Objective. To identify bacterial interactions at the site of infection in cystic fibrosis patients and to assess their possible effects on the course of infection. Materials and Methods. The following strains were used in this study: Alcaligenes faecalis LGBP strain, isolated from the environment; clinical isolates of Pseudomonas aeruginosa; Achromobacter xylosoxidans, Acinetobacter baumannii, Alcaligenes faecalis, and Bacillus subtilis strains; the standard laboratory P. aeruginosa PAO1 strain and its lysogens by temperate bacteriophages of various species, and its phageresistant mutants. Imaging and evaluation of the effects of bacterial interaction was performed in an in vitro co-infection with A. faecalis LGBP and the tested strains. Results. The bacteria of A. faecalis which are often involved in the lung co-infection in cystic fibrosis have been shown to stimulate the growth of most of the tested P. aeruginosa strains, as well as bacteria of some other species (for example, B. subtilis). The interspecies interactions pattern depends primarily on the strain of A. faecalis and physiological features of the infecting P. aeruginosa strains. When growing concurrently, the contacts between bacteria may change both the physical properties of the contacting bacteria surface (propagation rate) and the course of biochemical reactions in the contacting bacteria (occurrence of pigmentation, change in auto-plaquing pattern, reduction in alginate production). Conclusions. The results suggest that visually recognizable interactions are similar to the interactions of A. faecalis LGBP, exhibited in vitro with clinical isolates of P. aeruginosa, may influence on the course of chronic infections and their treatment results. Expanding of model studies of bacterial interspecies interactions may contribute to better understanding of their molecular mechanism that may be useful for optimizing therapy

Careful selection of bacteriophages for phage therapy is needed to avoid undesirable consequences. Different approaches to phage therapy are compared: from the use of multispecies industrially produced phage mixtures with wide range of antibacterial activity to the 'magistral phage' approach in which bacteriophages are selected for treating individual patients.

AIM:Evaluate antibacterial activity of an experimental mixture of phages, belonging to several well-studied species.MATERIALS AND METHODS:The study was carried out using a group of 55 clinical Pseudomonas aeruginosa strains of various origins,- 4 mono-species mixtures of 32 virulent bacteriophages (species phiKZ-, phiKMV-, phiPBl-, PaP3-like phages) and 2 novel phages, phiMK (species PaK-P2) and phiPerm5. Activity of preparations from mono-species mixtures of bacteriophages ofvarious species were compared with activity of 3 commercial mixtures. Standard methods of study of bacteriophages were used: determination of lytic activity by seeding onto bacterial lawns of P. aeruginosa, restriction analysis of phage DNA for confirmation of their be- longing to certain species.RESULTS:Cumulative activity of 6 mono-species mixtures of virulent phages was shown to be similar to lytic activity of commercial therapeutic mixtures used against P. aeruginosa infections. 54 of 55 strains of clinical isolates of P: aeruginosa showed sensitivity to experimental mixtures composed of mono-species mixtures of bacteriophages. 53 strains were lysed by commercial preparations. Wherein the possibility of accidental inclusion of moderate -bacteriophages in the experimental mixture is excluded.CONCLUSION:A possibility of creation of highly active therapeutic antibacterial preparations against P. aeruginosa using mono-species mixtures of 6 species of lytic bacteriophages is shown Use of such a mixture in therapy of lung infections reduces the risk of emergence of bacterial strains with increased virulence and patho- genicity during prolonged administration.

Aim. Evaluate antibacterial activity of an experimental mixture of phages, belonging to several well-studied species. Materials and methods. The study was carried out using a group of 55 clinical Pseudomonas aeruginosa strains of various origins, 4 mono-species mixtures of 32 virulent bacteriophages (species phiKZ-, phiKMV-, phiPBl-, РаРЗ-like phages) and 2 novel phages, phiMK (species PaK-P2) and phiPerm5. Activity of preparations from mono-species mixtures of bacteriophages of various species were compared with activity of 3 commercial mixtures. Standard methods of study of bacteriophages were used: determination of lytic activity by seeding onto bacterial lawns of P. aeruginosa, restriction analysis of phage DNA for confirmation of their belonging to certain species. Results. Cumulative activity of 6 mono-species mixtures of virulent phages was shown to be similar to lytic activity of commercial therapeutic mixtures used against P. aeruginosa infections. 54 of 55 strains of clinical isolates of P. aeruginosa showed sensitivity to experimental mixtures composed of mono-species mixtures of bacteriophages. 53 strains were lysed by commercial preparations. Wherein the possibility of accidental inclusion of moderate bacteriophages in the experimental mixture is excluded. Conclusion. A possibility of creation of highly active therapeutic antibacterial preparations against P. aeruginosa using mono-species mixtures of 6 species of lytic bacteriophages is shown. Use of such a mixture in therapy of lung infections reduces the risk of emergence of bacterial strains with increased virulence and pathogenicity during prolonged administration.

Приведены результаты изучения нового вирулентного фага phiPMG1, активного на бактериях вида Pseudomonas aeruginosa. Показано, что phiPMG1 проявляет значительную гомологию и сходство в общей структуре генома с умеренным конвертирующим фагом D3. Фаг phiPMG1 отличается от D3 тем, что не способен осуществлять стабильную лизогенизацию и растет на штаммах с плазмидами, подавляющими развитие фага D3 и некоторых других фагов. Это понижает вероятность горизонтального генетического переноса фагом phiPMG и делает возможным его использование в фаготерапии. Сравнение структуры геномов фагов phiPMG1 и D3 выявило не только значительную гомологию 65 генов, но и присутствие в геноме phiPMG1 16 генов, не представленных в базе данных NCBI. Мы предполагаем, что эволюция геномов фагов этого вида в значительной степени связана с миграциями в другие виды бактерий и рекомбинациями с фагами других видов (например, F116). Подробный анализ структуры одной из областей геномов, в которой проявляется существенная негомология у трех D3-подобных фагов (D3, phiPMG1 и PAJU2), показал, что возможно геном phiPMG1 ближе других к геному гипотетического предкового фага этого вида.

Results of studying the novel virulent phage phiPMG1 active on Pseudomonas aeruginosa are presented. It is shown that phiPMG1 exhibits significant homology and the similarity in the overall structure with the genome of a temperate phage converts D3. Phage phiPMG1 differs from D3 in that it fails to stably lysogenize bacteria and can grow on strains carrying plasmids that cause growth inhibition of phage D3 and some other phages. This significantly diminishes the probability of horizontal gene transfer with phage phiPMG1 and suggests the possible employment of this phage in phage therapy. A comparison of phages phiPMG1 and D3 structures of genomes in demonstrated not only high homology of 65 genes, but also the presence of 16 genes in the phiPMG1 genome that were not included in the in NCBI database. Apparently, the evolution of genomes in phages of this species is mostly associated with migrations into other species of bacteria, and recombinations with phages of other species (for example, F116). A detailed analysis of structure of one region genomes, which significant nonhomology for the three D3-like phages (D3, phiPMG1 and PAJU2), revealed that the phiPMG1 genome possible closest to a hypothetical genome of ancestral phage of this species.

Продолжено изучение явления псевдолизогении у бактерий Pseudomonas aeruginosa, инфицированных phiKZ-подобными фагами вида EL. Проведен анализ группы вновь изолированных vir-мутантов фагов вида EL (EL и RU), проявляющих вирулентность (способность лизировать бактерии, зараженные фагом дикого типа) и пониженный уровень опалесценции негативных колоний (НК). Выявление рекомбинантов дикого типа в скрещиваниях vir-мутантов фагов EL и RU подтверждает полигенный контроль этого признака. Делеционный характер мутации у одного из vir-мутантов фага EL, а также высокий уровень генетической нестабильности другого мутанта предполагают возможное участие в мутагенезе мобильного генетического элемента. Возникновение в бактерии псевдолизогенного состояния создает условия для горизонтального переноса генов между разными штаммами бактерий. Таким образом, только секвенирования геномов фагов и доказательства отсутствия у них генопродуктов с токсическим эффектом оказывается недостаточным для включения фагов в лечебные смеси. Обязательным условием использования живых фагов является детальное изучение возможных результатов их взаимодействия с бактериальным хозяином.

Приведены результаты исследования генома умеренного ореолобразующего бактериофага Pseudomonas aeruginosa phi297 и литической активности его вирулентного мутанта. Определение полной последовательности генома этого бактериофага обнаружило его мозаичную структуру. Часть последовательности генома phi297 гомологична последовательности генома фагов D3 и F116. Особенности морфологии негативных колоний (НК) умеренного бактериофага P. aeruginosa phi297 позволили предположить, что он обладает высокой литической активностью. Был выделен вирулентный мутант фага phi297vir, способный лизировать бактерии, лизогенные по фагу дикого типа. Сравнение литической активности phi297vir с литической активностью фагов из коммерческих смесей двух производителей (предприятий Нижнего Новгорода и Перми) показало, что phi297vir способен лизировать мутанты бактерий РАО1, устойчивые к фагам из коммерческих препаратов, но спектр литической активности phi297vir уже, чем у фагов коммерческих препаратов. Проведенное исследование показало, что применение неревертирующих вирулентных мутантов некоторых умеренных бактериофагов может быть целесообразно в лечении инфекций, вызванных P. aeruginosa.

Выделенный бактериофаг Pseudomonas aeruginosa имеет отличительные особенности в литическом инфекционном цикле и образует ореол (зону просветления) вокруг негативных колоний. Фаг был изучен с точки зрения потенциального использования в терапевтических фаговых препаратах и как источник пептидогликан- и липополисахариддеградирующих ферментов. Частичное секвенирование генома показало высокую степень его гомологии с известным умеренным бактериофагом D3. В геноме была идентифицирована открытая рамка считывания, кодирующая литическую трансгликозилазу. Этот фермент был получен в рекомбинантном виде, изучены его активность и субстратная специфичность.

The properties of the isolated Pseudomonas aeruginosa bacteriophage phiPMG1 include the lytic infection cycle, and the formation of a broad halo (semi-transparent zone) around the plaques. We consider phiPMG1 as a potential member of therapeutic cocktails of live phages, and as a source of peptidoglycan and lipopolysaccharide degrading enzymes. Partial sequencing of phiPMG1 genome has revealed high similarity with known temperate P. aeruginosa phage D3. An open reading frame encoding lytic transglycosilase was identified in the genome. This enzyme PMG MUR was obtained in recombinant form, and its activity and substrate specificity has been studied.

The genome of halo-forming temperate Pseudomonas aeruginosa phage phi297 and lytic activity of its virulent mutant were studied. A mosaic structure was revealed for phi297 genome by its complete sequencing. The phi297 genome was partly homologous to the genomes of phages D3 and F116. High lytic activity was assumed for temperate P. aeruginosa bacteriophage phi297 on the basis of morphological features of negative colonies. Virulent mutant phi297vir, which was capable of lysing the wild-type phage bacteria, was isolated. Lytic activity was compared for phi297 and the phages from commercial mixtures of two manufacturers (facilities of Nizhnii Novgorod and Perm’). Phage phi297 caused lysis of the mutant PAO1 bacteria that were resistant to the phages from commercial preparations, but the lytic activity spectrum of phi297 was narrower that the spectra of the commercial phages. The use of nonreverting virulent mutants of certain temperate bacteriophages was proposed for the treatment of P. aeruginosa infections.

The article continues a study of pseudolysogeny in Pseudominas aeruginosa infected with phiKZ-like phages of the EL species. Analysis was performed for several newly isolated vir mutants of EL phages (EL and RU) that were virulent (capable of causing lysis of bacteria infected with the wild-type phage) and a lower extent of opalescence of negative colonies (NCs). Wile-type recombinants were detected in crosses of virulent mutants of phages EL and RU to confirm the polygenic control of virulence. Since a deletion mutation was found in one of the virulent EL mutants and high genetic instability was characteristic of another mutant, a mobile genetic element was assumed to play a role in mutagenesis. Pseudolysogeny of bacteria provides for horizontal gene transfer between different bacterial strains. Hence, sequencing of the phage genome and demonstration of the lack of toxic gene products are insufficient for the phage to be included into a therapeutic mixture. To use live phages, it is essential to study in detail the possible consequences of their interaction with host bacteria.

The ϕPMG1 Pseudomonas aeruginosa bacteriophage was isolated. It is characterized by certain peculiarities of the lytic infection cycle and forms a halo (clear zone) around negative colonies. The phage was studied with regard to its potential use in therapeutic phage preparations and as a source of peptidoglycan- and lipopolysacchraide-degrading enzymes. Partial sequencing of the ϕPMG1 genome revealed a high degree of homology with the D3 moderate bacteriophage. An open reading frame coding for a lytic transglycosylase has been identified in ϕPMG1 genome. The enzyme has been obtained in a recombinant form, and its activity and substrate specificity have been characterized.

The properties of new virulent bacteriophage TL of Pseudomonas aeruginosa belonging to the family Podoviridae (genome size of 46 kb) were investigated. This bacteriophage is capable of lysing the bacterial lawn in halo zones around negative colonies (NC) of other bacteriophages. TL forms large NC, that are hardly distinguishable on the lawn of P. aeruginisa PAO1. At the same time, on the lawns of some phage-resistant PAO1 mutants, as well as on those produced by a number of clinical isolates, TL forms more transparent NC. It is suggested that more effective growth of the bacteriophage TL NC is associated with the differences in outer lipopolysaccharide (LPS) layer of the cell walls of different bacterial strains, as well as of the bacteria inside and outside of the halos. This TL property was used to optimize selection of bacteriophages producing halos around NC on the lawn of P. aeruginosa PAO1. As a result, a group of bacteriophages differing in the patterns of interaction between their halos and TL bacteriophage, as well as in some characters was identified. Taking into consideration the importance of cell-surfaced structures of P. aeruginosa in manifestation of virulence and pathogenicity, possible utilization of specific phage enzymes, polysacchadide depolymerases, for more effective treatment of P. aeruginosa infections is discussed.

ABSTRACT We report the selection and amplification of the broad-host-range Salmonella phage phi PVP-SE1 in an alternative nonpathogenic host. The lytic spectrum and the phage DNA restriction profile were not modified upon replication in Escherichia coli Bl21, suggesting the possibility of producing this phage in a nonpathogenic host, contributing to the safety and easier approval of a product based on this Salmonella biocontrol agent.

В настоящей работе впервые получено окончательное доказательство возможности перехода бактерий P. aeruginosa в псевдолизогенное состояние после инфекции phiKZ-подобными фагами. Показано, что решающим фактором в этом процессе является множественное заражение бактерий бактериофагами этого рода. В ходе работы были обнаружены стабильные клинические изоляты бактерий, выделяющие новые бактериофаги этого рода (Che2/2 и Che21/5), отнесенные по фенотипическим признакам и результатам анализа ДНК к видам phiKZ и EL, соответственно. Для трех бактериофагов вида EL – EL, RU и Che21/5 удалось выделить мутанты с нарушенной способностью к псевдолизогенизации. Один из мутантов фага EL имеет свойства вирулентных мутантов типичных умеренных фагов (“vir” – мутант). Он не образует псевдолизогенов и, более того, проявляет эффект доминантности при коинфекции бактерий с фагом дикого типа EL и не проявляет его при совместной инфекции бактерий с фагами дикого типа видов phiKZ и Lin68. Предполагается, что эффект псевдолизогении может быть связан с функционированием генов фагов phiKZ и EL, контролирующих продукты, сходные с репрессорами других фагов. Учитывая обнаруженное нами ранее присутствие в коммерческих фаготерапевтических препаратах phiKZ-подобных фагов дикого типа, что может создавать определенные проблемы, представляется целесообразным использовать вместо фагов дикого типа этого рода их вирулентные мутанты.

In this work, a final piece of evidence proving that bacteria Pseudomonas aeruginosa are capable of transition to the pseudolysogenic state after infection with φKZ-like phages has been produced. It was shown that the decisive factor in this process is multiple infection of bacteria with bacteriophages belonging to this genus. In the course of this work, stable clinical isolates of bacteria liberating novel bacteriophages of this genus (Che2/2 and Che21/5) were detected and attributed to species φKZ and EL, respectively, according to their phenotypic characters and the results of DNA analysis. For three bacteriophages belonging to species EL (EL, RU, and Che21/5), mutants with disorders in the capability for pseudolysogenization were isolated. One of the mutants of phage EL possesses properties of virulent mutants of typical temperate phages (vir mutant). This mutant fails to form pseudolysogens and, moreover, provides the effect of dominance upon coinfection of bacteria with the wild-type phage EL, but however is unable to exhibit this effect upon joint infection of bacteria with wild-type phages of species φKZ and Lin68. It is assumed that the effect of pseudolysogeny may be connected with functioning of φKZ and EL genes that control the products similar to repressors of other phages. Because earlier wild-type φKZ-like phages were shown to be present in commercial phage-therapeutic preparations (which represents certain problems), it is expedient to use virulent mutants of phages belonging to this genus rather than phages of the wild type.

The influence of infection of natural isolates of Salmonella enterica with lytic (nonlysogenic) phages on the expression of resident cryptic or defective prophages in host bacteria was studied. The induction of defective/cryptic phages after infection with nonlysogenic phages and packaging of bacterial chromosomal fragments in capsids of defective phages is demonstrated. This may lead to migration and wide distribution of both the genomes of defective phages per se and various fragments of the bacterial chromosome (including pathogenic islands) in new bacterial strains with concomitant change of their properties, the acquired new features of pathogenicity among them.

Some properties of bacteriophages with large (200 kb and more) sequenced genomes have been compared. In contrast to other large bacteriophages from different families, bacteriophages active on pseudomonads of various species (phiKZ-like bacterio phages) have some common features, which suggests their phylogenetic relationship and independence of their evolution as a result of migration among bacteria of this family. Among such common features are the absence in the genomes of these phages of sites sensitive to endonuclease PstI, the absence of genes encoding DNA polymerases that are similar to the known enzymes of this type, possible dependence of replication of the phage genome on bacterial DNA polymerase, and a considerably larger average gene size as compared to that for other phages. Criteria are suggested for searching for novel phiKZ-like bacteriophages: the size of a phag e particle, production by bacteria infected with such phages of a large amount of highly viscous mucus. Taking into account the use of these bacteriophages in therapeutic preparations (due to a broad spectrum of lytic activity) and a poor knowledge of a majority of their gene products, it seems necessary to perform a more comprehensive genetic analysis of phages of this genus or their mutants for selecting those adequate for phage therapy.