Publicação
Control of Salmonella Enteritidis biofilms present on different food contact surfaces using bacteriophages
| Resumo: | Salmonella, a human pathogenic bacterium, is still, nowadays, one of the main causes of food-related outbreaks, that result in thousands of illnesses and hospitalizations each year, around the globe. Contaminated poultry, fruit, and fresh vegetables are the key vehicles of human contamination, which could be prevented by the application of good processing practices and appropriate washing of food products. Besides this, the fact that Salmonella can form biofilms on food working surfaces and equipments, makes this microorganism even more difficult to eliminate with the commonly used chemical and physical cleaning procedures. Hence, the development of new Salmonella control strategies is imperative. Virulent bacterio(phages), viruses that exclusively infect bacteria, resulting in their lysis, are regarded as good alternatives for the elimination of Salmonella biofilms and adhered cells from contaminated surfaces and food products. Therefore, the main objective if this thesis was the application of phages for the control of S. Enteritidis biofilms formed on different food contact surfaces. In this work, a Salmonella phage, named PVP-SE2, was genomically analysed, and based on the nowadays gene information available in databases, this phage does not encode for known toxins or antibiotic resistance, making it a great candidate for the biocontrol of Salmonella. Other characterizations were also completed, such as determination of its growth parameters and phage infectivity towards S. Enteritidis biofilm communities after artificial contamination of surfaces (e.g. polystyrene, stainless steel, and poultry skins). Results showed that this phage was capable of reducing the number of Salmonella cells adhered to these surfaces, and also of significantly reducing the bacterial loads present in the formed biofilms. These results reaffirmed PVP-SE2 potential to be used in the control of this pathogen. Like most phage genomes, PVP-SE2 genome presented many open reading frames (ORFs) that encoded for proteins with unknown function. This comes as an obstacle since some of these ORFs may encode for proteins with toxic or other undesired properties. To be accepted in a possible phage-based product, the deletion of all of the ORFs with unknown and unnecessary functions would be ideal. Therefore, a strategy was designed: to use Bacteriophage Recombineering of Electroporated DNA to create a phage genome devoid of the ORFs with unknown and unnecessary functions. The first ORF with unknown function considered for deletion was Orf_01, which was successfully achieved. The stability of this mutation was evaluated, and the results showed that Orf_01 deletion was stable for at least 11 generations. The new recombinant phage, named PVP-SE2ΔOrf_01, was characterized regarding its replication parameters and ability to infect S. Enteritidis planktonic cells in the exponential and stationary phases. It was shown that, although PVP-SE2ΔOrf_01’s kinetics of infection were substatially different from the wild type phage, the reductions of Salmonella planktonic cells in both exponential and stationary phases were identical to the ones obtained for PVP-SE2, most likely due to the faster latent period of this phage compared to its parental phage. This work showed that BRED is a suitable method to genetically modify phages, and also that Orf_01 is not essential for PVP-SE2 replication and infection abilities. Escherichia coli, another human pathogen commonly related to foodborne outbreaks, was previously shown to be present together with Salmonella in food processing facilities. This information led to the idea that these two bacteria could be forming mixed biofilms when contaminating food working surfaces. Hence, the last chapter of this thesis was dedicated to the study of interactions found to happen between E. coli and S. Enteritidis when forming dual-species biofilms, and how this relationship could affect the ability of a cocktail composed by two phages, DaIca, an E. coli phage, and 135, a Salmonella phage, to control these mixed biofilm populations. First, kinetics formation of mono- and dual-species biofilms were characterized, showing that both bacteria grew more when alone than in mixed biofilms. Then, confocal laser microscopy imaging was used to characterize spatial distribution of both species when forming single and mixed biofilms, which showed that spatial distribution of cells in dual-species biofilms resembles the distribution of E. coli and S. Enteritidis, when in single biofilms. FTIR-ATR biofilm extracellular polymeric substances (EPS) matrices analyses were performed and showed that the EPS spectra of mixed species biofilms can either be a mixture of both species EPS, or that the EPS of one strains predominates. DaICa and 135 phages were used to challenge both mono- and dual-species biofilms, and results showed that both phages had a better antibiofilm capacity against E. coli and S. Enteritidis, respectively, when these species formed single-species biofilms, rather than mixed biofilms. This could be explained by the alterations in biofilm structure, EPS composition when the two species are forming a mixed biofilm, by the differences in phage growth characteristics in the two strains of each species tested, and also the distinct growth characteristics of the strains used in this work. In conclusion, this work has emphasized the importance of the control of S. Enteritidis biofilms using phages as an alternative to the usually used cleaning processes so cross-contamination of food products does not occur. However, it has been shown that, although phages can effectively reduce the number of viable cells present on different types of food contact surfaces, this is not enough. Therefore, the need to genetically modify phages so that they become more effective towards biofilms, which can be accomplished by the insertion, for example, of genes encoding for enzymes with antibiofilm properties, or genome engineered phages that contain only essential genes for their replication and infectivity, so they become safer to be used in a possible phage-based product, is a reality and it should be pursued. Furthermore, since biofilms in nature are rarely composed just by one bacterial species, more studies of species-species interactions that can influence phage’s antibiofilm properties must be done in a more exhaustive manner. |
|---|---|
| Autores principais: | Milho, Catarina |
| Assunto: | Engenharia e Tecnologia::Engenharia Química |
| Ano: | 2019 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Salmonella, a human pathogenic bacterium, is still, nowadays, one of the main causes of food-related outbreaks, that result in thousands of illnesses and hospitalizations each year, around the globe. Contaminated poultry, fruit, and fresh vegetables are the key vehicles of human contamination, which could be prevented by the application of good processing practices and appropriate washing of food products. Besides this, the fact that Salmonella can form biofilms on food working surfaces and equipments, makes this microorganism even more difficult to eliminate with the commonly used chemical and physical cleaning procedures. Hence, the development of new Salmonella control strategies is imperative. Virulent bacterio(phages), viruses that exclusively infect bacteria, resulting in their lysis, are regarded as good alternatives for the elimination of Salmonella biofilms and adhered cells from contaminated surfaces and food products. Therefore, the main objective if this thesis was the application of phages for the control of S. Enteritidis biofilms formed on different food contact surfaces. In this work, a Salmonella phage, named PVP-SE2, was genomically analysed, and based on the nowadays gene information available in databases, this phage does not encode for known toxins or antibiotic resistance, making it a great candidate for the biocontrol of Salmonella. Other characterizations were also completed, such as determination of its growth parameters and phage infectivity towards S. Enteritidis biofilm communities after artificial contamination of surfaces (e.g. polystyrene, stainless steel, and poultry skins). Results showed that this phage was capable of reducing the number of Salmonella cells adhered to these surfaces, and also of significantly reducing the bacterial loads present in the formed biofilms. These results reaffirmed PVP-SE2 potential to be used in the control of this pathogen. Like most phage genomes, PVP-SE2 genome presented many open reading frames (ORFs) that encoded for proteins with unknown function. This comes as an obstacle since some of these ORFs may encode for proteins with toxic or other undesired properties. To be accepted in a possible phage-based product, the deletion of all of the ORFs with unknown and unnecessary functions would be ideal. Therefore, a strategy was designed: to use Bacteriophage Recombineering of Electroporated DNA to create a phage genome devoid of the ORFs with unknown and unnecessary functions. The first ORF with unknown function considered for deletion was Orf_01, which was successfully achieved. The stability of this mutation was evaluated, and the results showed that Orf_01 deletion was stable for at least 11 generations. The new recombinant phage, named PVP-SE2ΔOrf_01, was characterized regarding its replication parameters and ability to infect S. Enteritidis planktonic cells in the exponential and stationary phases. It was shown that, although PVP-SE2ΔOrf_01’s kinetics of infection were substatially different from the wild type phage, the reductions of Salmonella planktonic cells in both exponential and stationary phases were identical to the ones obtained for PVP-SE2, most likely due to the faster latent period of this phage compared to its parental phage. This work showed that BRED is a suitable method to genetically modify phages, and also that Orf_01 is not essential for PVP-SE2 replication and infection abilities. Escherichia coli, another human pathogen commonly related to foodborne outbreaks, was previously shown to be present together with Salmonella in food processing facilities. This information led to the idea that these two bacteria could be forming mixed biofilms when contaminating food working surfaces. Hence, the last chapter of this thesis was dedicated to the study of interactions found to happen between E. coli and S. Enteritidis when forming dual-species biofilms, and how this relationship could affect the ability of a cocktail composed by two phages, DaIca, an E. coli phage, and 135, a Salmonella phage, to control these mixed biofilm populations. First, kinetics formation of mono- and dual-species biofilms were characterized, showing that both bacteria grew more when alone than in mixed biofilms. Then, confocal laser microscopy imaging was used to characterize spatial distribution of both species when forming single and mixed biofilms, which showed that spatial distribution of cells in dual-species biofilms resembles the distribution of E. coli and S. Enteritidis, when in single biofilms. FTIR-ATR biofilm extracellular polymeric substances (EPS) matrices analyses were performed and showed that the EPS spectra of mixed species biofilms can either be a mixture of both species EPS, or that the EPS of one strains predominates. DaICa and 135 phages were used to challenge both mono- and dual-species biofilms, and results showed that both phages had a better antibiofilm capacity against E. coli and S. Enteritidis, respectively, when these species formed single-species biofilms, rather than mixed biofilms. This could be explained by the alterations in biofilm structure, EPS composition when the two species are forming a mixed biofilm, by the differences in phage growth characteristics in the two strains of each species tested, and also the distinct growth characteristics of the strains used in this work. In conclusion, this work has emphasized the importance of the control of S. Enteritidis biofilms using phages as an alternative to the usually used cleaning processes so cross-contamination of food products does not occur. However, it has been shown that, although phages can effectively reduce the number of viable cells present on different types of food contact surfaces, this is not enough. Therefore, the need to genetically modify phages so that they become more effective towards biofilms, which can be accomplished by the insertion, for example, of genes encoding for enzymes with antibiofilm properties, or genome engineered phages that contain only essential genes for their replication and infectivity, so they become safer to be used in a possible phage-based product, is a reality and it should be pursued. Furthermore, since biofilms in nature are rarely composed just by one bacterial species, more studies of species-species interactions that can influence phage’s antibiofilm properties must be done in a more exhaustive manner. |
|---|