Genome sequence of the broad-host-range Pseudomonas phage Phi-S1

The broad-host-range lytic Pseudomonas phage Φ-S1 possess a 40,192 bp double-stranded DNA (dsDNA) genome of 47 open reading frames (ORFs) and belongs to the family Podoviridae, subfamily Autographivirinae, genus T7likevirus.S.S. acknowledges the financial support from the Portuguese Foundation for Science and Technology (FCT) (grant SFRH/BPD/48803/2008) and the FCT project PTDC/EBB-BIO/114760/2009

The use of bacteriophages to control biofilms

Este resumo faz parte de: Book of abstracts of the Meeting of the Institute for Biotechnology and Bioengineering, 2, Braga, Portugal, 2010. A versão completa do livro de atas está disponível em: http://hdl.handle.net/1822/1096

Utilização de bacteriófagos no controlo de células suspensas e biofilmes de Pseudomonas fluorescens

Os ambientes industriais são propícios à formação de biofilmes devido à presença de microrganismos e às condições favoráveis para o seu desenvolvimento. Os biofilmes são responsáveis por perdas económicas elevadas e alterações da qualidade dos produtos industriais. Devido a estes factores têm surgido diversos estudos com o intuito de se obter a melhor metodologia para a sua eliminação. A maior parte destes estudos baseia-se na utilização de agentes químicos que causam impactos ambientais adversos devido à sua toxicidade e para além disso têm demonstrado fraca eficiência na remoção de biofilmes das superfícies industriais. Assim, é fundamental investigar agentes alternativos para a erradicação de biofilmes. Neste trabalho estudou-se a utilização de vírus bacterianos, os bacteriófagos (fagos), na eliminação de células de Pseudomonas fluorescens. Os fagos são ubíquos na natureza, bastantes específicos e não tóxicos para o Homem e animais. Esta dissertação encontra-se dividida em diferentes capítulos. Após uma breve introdução teórica sobre fagos e biofilmes (Capítulo 1), encontram-se descritos, no Capítulo 2, todos os métodos desenvolvidos e utilizados neste trabalho. Seguem-se os capítulos referentes aos estudos de infecção fágica realizados com células suspensas (Capítulo 3), biofilmes (Capítulo 4) e células aderidas a superfícies (Capítulo 5). A finalizar encontram-se as conclusões e propostas para trabalho futuro (Capítulo 6). De acordo com os resultados deste trabalho, os fagos, sob condições óptimas e na presença de hospedeiros com qualidade (crescidos à temperatura óptima e meio de crescimento óptimo) são bastante eficientes na remoção de biofilmes no estado inicial de formação (células aderidas a superfícies) e biofilmes maduros, tendo-se obtido percentagens de remoção acima dos 80%. No entanto, verificou-se que a infecção fágica é condicionada por diversos factores, tais como a temperatura, fase e meio de crescimento do hospedeiro, meio de infecção e concentração de fago. Este facto poderá limitar a aplicabilidade destes agentes biológicos no controlo bacteriano em ambientes industriais devido à diversidade das condições ambientais.In industrial environments the development of biofilms is favorable due to the presence of microorganisms and the existing conditions. These biofilms are responsible for severe economic losses and alterations of the industrial products quality. Due to these factors, several studies have been made in order to develop a good methodology for their removal. The majority of these studies involve the use of chemical agents that are responsible for negative environmental impacts due to their toxicity and have poor biofilm removal action. Therefore, it is of utmost importance the investigation of alternative strategies to eradicate biofilms. In this study, bacteriophages (phages) were used to eliminate Pseudomonas fluorescens cells. Phages are ubiquitous in nature, highly specific and non-toxic for humans and animals. This Thesis is divided in different chapters. After a brief introduction to the phage subject, the methods developed and used in this work are described (Chapter 2). This follows of the chapters considering phage infection of planktonic cells (Chapter 3), biofilms (Chapter 4) and biofilms in the early stage of formation (Chapter 5). Finally Chapter 6 consists of the conclusions and proposals for future work. According to the results of this work, phages are very effective in the removal of planktonic cells, biofilms in the early stage of formation and 5 days old biofilms, when optimal conditions are gathered. In optimal conditions the removal percentages obtained were higher than 80%. Nevertheless, phage infection of cells revealed to be conditioned due to several factors, such as temperature, growth stage and media, infection media and phage concentration. Biological control of P. fluorescens cells using phages is limited by these factors.Fundação para a Ciência e a Tecnologia - POCTI/BIO/35683/99 "Controlo Químico e Biológico de Biofilmes"

Seeking a phage-based therapy to control otitis media

info:eu-repo/semantics/publishedVersio

Use of bacteriophages to control biofilms

Tese de doutoramento em Engenharia Química e BiológicaAfter several years of abandonment, the use of bacteriophages (phages) for killing bacteria has withdrawn recent attention and reappraisal. This has led to a vast phage research, in varied fields, with impressive outcomes and currently several studies are ongoing with animals, horticulture and agriculture products, and even with humans. Despite this enthusiasm, there is a lack of research conserning phage utilization to reduce bacteria living on surfaces in a lifeform known as biofilms. This work explores the potential of phages in controlling bacteria present in single and dual species biofilms. Gram-negative Pseudomonas fluorescens and Gram-positive Staphylococcus lentus, widespread inhabitants of dairy plant surfaces and products were the studied bacterial hosts. P. fluorescens is the dominant microorganism present in the microflora of raw or pasteurized milk at the time of spoilage and commonly isolated from contaminated fresh meats and refrigerated products. S. lentus is coagulase-negative staphylococci (CoNS) and a member of the S. sciuri group. CoNS are the bacteria most frequently recovered from infected bovine and caprine mammary glands. Before performing biofilm studies, special emphasis was given to isolation of new lytic phages and to their thorough characterization. Several phages were isolated amongst which φIBB-PF7A and φIBB-SL58B were selected for the control experiments. Both belong to the Podoviridae family and can kill a wide host range. Phage φIBB-PF7A resembles, morphologically, physico-chemically and genomically, to T7-like phages while φIBB-SL58B cannot, so far, be assigned to any known Podoviridae group and is possibly a novel type of phage. After the characterization of the phage candidates, their ability to infect biofilms was investigated. P. fluorescens biofilms exhibited cells with two distinct morphologies that, independently of the age of the biofilm infected, were efficiently killed by phage φIBBPF7A. Unexpectedly, the best infection by S. lentus phage φIBB-SL58B was found to be the killing of their hosts formed under severe starvation conditions. Furthermore, biofilms of S. lentus colonized better the substratum than P. fluorescens cells, and in effect S. lentus was the predominant bacterium in most dual species biofilms investigated. The dual species biofilms were challenged using two approaches. A phage cocktail, for each of the hosts of the dual species biofilms, decreased efficiently not only the cell number in the biofilm, but also the cells which were released to the planktonic phase. The use of a single phage, for the less predominant bacterium, revealed that the applied phage (φIBBPF7A) can efficiently reach the host and reduce their cell number in the biofilm, but also may cause the release of the non-susceptible species to the planktonic phase. Interestingly and contrarily to what is commonly described in literature, one of the studied phages (φIBB-PF7A) proved to be excellent in killing stationary phase host cells. The conditions under which the phages are applied are important factors to be considered. Rotary agitation and medium conditions (exchange or non-exchange) influenced vastly phage killing of biofilm cells. Additionaly, rotary agitation, temperature, host cell length and growth rate influenced planktonic cell lysis by phage (φIBB-PF7A). Nevertheless, the optimal conditions for phage infection are highly depended on the phage-host system investigated, as demonstrated in φIBB-PF7A and φIBB-SL58B infection experiments performed with their respective hosts.Após vários anos de abandono, assistiu-se, recentemente, ao crescente interesse científico e reconhecimento das notáveis capacidades dos bacteriófagos (fagos) no controlo de bactérias. São diversos os domínios de aplicação de fagos como agentes de controlo de bactérias, nomeadamente na produção animal, horticultura e agricultura e existem inclusive investigações a decorrer com humanos. Apesar deste recente entusiasmo, há uma grande lacuna relativamente à utilização de fagos para a redução de bactérias aderidas a superfícies ou mais propriamente biofilmes. Este trabalho explora a utilização de fagos no controlo de bactérias presentes em biofilmes formados por uma única espécie bacteriana ou mistos (constituidos por várias espécies). As bactérias Gram-negativa Pseudomonas fluorescens e Gram-positiva Staphylococcus lentus são ubíquas em superfícies e produtos da indústria dos lacticínios. P. fluorescens é a bactéria dominante da microflora dos leites, não-pasteurizados e pasteurizados, estragados e frequentemente isolada de carnes e produtos refrigerados estragados. S. lentus, é uma bactéria coagulase-negativa (CoNS), pertence ao grupo de S. sciuri e pode causar doenças em animais. As CoNS são, de facto, as bactérias mais frequentemente isoladas de bovinos e caprinos com infecção das glândulas mamárias. Antes de serem iniciados os estudos de infecção de biofilmes, procedeu-se deu-se ao isolamento de novos fagos e à sua rigorosa caracterização. Foram isolados vários fagos para P. fluorescens e S. lentus e do conjunto total de fagos purificados, os fagos φIBBPF7A e φIBB-SL58B, foram seleccionados para os estudos de controlo de biofilmes. Ambos pertencem à familia Podoviridae e têm um espectro lítico alargado. O fago φIBBPF7A assemelha-se morfologicamente e genomicamente a fagos do tipo T7, enquanto que o fago φIBB-SL58B poderá ser um novo tipo de fago uma vez não há semelhanças morfológicas, genómicas nem fisico-quimicas com nenhum fago descrito na literatura. O fago φIBB-PF7A foi capaz de lisar com grande eficiência os biofilmes de P. fluorescens, independentemente da idade do biofilme. Inesperadamente, este estudo revelou que a melhor infecção dos biofilmes de S. lentus por perte do fago φIBB-SL58B foi conseguida em biofilmes formados sem renovação do meio de crescimento. No que respeita à infecção de biofilmes mistos, formados por P. fluorescens e S. lentus, utilizouse duas estratégias distintas: o uso de um cocktail formado pelos dois fagos e a aplicação de apenas um fago específico para a bactéria minoritariamente presente nos biofilmes mistos, a P. fluorescens. A primeira estratégia revelou uma redução no número de células viáveis presentes nos biofilmes e de células libertadas dos biofilmes para a fase planctónica. A utilização de apenas um fago foi eficaz no controlo das células de P.fluorescens presentes no biofilme, assistindo-se a uma destruição parcial do biofilme e consequentemente à libertação para a fase planctónica da bactéria não susceptível ao fago. Contrariamente ao que é frequentemente descrito na literatura, um dos fagos estudados (φIBB-PF7A) revelou uma elevada eficácia na lise de células na fase estacionária de crescimento. A avaliação de diferentes condições de ifecção é fundamental de forma a se conseguir optimizar a eficiência fágica no controlo de biofilmes e células planctónicas. Este trabalho evidencia que a aplicação de fagos no controlo dos hospedeiros é influenciada por factores tais como: a velocidade de agitação, temperatura e vários parâmetros relacionados com o hospedeiro tais como o seu tamanho, taxa específica de crescimento e com as condições usadas na formação de biofilmes nomeadamente a renovação ou não de meio de crescimento. No entanto, este trabalho revela também que as condições óptimas de infecção fágica dependem bastante do fago e hospedeiro investigados como verificado em ensaios de infecção realizados com os fagos φIBB-PF7A e φIBB-SL58B e os seus respectivos hospedeiros.Portuguese Foundation for Science and Technology through the grant SFRH/BD/18485/200

Phage control of dual species biofilms of Pseudomonas fluorescens and Staphylococcus lentus

Despite the recent enthusiasm for using bacteriophages as bacterial control agents, there are only limited studies concerning phage interaction with their respective hosts residing in mixed biofilm consortia and especially in biofilms where the host species is a minor constituent. In the present work, a study was made of mono and dual species biofilms formed by Pseudomonas fluorescens (Gram-negative) and/or Staphylococcus lentus (Gram-positive) and their fate after infection with phages. The dual species biofilms consisted predominantly of S. lentus. The exposure of these biofilms to a cocktail containing both P. fluorescens and S. lentus phages effectively killed and removed the hosts from the substratum. Additionally, this cocktail approach also controlled the hosts released from the biofilms to the planktonic phase. The ability of phages to control a host population present in minority in the mixed species biofilm was also assessed. For this objective, the biofilms were challenged only with phage øIBB-PF7A, specific for P. fluorescens and the results obtained were to some extent unpredicted. First, øIBB-PF7A readily reached the target host and caused a significant population decrease. Secondly, and surprisingly, this phage was also capable of causing partial damage to the biofilms leading to the release of the non-susceptible host (S. lentus) from the dual species biofilms to the planktonic phase. The efficiency of phage treatment of biofilms was to some extent dependent on the number of cells present and also conditioned by the infection strategy (dynamic or static) utilized in the infection of the biofilms. Nevertheless, in most circumstances phages were well capable of controlling their target hosts.Fundação para a Ciência e a Tecnologia (FCT

The use of bacteriophages for P. aeruginosa biofilm control

Pseudomonas aeruginosa is a relevant opportunistic pathogen frequently associated with several nosocomial infections and, worryingly, this bacterium shows a low antibiotic susceptibility. One of its virulence factors is related with the ability to adhere to surfaces and also human epithelium and form virulent biofllms. This work describes the isolation and characterization of lytic phages capable to infect antibiotic resistant P. aeruginosa strains. It is also described herein the potential of the new isolated phages for planktonic cells and biofilm control. According to the results of this work, the isolated phages showed different spectra of activity and most of them were efficient even against P. aeruginosa multidrug resistant strains. Furthermore, the biofilm infection assays revealed that phages can be a good strategy to combat virulent biofilms achieving significant reductions in the number of biofilm cells.(undefined

Isolation and characterization of a T7-like lytic phage for Pseudomonas fluorescens

Background: Despite the proven relevance of Pseudomonas fluorescens as a spoilage microorganism in milk, fresh meats and refrigerated food products and the recognized potential of bacteriophages as sanitation agents, so far no phages specific for P. fluorescens isolates from dairy industry have been closely characterized in view of their lytic efficiency. Here we describe the isolation and characterization of a lytic phage capable to infect a variety of P. fluorescens strains isolated from Portuguese and United States dairy industries. Results: Several phages were isolated which showed a different host spectrum and efficiency of lysis. One of the phages, phage ϕIBB-PF7A, was studied in detail due to its efficient lysis of a wide spectrum of P. fluorescens strains and ribotypes. Phage ϕIBB-PF7A with a head diameter of about 63 nm and a tail size of about 13 × 8 nm belongs morphologically to the Podoviridae family and resembles a typical T7-like phage, as analyzed by transmission electron microscopy (TEM). The phage growth cycle with a detected latent period of 15 min, an eclipse period of 10 min, a burst size of 153 plaque forming units per infected cell, its genome size of approximately 42 kbp, and the size and N-terminal sequence of one of the protein bands, which gave similarity to the major capsid protein 10A, are consistent with this classification. Conclusion: The isolated T7-like phage, phage ϕIBB-PF7A, is fast and efficient in lysing different P. fluorescens strains and may be a good candidate to be used as a sanitation agent to control the prevalence of spoilage causing P. fluorescens strains in dairy and food related environments.Quebec, Canada; Portuguese Foundation for Science and Technology (FCT).Cornell University, N.Y., U.S.A.; Laval University