Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Characterization and efficacy of Salmonella phage cocktail PHA46 in the control of Salmonella Newport and Typhimurium internalized into cherry tomatoes.

Non-typhoid Salmonella enterica causes salmonellosis illness, and this bacterium can contaminate food throughout the production chain, including those that are consumed as raw products. Salmonella enterica can adhere to and internalize into fresh produce such as cherry tomatoes. It has been reported that lytic bacteriophages (phages) can be used as a biocontrol agent in the agricultural field, being an alternative for the control of Salmonella in red meat, fish, lettuce, and cabbage. The aim of this study was to characterize the two phages present in the PHA46 cocktail to determine their morphology, genome, host range, and resistance to different temperatures and pHs values; and later evaluate their lytic activity to reduce the adherence to and internalization of Salmonella enterica serovars Newport and Typhimurium into cherry tomatoes. In addition, in this work, we also explored the effect of the PHA46 cocktail on the virulence of S. Newport-45 and S. Typhimurium SL1344, recovered from the interior of cherry tomatoes, on the lifespan of the animal model Caenorhabditis elegans. The nematode C. elegans, recently has been used to test the virulence of Salmonella and it is easy to maintain and work with in the laboratory. The results revealed that the morphology obtained by Transmission Electron Microscopy of two phages from the PHA46 cocktail correspond to a myovirus, the analyses of their genomes sequences did not report virulence or antimicrobial resistance genes. The PHA46 sample is specific for 33 different serovars from different Salmonella strains and shows stability at 7 °C and pH 6. Also, the PHA46 cocktail was effective in reducing the adherence of S. Newport-45 and S. Typhimurium SL1344 to cherry tomatoes, at an average of 0.9 log10, respectively. Regarding internalized bacteria, the reduction was at an average of 1.2 log10, of the serovars mentioned above. The lifespan experiments in C. elegans showed by itself, that the PHA46 cocktail was harmless to the nematode, and the virulence from both Salmonella strains grown in vitro is diminished in the presence of the PHA46 cocktail. In conclusion, these results showed that the PHA46 cocktail could be a good candidate to be used as a biocontrol agent against Salmonella enterica.

Long-Term Interactions of Salmonella Enteritidis With a Lytic Phage for 21 Days in High Nutrients Media.

Salmonella spp. is a relevant foodborne pathogen with worldwide distribution. To mitigate Salmonella infections, bacteriophages represent an alternative to antimicrobials and chemicals in food animals and food in general. Bacteriophages (phages) are viruses that infect bacteria, which interact constantly with their host. Importantly, the study of these interactions is crucial for the use of phages as a mitigation strategy. In this study, experimental coevolution of Salmonella Enteritidis (S. Enteritidis) and a lytic phage was conducted in tryptic soy broth for 21 days. Transfer to fresh media was conducted daily and every 24 hours, 2 mL of the sample was collected to quantify Salmonella OD600 and phage titter. Additionally, time-shift experiments were conducted on 20 colonies selected on days 1, 12, and 21 to evaluate the evolution of resistance to past (day 1), present (day 12), and future (day 21) phage populations. The behavior of the dynamics was modeled and simulated with mathematical mass-action models. Bacteria and phage from days 1 and 21 were sequenced to determine the emergence of mutations. We found that S. Enteritidis grew for 21 days in the presence and absence of the phage and developed resistance to the phage from day 1. Also, the phage was also able to survive in the media for 21 days, however, the phage titer decreased in approx. 3 logs PFU/mL. The stability of the lytic phage population was consistent with the leaky resistance model. The time-shift experiments showed resistance to phages from day 1 of at least 85% to the past, present, and future phages. Sequencing of S. Enteritidis showed mutations in genes involved in lipopolysaccharide biosynthesis genes rfbP and rfbN at day 21. The phage showed mutations in the tail phage proteins responsible for recognizing the cell surface receptors. These results suggest that interactions between bacteria and phage in a rich resource media generate a rapid resistance to the infective phage but a fraction of the population remains susceptible. Interactions between Salmonella and lytic phages are an important component for the rational use of phages to control this important foodborne pathogen.

Efficacy of Salmonella Bacteriophage S1 Delivered and Released by Alginate Beads in a Chicken Model of Infection.

Modern bacteriophage encapsulation methods based on polymers such as alginate have been developed recently for their use in phage therapy for veterinary purposes. In birds, it has been proven that using this delivery system allows the release of the bacteriophage in the small intestine, the site of infection by Salmonella spp. This work designed an approach for phage therapy using encapsulation by ionotropic gelation of the lytic bacteriophage S1 for Salmonella enterica in 2% w/v alginate beads using 2% w/v calcium chloride as crosslinking agent. This formulation resulted in beads with an average size of 3.73 ± 0.04 mm and an encapsulation efficiency of 70%. In vitro, the beads protected the bacteriophages from pH 3 and released them at higher pH. To confirm that this would protect the bacteriophages from gastrointestinal pH changes, we tested the phage infectivity in vivo assay. Using a model chicken (Gallus gallus domesticus) infected with Salmonella Enteritidis, we confirmed that after 3 h of the beads delivery, infective phages were present in the chicken's duodenal and caecal sections. This study demonstrates that our phage formulation is an effective system for release and delivery of bacteriophage S1 against Salmonella Enteritidis with potential use in the poultry sector.

Two Phages of the Genera Felixounavirus Subjected to 12 Hour Challenge on Salmonella Infantis Showed Distinct Genotypic and Phenotypic Changes.

Salmonella Infantis is considered in recent years an emerging Salmonella serovar, as it has been associated with several outbreaks and multidrug resistance phenotypes. Phages appear as a possible alternative strategy to control Salmonella Infantis (SI). The aims of this work were to characterize two phages of the Felixounavirus genus, isolated using the same strain of SI, and to expose them to interact in challenge assays to identify genetic and phenotypic changes generated from these interactions. These two phages have a shared nucleotide identity of 97% and are differentiated by their host range: one phage has a wide host range (lysing 14 serovars), and the other has a narrow host range (lysing 6 serovars). During the 12 h challenge we compared: (1) optical density of SI, (2) proportion of SI survivors from phage-infected cultures, and (3) phage titer. Isolates obtained through the assays were evaluated by efficiency of plating (EOP) and by host-range characterization. Genomic modifications were characterized by evaluation of single nucleotide polymorphisms (SNPs). The optical density (600 nm) of phage-infected SI decreased, as compared to the uninfected control, by an average of 0.7 for SI infected with the wide-host-range (WHR) phage and by 0.3 for SI infected with the narrow-host-range (NHR) phage. WHR phage reached higher phage titer (7 × 1011 PFU/mL), and a lower proportion of SI survivor was obtained from the challenge assay. In SI that interacted with phages, we identified SNPs in two genes (rfaK and rfaB), which are both involved in lipopolysaccharide (LPS) polymerization. Therefore, mutations that could impact potential phage receptors on the host surface were selected by lytic phage exposure. This work demonstrates that the interaction of Salmonella phages (WHR and NHR) with SI for 12 h in vitro leads to emergence of new phenotypic and genotypic traits in both phage and host. This information is crucial for the rational design of phage-based control strategies.

Host Resistance, Genomics and Population Dynamics in a Salmonella Enteritidis and Phage System.

Bacteriophages represent an alternative solution to control bacterial infections. When interacting, bacteria and phage can evolve, and this relationship is described as antagonistic coevolution, a pattern that does not fit all models. In this work, the model consisted of a microcosm of Salmonella enterica serovar Enteritidis and φSan23 phage. Samples were taken for 12 days every 48 h. Bacteria and phage samples were collected; and isolated bacteria from each time point were challenged against phages from previous, contemporary, and subsequent time points. The phage plaque tests, with the genomics analyses, showed a mutational asymmetry dynamic in favor of the bacteria instead of antagonistic coevolution. This is important for future phage-therapy applications, so we decided to explore the population dynamics of Salmonella under different conditions: pressure of one phage, a combination of phages, and phages plus an antibiotic. The data from cultures with single and multiple phages, and antibiotics, were used to create a mathematical model exploring population and resistance dynamics of Salmonella under these treatments, suggesting a nonlethal, growth-inhibiting antibiotic may decrease resistance to phage-therapy cocktails. These data provide a deep insight into bacterial dynamics under different conditions and serve as additional criteria to select phages and antibiotics for phage-therapy.

Backyard Farms Represent a Source of Wide Host Range Salmonella Phages That Lysed the Most Common Salmonella Serovars.

The genus Salmonella has more than 2,600 serovars, and this trait is important when considering interventions for Salmonella control. Bacteriophages that are used for biocontrol must have an exclusively lytic cycle and the ability to lyse several Salmonella serovars under a wide range of environmental conditions. Salmonella phages were isolated and characterized from 34 backyard production systems (BPSs) with a history of Salmonella infections. BPSs were visited once, and cloacal or fecal samples were processed for phage isolation. Four hosts, Salmonella serovars Enteritidis, Heidelberg, Infantis, and Typhimurium, were used for phage isolation. The host range of the phages was later characterized with a panel of 23 Salmonella serovars (serovar diversity set) and 31 isolates obtained from the same farms (native set). Genetic relatedness for 10 phages with a wide host range was characterized by restriction fragment length polymorphism, and phages clustered based on the host range. We purified 63 phages, and 36 phage isolates were obtained on Salmonella Enteritidis, 16 on Salmonella Heidelberg, and 11 on Salmonella Infantis. Phages were classified in three clusters: (i) phages with a wide host range (cluster I), (ii) phages that lysed the most susceptible Salmonella serovars (serogroup D) and other isolates (cluster II), and (iii) phages that lysed only isolates of serogroup D (cluster III). The most susceptible Salmonella serovars were Enteritidis, Javiana, and Dublin. Seven of 34 farms yielded phages with a wide host range, and these phages had low levels of genetic relatedness. Our study showed an adaptation of the phages in the sampled BPSs to serogroup D Salmonella isolates and indicated that isolation of Salmonella phages with wide host range differs by farm. A better understanding of the factors driving the Salmonella phage host range could be useful when designing risk-based sampling strategies to obtain phages with a wide lytic host range for biocontrol purposes.

Salmonella phages and prophages: genomics, taxonomy, and applied aspects.

Since this book was originally published in 2007 there has been a significant increase in the number of Salmonella bacteriophages, particularly lytic virus, and Salmonella strains which have been fully sequenced. In addition, new insights into phage taxonomy have resulted in new phage genera, some of which have been recognized by the International Committee of Taxonomy of Viruses (ICTV). The properties of each of these genera are discussed, along with the role of phage as agents of genetic exchange, as therapeutic agents, and their involvement in phage typing.

Identification of the Salmonella phage epsilon 34 tailspike gene.

To understand the interaction between lipopolysaccharide (LPS) and proteins in molecular detail, a molecular genetic approach has been employed, using phage as a model system. The phage epsilon(34) is a Salmonella phage whose tailspike protein (TSP) uses the host LPS as its initial host cell receptor. Previous studies indicated that there was a similarity between the well-studied tail protein of Salmonella phage P22 and the epsilon(34). This study reports the identification of the gene for the epsilon(34) TSP as well as its initial characterization. In addition, some aspects of the structure of the epsilon(34) TSP have been deduced.

Conservation of the N-terminus of some phage tail proteins.

To study the interaction between lipopolysaccharide and protein, a comparative approach was employed using seven Salmonella enterica serovar Typhimurium typing phages as the protein model systems. This interaction has been studied in detail in the Salmonella enterica serovar Typhimurium phage P22 system and involves only the viral tailspike protein. Similarity between these phages and phage P22 was monitored in this Report by assaying restriction endonuclease digestions, capsid size, reactivity to the P22 tailspike protein monoclonal antibody, mAb92, which reacts with the N-terminus of the P22 tail protein and the ability to produce a PCR fragment using primers made to the ends of the P22 tailspike gene. The data indicate that tailspike similarity exists between most of these phages and a scheme reclassifying them is presented and that the N-terminus of the P22 tailspike protein may be a motif for many phage systems and may serve as a aid in the taxonomy of phages. The data suggest a classification scheme in which the N-terminus of some tailspike proteins (head-binding region in some tail proteins) may play a critical element role in the classification of Salmonella viruses.