Silica vesicles increase stability of Salmonella-specific phages isolated from chicken in environments mimicking the gastrointestinal tract

Non-typhoidal Salmonella (NTS) enterica serovar Enteritidis is one of the major causes of foodborne infections worldwide. This NTS serovar is mainly transmitted to humans through poultry products. Bacteriophages (phages) are a promising alternative to antibiotics to reduce NTS incidences in poultry farms. The ability to survive the harsh environment encountered in the chicken gastrointestinal tract (GIT), such as low pH, high temperature and enzymatic digestion, can be valuable in selecting phages with high therapeutic potential. In this study, we characterized 13 newly isolated Kenyan S. Enteritidis-specific phages for their ability to survive in pH-adjusted media, different temperatures, and simulated gastric and intestinal fluids (SGF and SIF, respectively). Furthermore, we evaluated the possibility of using silica vesicles (SV) to increase the stability of these phages in these environments. All phages were relatively stable from pH 4 to 12 and from 25℃ to 42℃ following 12 hours of incubation. At pH 3, phages lost up to 3 logs in viral titres after three hours of incubation. They remained more stable at pH 9, with phage titres 2 logs higher than at pH 3. In SGF, they were stable for 20 minutes; afterwards, they started losing their viability up to 5 logs, while they were relatively stable in SIF for up to two hours. Moreover, significant differences were observed among the different phages in surviving these environments. Encapsulating phages with SV demonstrated a slow but long rate of phage release upon adsorption for 96 hours. Preliminary data indicate that SV 140 C18 can protect phages longer than other silica vesicles tested. In contrast, free phages in SGF had an average reduction of 7 logs PFU/ml after 60 minutes of incubation. These data suggest that a number of these phages can potentially survive through the chicken GIT and that SV can be an ideal technology to prolong the stability of phages in acidic environments

Phages for Africa: The Potential Benefit and Challenges of Phage Therapy for the Livestock Sector in Sub-Saharan Africa

One of the world’s fastest-growing human populations is in Sub-Saharan Africa (SSA), accounting for more than 950 million people, which is approximately 13% of the global population. Livestock farming is vital to SSA as a source of food supply, employment, and income. With this population increase, meeting this demand and the choice for a greater income and dietary options come at a cost and lead to the spread of zoonotic diseases to humans. To control these diseases, farmers have opted to rely heavily on antibiotics more often to prevent disease than for treatment. The constant use of antibiotics causes a selective pressure to build resistant bacteria resulting in the emergence and spread of multi-drug resistant (MDR) organisms in the environment. This necessitates the use of alternatives such as bacteriophages in curbing zoonotic pathogens. This review covers the underlying problems of antibiotic use and resistance associated with livestock farming in SSA, bacteriophages as a suitable alternative, what attributes contribute to making bacteriophages potentially valuable for SSA and recent research on bacteriophages in Africa. Furthermore, other topics discussed include the creation of phage biobanks and the challenges facing this kind of advancement, and the regulatory aspects of phage development in SSA with a focus on Kenya

Impact of temperate phages P22 and BTP1 on the behavior of Salmonella Typhimurium

Salmonella enterica significantly contributes to the infectious disease burden in both developed and developing countries. While non-typhoidal S. enterica (NTS) serovars (e.g. serovar Typhimurium and Enteritidis) typically are an important cause of (self-limiting) gastro-enteritis in developed countries, recent reports warn for the dramatic rise and fatality of invasive NTS (iNTS) strains in sub-Saharan Africa. The latter strains form part of a recently described sequence type of S. Typhimurium (i.e. ST313, with D23580 as its representative type strain), and seem to be equipped with a distinctive set of prophages. Since the individual evolution and physiology of Salmonella strains are often shaped by laterally acquired genetic elements such as prophages, this dissertation aimed to focus on the impact of novel prophages present in iNTS.Isolation and preliminary characterization of a number of prophages from iNTS isolates underscored the prevalence of BTP1-like prophages that share an unusually high degree of spontaneous induction. Moreover, these prophages also harbor a genetic region identical to the pid locus of the temperate Salmonella phage P22 that was previously discovered and characterized by our research group. In fact, expression of the Pid protein during P22 infection was previously found to specifically derepress the S. Typhimurium dgoRKDAT operon that encodes proteins required for galactonate metabolism. Surprisingly, however, despite harboring the exact same pid locus, phage BTP1 did not support the Pid/dgo interaction during infection of S. Typhimurium, highlighting regulatory differences between P22 and BTP1 on this front. Subsequently, a parS/ParB-based fluorescent labeling of the incoming phage chromosome in combination with detection by time-lapse fluorescence microscopy was used to study BTP1 infection and transmission dynamics throughout a S. Typhimurium population at single cell resolution, and to compare these dynamics with those of P22. However, while this labelling method could successfully track the cellular whereabouts of the P22 chromosome during all stages of infection, it seemed that efforts to fluorescently track the BTP1 chromosome somehow affected its normal infection behavior. Nevertheless, careful time-lapse microscopic analysis of wild-type BTP1 infection dynamics suggested that this phage (like P22) was also able to commit to a carrier state association in which the phage pseudolysogenizes its S. Typhimurium host cell without integration into the host chromosome or lytic development.To further study how phage BTP1 could potentially affect the phenotypic behavior of its host, a plasmid-borne BTP1 shotgun library was constructed downstream of a conditional promoter, subsequently expressed in S. Typhimurium, and finally screened for BTP1-borne genetic determinants able to affect the growth of this host. As such, several BTP1 loci could be discovered conferring a lethal interference with host physiology upon overexpression, with subsequent efforts aimed at dissecting the mechanism and potential impact of this toxicity. The screening of this library also revealed that plasmid-borne overexpression of the BTP1 pid locus was able to properly trigger the Pid/dgo interaction in S. Typhimurium, which was previously found absent during BTP1 phage infection.In order to better understand the premise of this Pid/dgo interaction, the P22/BTP1 pid promoter was studied in more detail by mutagenesis and fluorescent reporter fusions. This analysis revealed an unusually large 5’ untranslated region to precede the pid open reading frame on the pid transcript, although its potential regulatory impact requires further study. Further scrutiny also revealed that a specific mutation in the S. Typhimurium rpoB gene (which encodes a subunit of the RNA polymerase) could impair the P22 imposed Pid/dgo interaction, although the underlying mechanism so far remains to be further elucidated. In essence, this dissertation has uncovered a number of interesting characteristics of the BTP1 phage and of the pid locus that it shares with phage P22, although the potential contribution of BTP1 to the emergence of iNTS strains still remains to be established. Further study of the interactions between S. enterica and its laterally acquired genetic elements is bound to yield important clues regarding the behavior of this notorious pathogen.status: publishe

Xanthomonas bacteriophages: A review of their biology and biocontrol applications in agriculture

Phytopathogenic bacteria are economically important because they affect crop yields and threaten the livelihoods of farmers worldwide. The genus Xanthomonas is particularly significant because it is associated with some plant diseases that cause tremendous loss in yields of globally essential crops. Current management practices are ineffective, unsustainable and harmful to natural ecosystems. Bacteriophage (phage) biocontrol for plant disease management has been of particular interest from the early nineteenth century to date. Xanthomonas phage research for plant disease management continues to demonstrate promising results under laboratory and field conditions. AgriPhage has developed phage products for the control of Xanthomonas campestris pv. vesicatoria and Xanthomonas citri subsp. citri. These are causative agents for tomato, pepper spot and speck disease as well as citrus canker disease. Phage-mediated biocontrol is becoming a viable option because phages occur naturally and are safe for disease control and management. Thorough knowledge of biological characteristics of Xanthomonas phages is vital for developing effective biocontrol products. This review covers Xanthomonas phage research highlighting aspects of their ecology, biology and biocontrol applications

P22 mediated recombination of frt-sites

Flp mediated site specific recombination of frt-sites is frequently used in genetic engineering to excise, insert or invert DNA-cassettes in the chromosome. While constructs flanked by frt-sites are generally considered to be stable in the absence of the Flp enzyme, we observed that P22 chromosomes exceeding wild-type length tend to lose frt-flanked insertions via Flp independent recombination of frt-sites during phage propagation. This spontaneous recombination should be considered when engineering the chromosome of P22 and perhaps of other phages as well.publisher: Elsevier articletitle: P22 mediated recombination of frt-sites journaltitle: Virology articlelink: http://dx.doi.org/10.1016/j.virol.2014.06.015 content_type: article copyright: Copyright © 2014 Elsevier Inc. All rights reserved.status: publishe

Phages for Africa: The potential benefit and challenges of phage therapy for the livestock sector in sub-Saharan Africa

One of the world’s fastest-growing human populations is in Sub-Saharan Africa (SSA), accounting for more than 950 million people, which is approximately 13% of the global population. Livestock farming is vital to SSA as a source of food supply, employment, and income. With this population increase, meeting this demand and the choice for a greater income and dietary options come at a cost and lead to the spread of zoonotic diseases to humans. To control these diseases, farmers have opted to rely heavily on antibiotics more often to prevent disease than for treatment. The constant use of antibiotics causes a selective pressure to build resistant bacteria resulting in the emergence and spread of multi-drug resistant (MDR) organisms in the environment. This necessitates the use of alternatives such as bacteriophages in curbing zoonotic pathogens. This review covers the underlying problems of antibiotic use and resistance associated with livestock farming in SSA, bacteriophages as a suitable alternative, what attributes contribute to making bacteriophages potentially valuable for SSA and recent research on bacteriophages in Africa. Furthermore, other topics discussed include the creation of phage biobanks and the challenges facing this kind of advancement, and the regulatory aspects of phage development in SSA with a focus on Kenya

Phage–host interactions during pseudolysogeny: Lessons from the Pid/dgo interaction

Although the study of phage infection has a long history and catalyzed much of our current understanding in bacterial genetics, molecular biology, evolution and ecology, it seems that microbiologists have only just begun to explore the intricacy of phage-host interactions. In a recent manuscript by Cenens et al. we found molecular and genetic support for pseudolysogenic development in the Salmonella Typhimurium-phage P22 model system. More specifically, we observed the existence of phage carrier cells harboring an episomal P22 element that segregated asymmetrically upon subsequent divisions. Moreover, a newly discovered P22 ORFan protein (Pid) able to derepress a metabolic operon of the host (dgo) proved to be specifically expressed in these phage carrier cells. In this addendum we expand on our view regarding pseudolysogeny and its effects on bacterial and phage biology.status: publishe

Gender-responsive design of bacteriophage products to enhance adoption by chicken keepers in Kenya

Women and men keeping chickens in Kenya aspire to have a source of income, feed their families healthy food, and grow their businesses. Managing animal diseases and minimizing input costs enable their success. This study uses qualitative methods to recommend design opportunities for a veterinary product under development in Kenya that contains bacteriophages (phages) that target pathogenic Salmonella strains responsible for fowl typhoid, salmonellosis, and pullorum in chickens and foodborne illness in people. Our findings revealed the interplay between gender and two production systems: free-range and semi-intensive. Chicken keepers in both systems could benefit from phages combined with the orally administered Newcastle disease vaccine, one of the most commonly used preventive veterinary interventions, or phages as a treatment for fowl typhoid. Oral administration is less labor intensive, with greater benefits for women who have less control over family labor and reported doing more care tasks themselves. Men in free-range systems usually pay for veterinary inputs. In semi-intensive production systems, a phage-based product used prophylactically could be an alternative to expensive, intramuscular fowl typhoid vaccines. Keeping layers was common for women in semi-intensive systems, as they are more economically impacted by reduced laying caused by bacterial diseases. Awareness of zoonoses was low, but men and women were concerned about the negative health effects of drug residues in meat and eggs. Therefore, highlighting the lack of a withdrawal period for a phage product may appeal to customers. Antibiotics are used to both treat and prevent diseases, and phage products will need to do both to compete in the Kenyan market. These findings guide the ongoing design of a phage-based product with the goal of introducing a new veterinary product that meets the diverse needs of chicken keepers in Africa and serves as an alternative or complement to antibiotics