135 research outputs found
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
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
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
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