a staphylococci content of more than 5×102 CFU/ml. In hard cheeses which are realized in the retail network, Staphylococcus aureus was not isolated in an average of 25% of the samples, while approximately 30% of the samples were contaminated with this microorganism in the amount of more than 5×102 CFU/g, which exceeds the requirements of the standard. Two bacteriophages were selected that showed 75.0-80.0% lytic activity against cultures of Staphylococcus aureus from milk and hard cheeses. These bacteriophages were used in the technology of preserving hard Dutch cheese. The technology of Dutch cheese with staphylococcal bacteriophage has been developed to increase its safety during implementation. The use of lytic bacteriophages for the biocontrol of Staphylococcus aureus in rennet cheese makes it possible to almost completely neutralize them in the technology of production. Therefore, the use of lytic bacteriophages to increase the safety of dairy products is an environmentally clean way of restraining the development of pathogenic bacteria, thereby not influencing on the fermentation microflora of the product.

Isolation and characterization of lytic bacteriophages from various sources in Addis Ababa against antimicrobial-resistant diarrheagenic Escherichia coli strains and evaluation of their therapeutic potential

BackgroundEscherichia coli is a common fecal coliform, facultative aerobic, gram-negative bacterium. Pathogenic strains of such microbes have evolved to cause diarrhea, urinary tract infections, and septicemias. The emergence of antibiotic resistance urged the identification of an alternative strategy. The use of lytic bacteriophages against the control of pathogenic E. coli in clinics and different environmental setups (waste and drink water management) has become an alternative therapy to antibiotic therapy. Thus, this study aimed to isolate and characterize lytic bacteriophage from various sources in Addis Ababa, tested them against antimicrobial-resistant diarrheagenic E. coli strains and evaluated their therapeutic potential under in vitro conditions.MethodsA total of 14 samples were processed against six different diarrheagenic E. coli strains. The conventional culture and plaque analysis agar overlay method was used to recover lytic bacteriophage isolates. The phage isolates were characterized to determine their lytic effect, growth characteristics, host range activity, and stability under different temperature and pH conditions. Phage isolates were identified by scanning electron microscope (SEM), and molecular techniques (PCR).ResultsIn total, 17 phages were recovered from 84 tested plates. Of the 17 phage isolates, 11 (65%) were Myoviridae-like phages, and 6 (35%) phage isolates were Podoviridae and Siphoviridae by morphology and PCR identification. Based on the host range test, growth characteristics, and stability test 7 potent phages were selected. These phages demonstrated better growth characteristics, including short latent periods, highest burst sizes, and wider host ranges, as well as thermal stability and the ability to survive in a wide range of pH levels.ConclusionsThe promising effect of the phages isolated in this study against AMR pathogenic E. coli has raised the possibility of their use in the future treatment of E. coli infections.

Isolation, characterization, and application of lytic bacteriophages for controlling Enterobacter cloacae complex (ECC) in pasteurized milk and yogurt

Reducing bacterial pathogen contamination not only improves overall global public health but also diminishes food waste and loss. The use of lytic bacteriophages (phages) that infect and kill bacteria could be a beneficial tool for suppressing bacterial growth during dairy products storage time. Four Enterobacter cloacae (E. cloacae) complex isolates which were previously isolated from contaminated dairy products were used to identify lytic phages in wastewater. Phages specific to multi-drug resistant (MDR) E. cloacae complex 6AS1 were isolated from local sewage. Two novel phages vB_EclM-EP1 and vB_EclM-EP2 were identified as myoviral particles and have double-stranded DNA genome. Their host range and lytic capabilities were detected using spot test and efficiency of plating (EOP) against several bacterial isolates. The phages had a latent period of 30 min, and a large burst size of about 100 and 142 PFU/cell for vB_EclM-EP1 and vB_EclM-EP2, respectively. Both phages were viable at pH ranging 5–9 and stable at 70 °C for 60 min. The individual phages and their cocktail preparations (vB_EclM-EP1 and vB_EclM-EP2) reduced and inhibited the growth of E. cloacae complex 6AS1 during challenge test in milk and yogurt samples. These results indicate that the E. cloacae complex-specific phages (vB_EclM-EP1 and vB_EclM-EP2) have a potential application as microbicidal agents in packaged milk and milk derivatives during storage time. In addition, our environment is a rich sources of lytic phages which have potential use in eliminating multidrug-resistant isolates in food industry as well as in biocontrol.

Control of Escherichia coli O157:H7 using lytic bacteriophage and lactic acid on marinated and tenderized raw pork loins

Escherichia coli (E. coli) O157:H7- contaminated pork and production environments, are often a major source of foodborne outbreaks. This study evaluated the inhibition of E. coli O157:H7 on artificially contaminated raw pork loins using lytic bacteriophage (phage) and lactic acid (LA). Pork samples were inoculated with 106 CFU/mL of E. coli O157:H7 cocktail and stored at 4 °C for 30 min for the initial surface attachment. Inoculated pork loins were randomly assigned to a treatment group (Control, DI water, LA 2.5%, phage 5%, and LA 2.5% + phage 5%; n = 6/group/replication). Following antimicrobial treatments and marination for 1 h, surface microbial population was enumerated. Phage 5% significantly reduced (P < 0.05) the microbial load by 1.90 logs in pre-tenderized loins and > 2.50 logs in post-tenderized loins. Likewise, the combined treatment of phage 5% and LA 2.5% significantly reduced (P < 0.05) the microbial load by 1.89 logs in pre-tenderized loins and > 1.75 logs in post-tenderized loins. The data showed that the use of lytic bacteriophages and lactic acid as antimicrobials can result in a reduced risk of E. coli O157:H7 on the surface of pork loins.

Isolation and Characterization of Lytic Bacteriophage against Methicillin Resistant Staphylococcus aureus from Pyoderma in Dog

Background: Pyoderma in dog poses complexity owing to involvement of multi drug resistant bacterial infection particularly Staphylococcus species. This may result into failure of antibiotic therapy in pyoderma. Therapeutic use of lytic bacteriophage (phage) may be an alternative to deal antimicrobial resistance in veterinary medicine. There is no report available on isolation and characterization of bacteriophage from clinical cases of pyoderma in animals, hence the study was performed with aim to isolate and characterize lytic phage against methicillin resistant S. aureus (MRSA). Methods: The phage was isolated from pus sample of dog suffering from severe pyoderma. The phage was isolated using MRSA as a bacterial host and was named as Staphylococcus phage BVC1 (SPBVC1). The morphology of the phage was determined using TME and was characterised by determining the host range and lytic potential of the phage at range of temperature and pH. Result: The morphology of phage revealed an icosahedral head of diameter 81.31 nm. with sheath and a central tube and a tail of 92.08 nm. It showed strong lytic activity against Methicillin resistant S. aureus and was stable under a range of temperature varying from 4oC to 45oC and pH from 4 to 11. The phage has shown lytic activity against the MRSA however no lytic activity against the MSSA was shown by the phage. The high specificity of the phage for MRSA indicated its potential use as an alternative therapeutic approach against multidrug resistant staphylococcal infections.

Use of Cocktail of Bacteriophage for Salmonella Typhimurium Control in Chicken Meat.

Foodborne diseases are extremely relevant and constitute an area of alert for public health authorities due to the high impact and number of people affected each year. The food industry has implemented microbiological control plans that ensure the quality and safety of its products; however, due to the high prevalence of foodborne diseases, the industry requires new microbiological control systems. One of the main causative agents of diseases transmitted by poultry meat is the bacterium Salmonella enterica. Disinfectants, antibiotics, and vaccines are used to control this pathogen. However, they have not been efficient in the total elimination of these bacteria, with numerous outbreaks caused by this bacterium observed today, in addition to the increase in antibiotic-resistant bacteria. The search for new technologies to reduce microbial contamination in the poultry industry continues to be a necessity and the use of lytic bacteriophages is one of the new solutions. In this study, 20 bacteriophages were isolated for Salmonella spp. obtained from natural environments and cocktails composed of five of them were designed, where three belonged to the Siphoviridae family and two to the Microviridae family. This cocktail was tested on chicken meat infected with Salmonella Typhimurium at 10 °C, where it was found that this cocktail was capable of decreasing 1.4 logarithmic units at 48 h compared to the control.

Novel Salmonella Phage, vB_Sen_STGO-35-1, Characterization and Evaluation in Chicken Meat

Salmonellosis is one of the most frequently reported zoonotic foodborne diseases worldwide, and poultry is the most important reservoir of Salmonella enterica serovar Enteritidis. The use of lytic bacteriophages (phages) to reduce foodborne pathogens has emerged as a promising biocontrol intervention for Salmonella spp. Here, we describe and evaluate the newly isolated Salmonella phage STGO-35-1, including: (i) genomic and phenotypic characterization, (ii) an analysis of the reduction of Salmonella in chicken meat, and (iii) genome plasticity testing. Phage STGO-35-1 represents an unclassified siphovirus, with a length of 47,483 bp, a G + C content of 46.5%, a headful strategy of packaging, and a virulent lifestyle. Phage STGO-35-1 reduced S. Enteritidis counts in chicken meat by 2.5 orders of magnitude at 4 °C. We identified two receptor-binding proteins with affinity to LPS, and their encoding genes showed plasticity during an exposure assay. Phenotypic, proteomic, and genomic characteristics of STGO-35-1, as well as the Salmonella reduction in chicken meat, support the potential use of STGO-35-1 as a targeted biocontrol agent against S. Enteritidis in chicken meat. Additionally, computational analysis and a short exposure time assay allowed us to predict the plasticity of genes encoding putative receptor-binding proteins.

Short-term results of treatment of staphylococcal periprosthetic hip joint infection with combined antibiotics and bacteriophages treatment

Infectious complications after primary implantation of the hip joint are 0.5–3 %, and in the case of re-endoprosthetics, the risk of periprosthetic infection can reach 30 %. Also, we should not forget about the high percentage (16–20 %) of recurrence of periprosthetic infection of the hip joint, which leads to an unsatisfactory result of treatment up to amputation of a limb or even death of the patient. The reasons for the recurrence of the infectious process can be antibiotic resistance and antibiotic tolerance of microorganisms, as well as the ability of microorganisms to form biofilms on implants. In this regard, there is a constant need to search for alternative means of antimicrobial therapy, as well as to select the optimal ways of their delivery and deposition, which is of practical importance when performing surgical interventions in traumatology and orthopedics to protect the implantable structure from possible infection of the surgical site. One of the methods currently available to combat bacterial infections acquired antibiotic resistance and antibiotic tolerance is the use of natural viruses that infect bacterial bacteriophages. The above suggests a more effective suppression of periprosthetic infection, including persisters that deviate from antibiotics. It is, as a rule, associated with biofilms if used in conjunction with antibiotics and phages, when the use of bacteriophages predetermines the effectiveness of treatment. With the use of sensitive bacteriophages in the treatment of periprosthetic infections, a significant (p = 0.030) reduction in the rate of recurrence of infection (from 31 to 4.5 %) was observed. The use of lytic bacteriophages in traumatology and orthopedics is of great interest for phagotherapy of infections caused by antibiotic-resistant and biofilm-forming strains of bacteria. A clinical study using a single-stage surgical revision with simultaneous application of antibiotics and phages in the treatment of deep periprosthesis infection of the hip joint endoprosthesis, followed by 12 months follow-up for periprosthetic infection recurrence, demonstrated the effectiveness of the use of combined antibiotic and bacteriophages treatment.

Isolation and Identification of Escherichia coli O157:H7 Lytic Bacteriophage from Environment Sewage

Escherichia coli O157:H7 is one of the pathogenic bacteria causing foodborne disease. The use of lytic bacteriophages can be a good solution to overcome the disease. This study is aimed at isolating lytic bacteriophages from environmental sewage with E. coli O157:H7 bacterial cells. The sample used in this study was eight bacteriophages, and the technique used in identifying E. coli O157:H7 carriers of the stx1 and stx2 genes was PCR. The double layer plaque technique was used to classify bacteriophages. Plaque morphology, host specificity, and electron micrograph were used to identify the bacteriophages. The result obtained plaque morphology as a clear zone with the largest diameter size of 3.5 mm. Lytic bacteriophage could infect E. coli O157:H7 at the highest titer of 10 × 108 PFU/mL. Bacteriophages have been identified as Siphoviridae and Myoviridae. Phage 3, phage 4, and phage 8 could infect Atypical Diarrheagenic E. coli 1 (aDEC1) due to their host specificity. The Friedman statistical tests indicate that lytic bacteriophage can significantly lyse E. coli O157:H7 (p = 0.012). The lysis of E. coli O157:H7 by phage 1, phage 2, phage 3, and phage 5 bacteriophages was statistically significant, according to Conover's posthoc test (p < 0.05). The conclusion obtained from this study is that lytic bacteriophages from environmental sewage could lyse E. coli O157:H7. Therefore, it could be an alternative biocontrol agent against E. coli O157:H7 that contaminates food causing foodborne disease.

Overcoming the growth-infectivity trade-off in a bacteriophage slows bacterial resistance evolution.

The use of lytic bacteriophages for treating harmful bacteria (phage therapy) is faced with the challenge of bacterial resistance evolution. Phage strains with certain traits, for example, rapid growth and relatively broad infectivity ranges, may enjoy an advantage in slowing bacterial resistance evolution. Here, we show the possibility for laboratory selection programs (“evolutionary training”) to yield phage genotypes with both high growth rate and broad infectivity, traits between which a trade‐off has been assumed. We worked with a lytic phage that infects the bacterium Pseudomonas fluorescens and adopted three types of training strategies: evolution on susceptible bacteria, coevolution with bacteria, and rotation between evolution and coevolution phases. Overall, there was a trade‐off between growth rate and infectivity range in the evolved phage isolates, including those from the rotation training programs. A small number of phages had both high growth rate and broad infectivity, and those trade‐off‐overcoming phages could slow or even completely prevent resistance evolution in initially susceptible bacterial populations. Our findings show the promise of well‐designed evolutionary training programs, in particular an evolution/coevolution rotation selection regime, for obtaining therapeutically useful phage materials.

Mycobacterium abscessus Strain Morphotype Determines Phage Susceptibility, the Repertoire of Therapeutically Useful Phages, and Phage Resistance.

Mycobacterium abscessus is an opportunistic pathogen whose treatment is confounded by widespread multidrug resistance. The therapeutic use of bacteriophages against Mycobacterium abscessus infections offers a potential alternative approach, although the spectrum of phage susceptibilities among M. abscessus isolates is not known. We determined the phage infection profiles of 82 M. abscessus recent clinical isolates and find that colony morphotype-rough or smooth-is a key indicator of phage susceptibility. None of the smooth strains are efficiently killed by any phages, whereas 80% of rough strains are infected and efficiently killed by at least one phage. The repertoire of phages available for potential therapy of rough morphotype infections includes those with relatively broad host ranges, host range mutants of Mycobacterium smegmatis phages, and lytically propagated viruses derived from integrated prophages. The rough colony morphotype results from indels in the glycopeptidolipid synthesis genes mps1 and mps2, negating reversion to smooth as a common route to phage resistance. Resistance is thus rare, and although mutations in polyketide synthesis, uvrD2, and rpoZ can confer resistance, these likely also impair survival in vivo The expanded therapeutic repertoire and the resistance profiles show that small cocktails or single phages could be suitable for controlling infections with rough strains.IMPORTANCEMycobacterium abscessus infections in cystic fibrosis patients are challenging to treat due to widespread antibiotic resistance. The therapeutic use of lytic bacteriophages presents a new potential strategy, but the great variation among clinical M. abscessus isolates demands determination of phage susceptibility prior to therapy. Elucidation of the variation in phage infection and factors determining it, expansion of the suite of therapeutic phage candidates, and a greater understanding of phage resistance mechanisms substantially advances the potential for broad implementation of new therapeutic options for M. abscessus infections.

The Combined Effect of Bacteriophages and Antibiotics on Pseudomonas aeruginosa Biofilm

Currently, the problem of nosocomial infections is of urgent concern. Pseudomonas aeruginosa is one of the key causative agents of this type of disease. The prospect of using bacteriophages in the prevention and control of infectious diseases is now being actively studied. The aim of the work is to study the effect of new bacteriophages on P.aeruginosa biofilm when used together with the antibiotic gentamicin. Cells of the laboratory reference strain P.aeruginosa PAO1 were grown in 96-well plates for a day, the resulting biofilms were treated with gentamicin in various concentrations, as well as bacteriophages AN14 and AN1. The degree of biofilm degradation was evaluated by staining the cells with crystal violet dye. The new lytic bacterio­phages AN14 (Siphoviridae family), AN1 (Myoviridae family) used in the study, showed pronounced antibiofilm activity on the first day of exposure to P.aeruginosa biofilm ( p &lt;0.001). The destructive effect of gentamicin on biofilms increased when the concentration of the antibiotic was increased in the range of 2—16 mg/ml. Addition of lytic bacteriophages AN14 and AN1 enhanced the effect of the antibiotic ( p =0.05). Thus, the combined use of lytic bacteriophages and antibiotics led to a more effective eradication of biofilms than when used separately.

Host Range of Bacteriophages Against a World-Wide Collection of Erwinia amylovora Determined Using a Quantitative PCR Assay.

Erwinia amylovora is a globally devastating pathogen of apple, pear, and other Rosaceous plants. The use of lytic bacteriophages for disease management continues to garner attention as a possible supplement or alternative to antibiotics. A quantitative productive host range was established for 10 Erwinia phages using 106 wild type global isolates of E. amylovora, and the closely related Erwinia pyrifoliae, to investigate the potential regional efficacy of these phages within a biopesticide. Each host was individually infected with each of the 10 Erwinia phages and phage production after 8 h incubation was measured using quantitative real time PCR (qPCR) in conjunction with a standardized plasmid. PCR amplicons for all phages used in the study were incorporated into a single plasmid, allowing standardized quantification of the phage genome copy number after the infection process. Nine of the tested phages exhibited a broad host range, replicating their genomes by at least one log in over 88% of tested hosts. Also, every Amygdaloideae infecting E. amylovora host was able to increase at least one phage by three logs. Bacterial hosts isolated in western North America were less susceptible to most phages, as the mean genomic titre produced dropped by nearly two logs, and this phenomenon was strongly correlated to the amount of exopolysaccharide produced by the host. This method of host range analysis is faster and requires less effort than traditional plaque assay techniques, and the resulting quantitative data highlight subtle differences in phage host preference not observable with typical plaque-based host range assays. These quantitative host range data will be useful to determine which phages should be incorporated into a phage-mediated biocontrol formulation to be tested for regional and universal control of E. amylovora.

Phage Therapy in the Twenty-First Century: Facing the Decline of the Antibiotic Era; Is It Finally Time for the Age of the Phage?

Burgeoning problems of antimicrobial resistance dictate that new solutions be developed to combat old foes. Use of lytic bacteriophages (phages) for the treatment of drug-resistant bacterial infections is one approach that has gained significant traction in recent years. Fueled by reports of experimental phage therapy cases with very positive patient outcomes, several early-stage clinical trials of therapeutic phage products have been launched in the United States. Eventual licensure enabling widespread access to phages is the goal; however, new paths to regulatory approval and mass-market distribution, distinct from those of small-molecule antibiotics, must be forged first. This review highlights unique aspects related to the clinical use of phages, including advantages to be reaped as well as challenges to be overcome.

Manipulation of the microbiota to eradicate multidrug-resistant Enterobacteriaceae from the human intestinal tract

Manipulation of the microbiota to eradicate multidrug-resistant Enterobacteriaceae from the human intestinal tract

Isolation, characterization and in vivo efficacy of Escherichia phage myPSH1131.

Phage therapy is the use of lytic bacteriophages to cure infections caused by bacteria. The aim of this study is to isolate and to characterize the bacteriophages against Escherichia coli isolated from clinical samples. For isolation of bacteriophages, water samples were collected from the Ganges River, and phage enrichment method was followed for phage isolation. Microbiological, genomic and lyophilization experiments were carried out to characterize the bacteriophage. Galleria mellonella was used to study the potential of phages against E. coli infection. Escherichia phage myPSH1131 belonging to Podoviridae family and found to have broad host range infectivity (n = 31) to infect Enterohemorrhagic E. coli (n = 9), Enteropathogenic E. coli (n = 6), Enterotoxigenic E. coli (n = 3), Enteroaggregative E. coli (n = 3), Uropathogenic E. coli (n = 9) and one unknown E. coli. The genome size is 76,163 base pairs (97 coding regions) and their genes show high similarity to SU10 phage. Lyophilization studies showed that the use of 1M sucrose, 2% gelatin and the combination of both 0.5M sucrose plus 1% gelatin could restore phage viability up to 20 months at 4°C. For in vivo studies, it was observed that a single phage dose can reduce the E. coli infection but to achieve 100% survival rate the infected larvae should be treated with three phage doses (20 μL, 103 PFU/mL) at 6 hours interval. The characterized Escherichia phage myPSH1131 was found to have broad host range activity against E. coli pathogens and in vivo studies showed that multiple doses are required for effective treatment.

A Lytic Bacteriophage for Controlling Pseudomonas lactis in Raw Cow's Milk.

The control of bacterial growth during milk processing is crucial for the quality maintenance of commercial milk and milk products. During a period of cold storage prior to heat treatments, some psychrotrophic bacteria grow and produce extracellular heat-resistant lipases and proteases that cause product defects. The use of lytic bacteriophages (phages) that infect and kill bacteria could be a useful tool for suppressing bacterial growth during this cold storage phase. In this study, we isolated a Pseudomonas lactis strain and a phage from raw cow's milk. Quantitative characterization of the phage was used to elucidate whether this phage was active under low temperatures and neutral pH and whether it was inactivated during pasteurization. Phage titer determination was possible under conditions ranging from pH 4 to 9 and from 3°C to 25°C; the phage was inactivated under pasteurization conditions at 63°C for 30 min. Furthermore, we showed that this phage reduced viable bacterial cell counts in both skim and whole milk. The results of this study represent the potential uses of phages for controlling psychrotrophic bacterial growth in raw cow's milk during cold storage.IMPORTANCE Suppression of bacterial growth in raw milk under cold storage is crucial for the quality control of commercially supplied milk. The use of lytic phages as low-cost microbicides is an attractive prospect. Due to strict host specificities, phages must be isolated from the raw milk where the host bacteria are growing. We first isolated the P. lactis bacterial strain and then the phage infecting that strain. Partial phage genomic analysis showed that this is a newly isolated phage, different from any previously reported. This study reports a lytic phage for P. lactis, and we have presented evidence here that this phage reduced viable bacterial cell counts not only in rich medium but also in skim and whole milk. As a result, we have concluded that the phage reported in this study would be useful in milk processing.

Isolation and characterization of a novel bacteriophage infecting Vibrio alginolyticus.

Vibrio alginolyticus is a common marine bacterium implicated in disease outbreaks in marine farmed fish and invertebrates. Due to the inappropriate use of antibiotics in aquaculture, alternative therapies have been proposed. One of the most promising options is the use of lytic bacteriophages to control pathogenic bacteria. This work describes the isolation and characterization of a lytic phage (VEN) against a V. alginolyticus strain (V2) isolated from a disease outbreak in common dentex (Dentex dentex) cultured at the Hellenic Centre for Marine Research (HCMR) in Crete, Greece. The bacteriophage is morphologically similar to phages from Podoviridae family and remained stable for 1year at 4°C and over 1h when kept at 50°C. VEN was able to lyse the host bacteria at several multiplicity of infection (MOI) (0.1-100) in liquid cultures. However, it was unable to infect other V. alginolyticus strains. Its genome consists of 44,603bp with a GC content of 43.5%, while sequence analysis revealed the presence of 54 potential ORFs with a T7-like genomic organization. Almost 65% of the predicted ORFs presented homology with proteins of the vibriophages Vc1 and phi-A318 infecting Vibrio cyclitrophicus and Vibrio alginolyticus, respectively. Phylogenetic analysis applying the amino acid sequence of the large terminase subunit confirmed the close relationship of these phages. Furthermore, the comparison of the RNA polymerase of these phages revealed that the motifs A, B and C related to the catalytic activity and the recognition loop related to promotor identification were also conserved. VEN has an obligate lytic life cycle demonstrated by experimental data and genomic analysis. These results suggest that VEN may provide a good candidate to control recurrent diseases caused by V. alginolyticus at HCMR.

Isolation of Salmonella Phage and its Ability to Control Pathogenic Salmonella in Peeled and Deveined Shrimp

Background: The use of lytic bacteriophages for the biocontrol of food borne pathogens is gaining interest. In recent years it has become widely recognized that bacteriophages have several potential applications in the food industry as a biocontrol agent. Seafood industry is still using chemicals to control the food borne bacterial pathogens which may adversely affect consumer health. Hence this study has been under taken to control food borne Salmonella infections in peeled and deveined (PD) shrimp using bacteriophage. Methods: Salmonella phage was isolated from raw sewage sample by using Salmonella typhimurium (MTCC 1264) as the host bacterium. The host range specificity of Salmonella specific bacteriophage isolated in the current study was checked using different bacterial strains by spotting technique. The efficacy of the isolated Salmonella phage as a biocontrol agent against Salmonella was checked by conducting challenge experiments in PD shrimp by collecting samples from all the controls and treatments at four time intervals viz.0th, 3rd, 6th, 9th h. Findings: The results showed that the log reduction between the positive and treatment trials at 0th and 3rd hour interval as 0.24 and 0.95 log unit respectively (p>0.05). But from 6th hour onwards the host bacterial count showed high reduction in Salmonella count when compared to 0th and 3rd hour (p<0.05). At the 6th hour, the Salmonella count obtained for the positive control was 3.08×103 cfu/ml and for the treatment, the bacterial count was 1.4 × 101. The log reduction in host bacterial count between the positive and treatment at 6th hour was 2.34 whereas at 9th hour the log reduction was 2. 51 log unit (p<0.05). Conclusion: Hence the study shows the potential effectiveness of this isolated Salmonella phage as a biocontrol agent of Salmonella typhimurium in PD shrimp.