Double Agar Layer Method Research Articles

Isolation, Antibacterial Activity and Molecular Identification of Avocado Rhizosphere Actinobacteria as Potential Biocontrol Agents of Xanthomonas sp.

Actinobacteria, especially the genus Streptomyces, have been shown to be potential biocontrol agents for phytopathogenic bacteria. Bacteria spot disease caused by Xanthomonas spp. may severely affect chili pepper (Capsicum annuum) crops with a subsequent decrease in productivity. Therefore, the objective of the study was to isolate rhizospheric actinobacteria from soil samples treated by physical methods and evaluate the inhibitory activity of the isolates over Xanthomonas. Initially, soil samples collected from avocado tree orchards were treated by dry heat air and microwave irradiation; thereafter, isolation was implemented. Then, antibacterial activity (AA) of isolates was evaluated by the double-layer agar method. Furthermore, the positive/negative effect on AA for selected isolates was evaluated on three culture media (potato-dextrose agar, PDA; yeast malt extract agar, YME; and oat agar, OA). Isolates were identified by 16S rRNA sequence analysis. A total of 198 isolates were obtained; 76 (series BVEZ) correspond to samples treated by dry heat and 122 strains (series BVEZMW) were isolated from samples irradiated with microwaves. A total of 19 dry heat and 25 microwave-irradiated isolates showed AA with inhibition zones (IZ, diameter in mm) ranging from 12.7 to 82.3 mm and from 11.4 to 55.4 mm, respectively. An increment for the AA was registered for isolates cultured on PDA and YME, with an IZ from 21.1 to 80.2 mm and 14.1 to 69.6 mm, respectively. A lower AA was detected when isolates were cultured on OA media (15.0 to 38.1 mm). Based on the 16S rRNA gene sequencing analysis, the actinobacteria belong to the Streptomyces (6) and Amycolatopsis (2) genera. Therefore, the study showed that microwave irradiation is a suitable method to increase the isolation of soil bacteria with AA against Xanthomonas sp. In addition, Streptomyces sp. BVEZ 50 was the isolate with the highest IZ (80.2 mm).

Evaluation of the application potential of Bdellovibrio sp. YBD-1 isolated from Yak faeces

Studies on Bdellovibrio and like organisms (BALOs), obligate predatory bacteria, have highlighted the possibility of regulating bacteria and biofilms; however, yak-derived BALOs are yet to be reported. We aimed to characterize the BALOs isolated and identified from yak (Bos grunniens) feces and examine application potential. BALOs were isolated from healthy yak fecal samples, with Escherichia coli (ATCC 25922) as prey using the double-layer agar method, identified by transmission electron microscopy (TEM), and the specific 16S rDNA sequencing analysis. Sequencing of the 16S rDNA gene indicated that this isolate was 91% similar to the Bdellovibrio sp. NC01 reference strain and was named YBD-1. Proportion of YBD-1 lysed bacteria is 12/13. The YBD-1 showed best growth at 25–40°C, 0–0.25% (w/v) NaCl, and pH 6.5–7.5. YBD-1 significantly reduced the planktonic cells and biofilms of E.coli in co-culture compared to the E.coli group. Additionally, SEM analysis indicated that YBD-1 significantly reduced biofilm formation in E. coli. Furthermore, quantitative Real Time-polymerase chain reaction (qRT-PCR) showed that the expression of the virulence genes fim and iroN and the genes pgaABC involved in biofilm formation went down significantly. We concluded that YBD-1 may have the potential to control bacterial growth and biofilm-associated bacterial illnesses.

Biological characterization and complete genome analysis of the newly isolated Serratia liquefaciens phage vB_SlqS_ZDD2.

A novel lytic phage named vB_SlqS_ZDD2 was isolated from hospital sewage using the double-layer agar method with Serratia liquefaciens ATCC 27592 as the host. BLASTn analysis showed that the genome sequence of phage vB_SlqS_ZDD2 did not resemble any other phages in the NCBI database. Phenotype and phylogeny analysis indicated that this phage might be a new member of the class Caudoviricetes. Phage vB_SlqS_ZDD2 has a dsDNA genome of 49,178 bp with 55% GC content and has 73 open reading frames. This phage exhibited strong lytic activity and a wide range of pH (3-12) and temperature tolerance (below 70℃).

Identification of a lytic bacteriophage against clinical isolates of Salmonella typhimurium in turkey poults.

The poultry products are known as a source of zoonotic and multi-drug resistant pathogens, especially Salmonella spp. The objective of this study was using bacteriophages as an alternative anti-microbial agent against Salmonella typhimurium isolate from turkey poults. The antibiotic susceptibility test was used to identify the antibiotic resistance pattern of the isolates. The bacteriophage was purified, enhanced and titrated using the Spot test and double layer agar (DLA) techniques after being isolated from a chicken slaughterhouse and sewage treatment facility. By determining the morphological characteristics of resulting plaque, the specificity and host range of the phage were studied on S. typhimurium isolates. A total number of 22 suspected Salmonella isolates were confirmed biochemically positive in sample by cultures method. Nine of these isolates (40.90%) were identified as S. typhimurium by polymerase chain reaction. All of isolates (100%) were resistant to chloramphenicol, doxycycline, kanamycin, florfenicol, rifampin, and erythromycin. Seven isolates (77.77%) were resistant to amoxicillin and nalidixic acid. The plaques were present with 3.00 ± 0.22 mm in diameter on the culture of 6 out of 9 (66.66%) isolates of S. typhimurium on brain heart infusion broth using DLA method. The amount of phage titer was 7.60 × 107 phage forming unit mL-1 and its multiplicity of infection value was calculated as 5.06 × 10-2 based on obtained results. In place of antibiotics, the multi-drug resistant (MDR) S. typhimurium was successfully destroyed by the isolated bacteriophage from wastewater. In vitro settings were used in this investigation to identify the efficient bacteriophages against MDR S. typhimurium.

2773. Brazilian Bacteriophage Activity Against Multidrug-Resistant (MDR) Klebsiella pneumoniae Isolated from Patients with Severe Nosocomial Infection

Abstract Background Phage therapy has re-emerged as a promising strategy for combatting antimicrobial resistance. Phages are ubiquitous viruses that infect and destroy bacteria and are easily isolated from environmental samples. An impressive reservoir of potentially efficacious bacteriophages has yet to be discovered, and the isolation of novel phages from diverse environments is of global importance. Methods A phage research lab was implemented in Salvador Bahia, Brazil, in collaboration with the Center for Innovative Phage Applications and Therapeutics (IPATH) located in San Diego, California. The goal of this project was to discover and isolate phages from water systems throughout the area. Samples were collected from sources including standing water surrounding residences in impoverished communities (favelas), sewage, and the intersection of seawater and wastewater along the coast. Protocols were followed for phage enrichment, spot assay, isolation, and purification utilizing the double-layer agar method. Phages isolated against laboratory strain K. pneumoniae (ATCC 10031) were then spot-tested against 30 clinical, multidrug-resistant strains of K. pneumoniae which were collected from a local hospital. Results A total of 28 phages were isolated from environmental samples with efficacy against the host strain, K. pneumoniae. All 28 phages were then tested against the 30 clinical strains of MDR K. pneumoniae, among which nine had at least intermediate activity against five or more MDR K. pneumoniae strains based on qualitative spot assay results. Two of the clinical K. pneumoniae samples were ESBL+ and showed at least intermediate sensitivity to three and four phages, respectively. Of the 30 clinical bacterial samples, six were completely resistant to all 28 phages. 25 of the tested phages revealed activity against at least one clinical K. pneumoniae strain. Spot assay demonstrating phage activity against host strain K. pneumoniae 19 of the 28 phages are shown spotted against a double-layer agar plate poured with host strain K. pneumoniae (ATCC 10031). All phage spots reveal disruption and clearing of the bacterial lawn, indicating bacterial sensitivity to each respective phage. Control spot, "SM" included only SM buffer and no phage for comparison. Brazilian Bacteriophage Activity Against Clinical K. pneumoniae Results of the spot assays performed with all clinical strain K. pneumoniae were qualitatively described as resistant, sensitive, or intermediate. The majority of tested bacteriophages (25) revealed some disruption of the bacterial lawn. Three bacteriophages (Jc(B), CP, and CB) did not demonstrate activity against any clinical bacterial samples. Phage C14C demonstrated activity against the greatest number of clinical bacteria (14 total intermediate/sensitive). Individual Sensitivity of Clinical Klebsiella pneumoniae (CKP) Samples to Brazilian Phages * Refers to ESBL-positive clinical Klebsiella pneumoniae. Six of the thirty CKP samples were resistant to all 28 bacteriophages. CKP29 was the most sensitive to the tested phages, with either intermediate or complete sensitivity to 17 phages. The majority of CKPs (20) were at least intermediately sensitive to between one and five phages. Conclusion There is potential to isolate phages from highly contaminated wastewaters in Brazil which have the capability to infect and kill distinct strains of K. pneumoniae in the laboratory setting. Continued international collaboration will be imperative in the global fight against antimicrobial resistance. Disclosures All Authors: No reported disclosures

Isolation, Characterization of Enterococcus phages and their application in control of E. faecalis in milk.

Isolation and characterization of Enterococcus phages and application of phage cocktail to control E. faecalis in milk. For phage isolations, double layer agar method was used. Host range of the phages were determined by the spot test. Twelve phages with varying host ranges were isolated. Phages PEF1, PEF7b, and PEF9 with different host ranges and lytic activities were selected for phage cocktails. Compared to two-phages cocktails tested, the cocktail containing all the three phages displayed stronger antibacterial and biofilm removal activities. The cocktail treatment reduced viable E. faecalis in biofilm by 6 log within 6h at both 30 and 4°C. In milk, the cocktail gradually reduced the viable count of E. faecalis and the count reached below the lower limit of detection at 48h at 4°C. The strong bactericidal and biofilm removal activities of the phage cocktail suggests the potential of this cocktail as a natural biocontrol agent for combating E. faecalis in milk. Enterococcus faecalis is an opportunistic pathogen being transmitted from food to humans and causes disease. This study demonstrated the effectiveness of the phage cocktail consisted of new isolated phages in controlling E. faecalis in food.

Isolation and characterization of a novel phage belonging to a new genus against Vibrio parahaemolyticus

BackgroundVibrio parahaemolyticus is a major foodborne pathogen that contaminates aquatic products and causes great economic losses to aquaculture. Because of the emergence of multidrug-resistant V. parahaemolyticus strains, bacteriophages are considered promising agents for their biocontrol as an alternative or supplement to antibiotics. In this study, a lytic vibriophage, vB_VpaM_R16F (R16F), infecting V. parahaemolyticus 1.1997T was isolated, characterized and evaluated for its biocontrol potential.MethodsA vibriophage R16F was isolated from sewage from a seafood market with the double-layer agar method. R16F was studied by transmission electron microscopy, host range, sensitivity of phage particles to chloroform, one-step growth curve and lytic activity. The phage genome was sequenced and in-depth characterized, including phylogenetic and taxonomic analysis.ResultsR16F belongs to the myovirus morphotype and infects V. parahaemolyticus, but not nine other Vibrio spp. As characterized by determining its host range, one-step growth curve, and lytic activity, phage R16F was found to highly effective in lysing host cells with a short latent period (< 10 min) and a small burst size (13 plaque-forming units). R16F has a linear double-stranded DNA with genome size 139,011 bp and a G + C content of 35.21%. Phylogenetic and intergenomic nucleotide sequence similarity analysis revealed that R16F is distinct from currently known vibriophages and belongs to a novel genus. Several genes (e.g., encoding ultraviolet damage endonuclease and endolysin) that may enhance environmental competitiveness were found in the genome of R16F, while no antibiotic resistance- or virulence factor-related gene was detected.ConclusionsIn consideration of its biological and genetic properties, this newly discovered phage R16F belongs to a novel genus and may be a potential alternate biocontrol agent.

Isolation and identification of specific lytic bacteriophages as a biocontrol agent against Serratia odorifera PBA-IAUF-1 and Kluyvera intermedia PBA-IAUF-6 causing bacterial canker in the grape and Siberian pear.

Bacterial canker, a prevalent disease among fruit trees, is a significant concern. The use of phage therapy is presently seen as a dependable biological strategy to control bacterial diseases in fruits. The objective of this research was to use various biochemical and molecular techniques to determine the types of bacteria responsible for causing cankers in various fruits. Additionally, their ability to cause disease in the fruit tissues was assessed, the specific bacteriophages targeting these bacteria were isolated and identified. The bacteria were separated from different parts of the infected fruits like grapes and Siberian pears. The selection of fruit tissues showing signs of canker disease was performed, and the validation of the isolates' pathogenicity was confirmed following Koch's principles. Subsequently, in order to establish a conclusive identification of the bacterial species, molecular identification was conducted through the sequencing of a specific fragment within the 16S rRNA following amplification by PCR by using universal primers, RW01 and DG74. Isolation and titration of phages specific to fruit spoilage bacteria was done by spot and double-layer agar method, and the growth curve of the isolated bacteriophage was drawn. The phages were detected by transmission electron microscopy (TEM). The results of the study proved the presence of canker causing agents, Kluyvera intermedia PBA-IAUF-6 with the code Sh6 in the Siberian pears, and Serratia odorifera PBA-IAUF-1 with the code Rz3 in the grape fruits, which were deposited in GenBank with the accession numbers of KU878579 and KU168605, respectively. Isolation of the specific bacteriophages to the S. odorifera PBA-IAUF-1 and K. intermedia PBA-IAUF-6 bacterial strains were done from the effluent of South Isfahan wastewater treatment plant and Caspian Sea water, respectively. The titer of the specific phage to S. odorifera PBA-IAUF-1 and K. intermedia PBA-IAUF-6 was detected in the samples as 2.2×10-5 and 5×10-11 PFU/ml, respectively. An electron micrograph of a bacteriophage that targets two different bacterial strains revealed phages with a geometrically shaped head and a flexible tail, which resembled viruses from the Siphoviridae family.

Morphological and Genetic Characterization of Eggerthella lenta Bacteriophage PMBT5.

Eggerthella lenta is a common member of the human gut microbiome. We here describe the isolation and characterization of a putative virulent bacteriophage having E. lenta as host. The double-layer agar method for isolating phages was adapted to anaerobic conditions for isolating bacteriophage PMBT5 from sewage on a strictly anaerobic E. lenta strain of intestinal origin. For this, anaerobically grown E. lenta cells were concentrated by centrifugation and used for a 24 h phage enrichment step. Subsequently, this suspension was added to anaerobically prepared top (soft) agar in Hungate tubes and further used in the double-layer agar method. Based on morphological characteristics observed by transmission electron microscopy, phage PMBT5 could be assigned to the Siphoviridae phage family. It showed an isometric head with a flexible, noncontractile tail and a distinct single 45 nm tail fiber under the baseplate. Genome sequencing and assembly resulted in one contig of 30,930 bp and a mol% GC content of 51.3, consisting of 44 predicted protein-encoding genes. Phage-related proteins could be largely identified based on their amino acid sequence, and a comparison with metagenomes in the human virome database showed that the phage genome exhibits similarity to two distantly related phages.

Double-Layer Agar (DLA) Modifications for the First Step of the Phage-Antibiotic Synergy (PAS) Identification

The research carried out so far for phage-antibiotic synergy (PAS) differs as regards the technique of modifying the double-layer agar (DLA) method to show the PAS effect on Petri plates, which may contribute to non-uniform research results. Therefore, there is a need to unify the method to effectively detect the PAS effect, at its most basic in vitro test. In this study, bacteriophage T45 and 43 antibiotics belonging to different antibiotic classes were used. Seven different DLA method modifications were tested, in terms of antibiotic addition placement and presence or absence of the base agar. The overall number of phage plaques per plate mainly depended on the antibiotic used. Differences in plaque quantity depended on the type of the DLA method modification. The largest total number of plaques was obtained by the addition of an antibiotic to a bottom agar with the presence of a top agar. This indicates that even though an antibiotic could manifest the PAS effect by a standard disk method, it would be worth examining if the effect is equally satisfactory when applying antibiotics directly into the agar, with regards to using the same bacteriophage and bacterial host.

Rapid detection of temperate bacteriophage using a simple motility assay.

Phage contamination is a common complication for the fermentation and pharmaceutical industries. The risk of bacteriophage contamination in laboratory processes increases with multiple rounds of genetic manipulation such as deletion and complementation. The contamination of temperate phages does not lead to immediate host cell lysis but could become a serious issue when the lytic cycle is activated under specific conditions. Our objective was to develop a quick and reliable detection method for checking possible temperate phage contamination. Here, using motility plates, we found that when the strain carries a newly acquired temperate phage, its presence can be easily detected by the formation of a clear 'lysis zone' when swimming against the original strain on the same swimming plates. Compared to the traditional double agar layer method and genomic sequencing-based methods, the duration of the motility-based assay is shorter and the procedure is simplified. More importantly, for the bacterial strains that already contain active prophages, this method can still easily detect the newly acquired phages without tedious phage identification procedure. These features make this method highly applicable to laboratory and industrial processes.

Isolation and Characterization of Shiga Toxin Bacteriophages.

Shiga toxin (Stx) phages can be induced from Stx-producing Escherichia coli strains (STEC) or can be isolated as free virions from different samples. Here we describe methods used for the detection, enumeration, and isolation of Stx bacteriophages. Stx phages are temperate phages located in the genome of STEC. Their induction from the host strain cultures is achieved by different inducing agents, mitomycin C being one of the most commonly used. Detection of infectious Stx phages requires the production of visible plaques in a confluent lawn of the host strain using a double agar layer method. However, as the plaques produced by Stx phages are often barely visible and there is a possibility that non-Stx phages can also be induced from the strain, a hybridization step should be added to recognize and properly enumerate the lysis plaques generated after induction. Molecular methods can also be used to identify and enumerate Stx phages. Real-time quantitative PCR (qPCR) is the most accurate method for absolute quantification, although it cannot determine the infectivity of Stx phages. qPCR can also be useful for the detection of free Stx phage virions in different sample types.Stx phages induced from lysogenic bacterial strains can be purified by cesium chloride density gradients; this protocol also helps to specifically discriminate Stx phages from other prophages present in the genome of the host strain by selecting the phages expressing the Stx gene. High titer suspensions of Stx phages obtained after induction of large volumes of bacterial cultures and lysate concentration permits phage characterization by electron microscopy studies and genomic analysis.

The morphological and biological characteristics of a virulent PI phage isolated from slaughterhouse sewage in Shiraz, Iran.

Background and Objectives:Foodborne pathogens are among the serious problems all around the world and thus a novel and natural strategy to control and to inhibit such pathogens is highly demanded nowadays. The aim of this study was to isolate a specific bacteriophage of Escherichia coli O157:H7 from sewage in Fars province, Iran to determine its morphological and antimicrobial activities.Materials and Methods:In order to isolate the bacteriophage of E. coli O157:H7, 10 samples of slaughterhouse wastewaters were used. Double-Layer Agar method was employed to isolate the bacteriophage. To identify the fine structure of the bacteriophage, electron microscope was employed. Host range and antibacterial activity of the phage was also investigated, in vitro.Results:The morphological and biological characteristics of a virulent Siphoviridae phage, PI, are reported. It was found that infection of E. coli O157:H7 strains with this specific bacteriophage produce clear plaques. In the one-step growth analysis, it was confirmed that the phage has been characterized with a very short rise period (around 15 min), an average burst size of 193 PFU/cell, high infectivity and potent lytic action. The bacteriolytic activity of PI was also investigated, in vitro. It was also clarified that at the MOI of 100, 10 and 1, the phage rapidly lysed the bacterial cells within 0.5 or 2 h.Conclusion:These results indicate that the phage PI is a newly discovered phage against E. coli O157:H7 in Iran which may be recommended to use as bio-control purposes.

Isolation and Host Range of Lytic Staphylophages on Clinical Isolates of Methicillin-Resistant Staphylococcus aureus

Background: Staphylococcus aureus (S. aureus) is a major cause of nosocomial infections in humans and animals. Because of the widespread resistance to antibiotics, microbiologists are trying to find other therapeutic interventions such as phage therapy for bacterial infections. Objectives: The present study aimed to isolate staphylophages with lytic effects on methicillin-resistant S. aureus (MRSA) clinical isolates as a potential alternative agent to antibiotic therapy. Methods: This experimental, descriptive study is performed in the Microbiology Laboratory of Shahrekord University (Iran) from September 2018 to March 2019. Two cocktails of staphylophages were isolated from Isfahan (Iran) urban sewage samples. The double-layer agar method was used to detect lytic phages. Morphology characteristic by transmission electron microscopy (TEM) images was used to identify staphylophages. One hundred and thirty three S. aureus were isolated from clinical samples of two teaching hospitals in Isfahan and Shiraz, Iran. Methicillin resistance and the presence of the mecA gene were determined by the disk diffusion method and polymerase chain reaction (PCR) assay, respectively. The phage susceptibility of mecA positive isolates was determined by plaque assay. Results: Two staphylophage cocktails were prepared, which had lytic effects on forty-four MRSA isolates. Cocktails 1 and 2 lysed 19 (14.2%) and 25 (18.7%) isolates, respectively. Of 133 S. aureus isolates, 88.7% carried the mecA gene. Conclusions: Different bacteriophages in two phage cocktails had relatively good lytic effects on S. aureus clinical isolates. Therefore, phage cocktails may be an appropriate alternative to antibiotics against S. aureus.

A newly isolated roseophage represents a distinct member of Siphoviridae family

BackgroundMembers of the Roseobacter lineage are a major group of marine heterotrophic bacteria because of their wide distribution, versatile lifestyles and important biogeochemical roles. Bacteriophages, the most abundant biological entities in the ocean, play important roles in shaping their hosts’ population structures and mediating genetic exchange between hosts. However, our knowledge of roseophages (bacteriophages that infect Roseobacter) is far behind that of their host counterparts, partly reflecting the need to isolate and analyze the phages associated with this ecologically important bacterial clade.MethodsvB_DshS-R4C (R4C), a novel virulent roseophage that infects Dinoroseobacter shibae DFL12T, was isolated with the double-layer agar method. The phage morphology was visualized with transmission electron microscopy. We characterized R4C in-depth with a genomic analysis and investigated the distribution of the R4C genome in different environments with a metagenomic recruitment analysis.ResultsThe double-stranded DNA genome of R4C consists of 36,291 bp with a high GC content of 66.75%. It has 49 genes with low DNA and protein homologies to those of other known phages. Morphological and phylogenetic analyses suggested that R4C is a novel member of the family Siphoviridae and is most closely related to phages in the genus Cronusvirus. However, unlike the Cronusvirus phages, R4C encodes an integrase, implying its ability to establish a lysogenic life cycle. A terminal analysis shows that, like that of λ phage, the R4C genome utilize the ‘cohesive ends’ DNA-packaging mechanism. Significantly, homologues of the R4C genes are more prevalent in coastal areas than in the open ocean.ConclusionsInformation about this newly discovered phage extends our understanding of bacteriophage diversity, evolution, and their roles in different environments.

Bacteriophage Isolation and Characterization: Phages of Escherichia coli.

Here we introduce methods for the detection, enumeration, and isolation of bacteriophages from Escherichia coli. In bacteria, horizontal gene transfer may be mediated by virulent and temperate phages. Strict virulent phages, able to propagate in a suitable strain following the lytic pathway, can be isolated directly from different natural environments. In temperate phages, the lytic cycle must be activated, and phages are detected after their induction. In both cases, detection is based on the production of visible plaques in a confluent lawn of the host strain using a double agar layer method. Further purification and characterization are achieved by density gradients, electron microscopy studies, and genomic analysis. This straightforward methodology can be applied to the detection, enumeration, and isolation of bacteriophages from any bacterial species, using the appropriate host strain, media, and culture conditions.

Genome Analysis of Two Novel Lytic Vibrio maritimus Phages Isolated from the Coastal Surface Seawater of Qingdao, China.

Two novel Vibrio phages, LP.1 and LP.2 that infected Vibrio maritimus R-40493, were isolated from surface seawater in Qingdao coastal area by the double-agar layer method. Morphological analysis by transmission electron microscope showed that the two phages displayed head-tail structures with icosahedral heads of 62.37 and 54.00nm in diameter and long non-contractile tails of 119.00 and 105.20nm in length, respectively, and can be grouped into the Siphoviridae family. Thermal and pH sensitivity tests exhibited that LP.1 was stable at temperature ranging from - 20 to 65°C and at pH ranging from 5 to 12, and LP.2 showed vitality over a wider range of temperature (- 20-75°C) and pH (3-12). Both LP.1 and LP.2 contained linear and double-stranded DNA genomes with a length of 46,791-bp and 37,128-bp, respectively. The genome of both phages can be classified into four functional groups, including DNA replication and regulation, phage packaging, phage structure, and additional function. The bioinformatic analysis demonstrated that the Vibrio phages LP.1 and LP.2 are novel phages. By conducting morphological, biochemical, and genomic analysis, our study provides useful information for further research on the interaction between Vibrio phages and their host.

The study of microbiological purity of ointment “Fitolan” during storage

Topicality. Mastopathy is a benign disease of the mammary glands, and it belongs to a number of hormone-dependent pathologies. The danger of mastopathy is not only in that it significantly reduces the quality of life of a woman, but also in the possibility of its rebirth and masking of the initial stage of breast cancer.Aim. Studies of microbiological purity of the developed semi-solid medicine for epicutaneous application on the basis of phytooils of sea buckthorn, amaranth, parsley and tea tree under the conditional name “Fitolan”.Materials and methods. The testing samples of semi-solid dosage form under the conditional name “Fitolan” was carried out at the Department of Biotechnology of NUPh in accordance with the requirements of the SPhU 2 ed. To test and eliminate the antimicrobial activity of the drug samples, dilutions 1 : 10 of the dosage form were used.Results and discussion. Determination of the microbiological purity of the samples of the medicine “Fitolan” for epicutaneous application using the two-layer seeding method showed that the total number of viable aerobic microorganisms (TAMC) is 20 CFU/g and the total number of yeast and mold fungi (TYMC) does not exceed 10 CFU/g within 27 months of storage, it meets the requirements of SPhU [1].Conclusions. According to the results of microbiological purity research of the studied samples of the semi-solid dosage form “Fitolan” using the double-layer agar method the ointment fully complies with the requirements of SPhU on the indicator “Microbiological purity of non-sterile drugs” within 27 months of storage.

Fast phage detection and quantification: An optical density-based approach.

Since 1959 with the proposal of Double Agar Layer (DAL) method for phage detection and quantification, many sophisticated methods have emerged meanwhile. However, many of them are either too complex/expensive or insensitive to replace routine utilization of DAL method in clinical, environmental and industrial environments. For that purpose, we have explored an alternative method for the detection and quantification of bacteriophages that fulfills the criteria of being rapid, simple and inexpensive. In this paper we have developed a method based on the analysis of optical density kinetics in bacterial cultures exposed to phage-containing samples. Although the decrease in optical density caused by cell lysis was one of the first observable consequences of the effect of viral infection in bacterial cultures, the potential of the method for the assessment of phage abundance has never been fully exploited. In this work we carry out a detailed study of optical density kinetics in phage-infected bacterial cultures, as a function of both, phage abundance and initial concentration of the host organisms. In total, 90 different combinations of bacteria/phage concentrations have been used. The data obtained provide valuable information about sensitivity ranges, duration of the assay, percentages of inhibition and type of lysing behavior for each phage concentration. The method described can detect, as few as 10 phage particles per assay volume after a phage incubation period of 3.5h. The duration of the assay can be shortened to 45min at the expense of losing sensitivity and increasing the limit of detection to 108 pfu/ml. Despite using non-sophisticated technology, the method described has shown sensitivity and response time comparable to other high-end methods. The simplicity of the technology and of the analytical steps involved, make the system susceptible of miniaturization and automation for high-throughput applications which can be implemented in routine analysis in many environments.

Specification of Bacteriophage Isolated Against Clinical Methicillin-Resistant Staphylococcus Aureus.

ObjectivesThe emergence of resistant bacteria is being increasingly reported around the world, potentially threatening millions of lives. Amongst resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is the most challenging to treat. This is due to emergent MRSA strains and less effective traditional antibiotic therapies to Staphylococcal infections. The use of bacteriophages (phages) against MRSA is a new, potential alternate therapy. In this study, morphology, genetic and protein structure of lytic phages against MRSA have been analysed.MethodsIsolation of livestock and sewage bacteriophages were performed using 0.4 μm membrane filters. Plaque assays were used to determine phage quantification by double layer agar method. Pure plaques were then amplified for further characterization. Sulfate-polyacrylamide gel electrophoresis and random amplification of polymorphic DNA were run for protein evaluation, and genotyping respectively. Transmission electron microscope was also used to detect the structure and taxonomic classification of phage visually.ResultsHead and tail morphology of bacteriophages against MRSA were identified by transmission electron microscopy and assigned to the Siphoviridae family and the Caudovirales order.ConclusionBacteriophages are the most abundant microorganism on Earth and coexist with the bacterial population. They can destroy bacterial cells successfully and effectively. They cannot enter mammalian cells which saves the eukaryotic cells from lytic phage activity. In conclusion, phage therapy may have many potential applications in microbiology and human medicine with no side effect on eukaryotic cells.