Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae

ONCE MORE ABOUT PHAGE THERAPY: WHAT IS RIGHT, AND WHAT IS NOT

A little more than 100 years ago the article “An investigation on the nature of ultra-microscopic viruses” by the English physician and researcher F. Twort was published in The Lancet. Twort described the phenomenon of transparent spots which occurred on the bacterial lawn when he worked with bacterial cultures using a petri dish. He examined the spots and found zones of dead bacteria. The spots were transmissible: they appeared on other dishes once transferred to a new lawn. Twort, failing to give a convincing explanation for this phenomenon, proposed three hypotheses:  it could be an unusual manifestation of the bacterial life cycle, or an enzyme produced by the bacteria themselves or some sort of “ultra-microscopic virus” which could cause the death of bacteria. Having voiced his hypotheses, Tvort let the matter drop, but many believe that with the article began the era of bacteriophages

THE STUDY THE EFFICACY AND SAFETY OF ANTIMICROBIAL AGENTS

Abstract:Effective treatment of patients with infectious and inflammatory diseases of the skin and mucous membranes often involves the use of antimicrobial agents.The purpose of the study was an in vitro estimation of cytotoxicity and the efficiency of national resources for local use: gel with bacteriophages («Otofag», «Fagogin», «Fagoderm», «Fagodent») and antiseptic — «Сhlorhexidine» and «Miramistin».Materials and Methods. To study the effectiveness of antimicrobial agents they used to provide crop strains of Staphylococcus aureus and Streptococcus pyogenes as one of the most common representatives of pathogens. The study of cell viability and cytotoxicity antimicrobials performed on cell lines KB — epidermoid carcinoma of the oral cavity of a human. For this purpose we use mikrotetrazoly test, which is widely used in the assessment of the effects on the cells of toxins, pharmaceuticals, adverse environmental factors, allowing to evaluate the toxicity of investigational drugs in vitro.The results showed that the efficacy against pathogens Staphylococcus aureus and Streptococcus pyogenes, has even a 10‑fold dilution of «Сhlorhexidine» 0.05% and gels with bacteriophages. Antiseptic «Miramistin» is effective only on the initial concentration. The study of cytotoxicity showed that the processing of epidermoid carcinoma cells with «Chlorhexidine» and «Мiramistin» invokes the irreversible reactions, while the composition processing of gels based on bacteriophages not further affect cell viability.Conclusions The results of the experiment confirmed the significant toxicity of tools such as «Сhlorhexidine» and «Miramistin» in proposed concentrations in the pharmacy network. Despite the high efficiency of these vehicles with regard to the studied pathogens, their long-term use in treatment of inflammatory diseases of the skin and mucous membranes can cause a slowing of repair processes. Gel means with bacteriophages «Fagodent» «Otofag» «Fagogin» and «Fagoderm» are highly effective and have no toxic effects on the cells. In this regard, they can be an effective and safe alternative to the popular antiseptic

STUDY OF FEATURES OF MICROFLORA OF PATIENTS WITH DISEASES OF THE ORAL MUCOSA. EVALUATION OF THE EFFICACY OF PRODUCTS BASED ON BACTERIOPHAGES "FAGODENT" IN THE TREATMENT OF CHRONIC APHTHOUS STOMA TITIS AND LICHEN PLANUS

Effective complex treatment of patients with pathology of the oral mucosa (OM) includes drug effects on pathogenic bacteria as a possible etiological factor in the development of these diseases.Purpose. Study of characteristics of oral microflora of patients with diseases of the OM, the efficiency of the national preventive agent for topical application "Fagodent" in the form of a gel based on bacteriophages in the treatment of chronic recurrent aphthous stomatitis and lichen planus.Materials and methods. In a pilot study (up to 3 months follow-up) they included 27 patients aged 19 to 80 years old with chronic recurrent aphthous stomatitis (CRAS) and lichen planus (LP). To monitor the efficiency of in vitro we carried out spot-testing for bacterial lawn.Results. Results showed the frequent detection of bacteria of the staphylococci and streptococci in smears obtained from the elements of defeat in the CRAS and LP, while the PCRdiagnostics detect the presence of rare parodontopatogenov on aftah and papules. The study of the effectiveness of products based on bacteriophages "Fagodent» in vitro showed a high sensitivity of the pathogens to bacteriophages gel "Fagodent" when exposed to cultures derived from patients with CRAS. The use of prophylactic agents based on bacteriophages in the treatment of chronic recurrent aphthous stomatitis significantly reduced pain, accelerated the relief of the inflammatory process to reduce the time of repair. Application means "Fagodent" in the complex treatment of lichen planus is not significantly affected the course of the disease.Conclusion. The laboratory research and clinical observations confirm the effectiveness and appropriateness of products based on bacteriophages "Fagodent" in the treatment of chronic aphthosous stomatitis

The Role of Moderate and Virulent Endogenous Bacteriophages in the Treatment of Pyoinflammatory Infections in Intensive Care Unit Patients

Abstract. In connection with the growth of resistance of pathogens of pyoinflammatory infections (PII) to antibiotics, physicians began to use bacteriophages, which are widespread where there are homologous bacteria. They are also found in the human body, possibly protecting against PII. It was found that mortality in patients with bacteriophages was lower than in patients without homologous endogenous bacteriophages. The most common were mild bacteriophages, which “protected” patients from infection, although it is believed that only virulent bacteriophages may do this.Aim of study. To study the effect of virulent and moderate endogenous bacteriophages on the course of pyoinflammatory infections in intensive care units (ICU) patients.Material and methods. The study included 33 patients with positive blood culture who were treated in the ICU. Of these, 12 (36.4%) had endogenous bacteriophages (10 men, 2 women).We isolated 16 strains of various bacteria. Bacteriological blood tests were performed using an automatic blood culture analyzer Bactec-9050. Identification of isolated microorganisms was carried out using an automatic microbiological analyzer WalkAway 40. Traditional virological methods on the basis of LLC SPC MicroMir were used for work with bacteriophages.Results and discussion. From 16 positive blood cultures, the following were isolated: Klebsiella pneumoniaе — in 7 samples, Acinetobacter spр. — in 4 samples, Staphylococcus spp. — in 2 samples, and one strain in each sample: Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis. Endogenous phages were isolated in 36.4% of patients, which were detected in all blood cultures. The exception was K. pneumoniae strains, for which in 42.9% of cases (3 cases out of 7) bacteriophages were absent.Out of 12 patients with isolated bacteriophages, 6 had positive treatment outcomes.Of 14 blood samples, 11 moderate bacteriophages were isolated, which amounted to 78.6%, virulent — 21.4%. Despite this, the protective effect of bacteriophages was noted.Conclusion. 1. All blood cultures contained homologous bacteriophages, except K. pneumoniaе strains, which had no endogenous bacteriophages in 42.9% of cases. 2. Most of the isolated endogenous bacteriophages (78.6%) were moderate. 3. In the absence of homologous bacteriophages in the blood of ICU patients, the likelihood of death increases (66.7%) compared to patients with bacteriophages (33.3%)

Роль умеренных и вирулентных эндогенных бактериофагов в лечении гнойно-воспалительных инфекций у пациентов отделений реанимации и интенсивной терапии

Abstract. In connection with the growth of resistance of pathogens of pyoinflammatory infections (PII) to antibiotics, physicians began to use bacteriophages, which are widespread where there are homologous bacteria. They are also found in the human body, possibly protecting against PII. It was found that mortality in patients with bacteriophages was lower than in patients without homologous endogenous bacteriophages. The most common were mild bacteriophages, which “protected” patients from infection, although it is believed that only virulent bacteriophages may do this.Aim of study. To study the effect of virulent and moderate endogenous bacteriophages on the course of pyoinflammatory infections in intensive care units (ICU) patients.Material and methods. The study included 33 patients with positive blood culture who were treated in the ICU. Of these, 12 (36.4%) had endogenous bacteriophages (10 men, 2 women).We isolated 16 strains of various bacteria. Bacteriological blood tests were performed using an automatic blood culture analyzer Bactec-9050. Identification of isolated microorganisms was carried out using an automatic microbiological analyzer WalkAway 40. Traditional virological methods on the basis of LLC SPC MicroMir were used for work with bacteriophages.Results and discussion. From 16 positive blood cultures, the following were isolated: Klebsiella pneumoniaе — in 7 samples, Acinetobacter spр. — in 4 samples, Staphylococcus spp. — in 2 samples, and one strain in each sample: Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis. Endogenous phages were isolated in 36.4% of patients, which were detected in all blood cultures. The exception was K. pneumoniae strains, for which in 42.9% of cases (3 cases out of 7) bacteriophages were absent.Out of 12 patients with isolated bacteriophages, 6 had positive treatment outcomes.Of 14 blood samples, 11 moderate bacteriophages were isolated, which amounted to 78.6%, virulent — 21.4%. Despite this, the protective effect of bacteriophages was noted.Conclusion. 1. All blood cultures contained homologous bacteriophages, except K. pneumoniaе strains, which had no endogenous bacteriophages in 42.9% of cases. 2. Most of the isolated endogenous bacteriophages (78.6%) were moderate. 3. In the absence of homologous bacteriophages in the blood of ICU patients, the likelihood of death increases (66.7%) compared to patients with bacteriophages (33.3%).Введение. В связи с развитием устойчивости возбудителей гнойно-воспалительных инфекций (ГВИ) к антибиотикам к лечению стали подключать бактериофаги, которые широко распространены в природе там, где есть гомологичные к ним бактерии. Они находятся и в организме человека, возможно, защищая от ГВИ. Установлено, что летальность у пациентов, имевших бактериофаги, была ниже, чем у пациентов без гомологичных эндогенных бактериофагов. Наиболее часто в биоматериалах (и в моче, и в крови) встречались умеренные бактериофаги, которые «защищали» пациентов от инфекции, несмотря на то, что считается, что эта роль присуща только вирулентным бактериофагам.Цель работы. Изучение влияния вирулентных и умеренных эндогенных бактериофагов на течение гнойно-воспалительных инфекций у пациентов отделений реанимации и интенсивной терапии (ОРИТ).Материал и методы. В исследование включены 33 пациента, находившиеся на лечении в ОРИТ, с положительной гемокультурой. Из них у 12 (36,4%) были выявлены эндогенные бактериофаги (мужчин — 10, женщин — 2). Из образцов выделено 16 гемокультур различных бактерий.Бактериологическое исследование крови проводили с помощью автоматического анализатора гемокультур Bactec-9050. Идентификацию выделенных микроорганизмов выполняли с использованием автоматического микробиологического анализатора WalkAway 40. Работу с бактериофагами выполняли традиционными вирусологическими методами на базе ООО НПЦ «МикроМир» .Результаты и обсуждение. Из 16 положительных гемокультур выделены: Klebsella pneumoniaе — в 7 пробах, Acinetobacter spр.— в 4, Staphylococcus spр. — в 2 и по одному штамму в каждой пробе: Pseudomonas aeruginosa, Staphylococcus aureus и Enterococcus faecalis.У 12 пациентов (36,4%) были выделены эндогенные фаги, которые были обнаружены ко всем гемокультурам. Исключение составили штаммы K. pneumoniaе, у которых в 42,9% наблюдений (3 случая из 7) бактериофаги отсутствовали.Из 14 проб крови выделено 11 умеренных бактериофагов, что составило 78,6%, вирулентных 21,4%. Несмотря на это, отмечено защитное действие бактериофагов.Выводы:Все гемокультуры содержали гомологичные к ним бактериофаги, за исключением штаммов K. pneumoniaе, у которых в 42,9% случаев эндогенные бактериофаги отсутствовали.Большинство выделенных эндогенных бактериофагов (78,6%) были умеренными.При отсутствии гомологичных бактериофагов в крови у пациентов отделения реанимации и интенсивной терапии повышается вероятность смертельного исхода (66,7%) по сравнению с исходами у пациентов, имеющих бактериофаги (33,3%)