Correction: Genome Dynamics of Campylobacter jejuni in Response to Bacteriophage Predation.

[This corrects the article DOI: 10.1371/journal.ppat.0030119.].

Galacto-oligosaccharides fed during gestation increase Rotavirus A specific antibodies in sow colostrum, modulate the microbiome, and reduce infectivity in neonatal piglets in a commercial farm setting.

Introduction: Rotavirus A is a major cause of acute dehydrating diarrhea in neonatal pigs resulting in significant mortality, morbidity, reduced performance and economic loss. Commercially available prebiotic galacto-oligosaccharides are similar to those of mammalian milk and stimulate the development of the microbiota and immune system in neonates. Little is known about the effects of supplementing sows' diets with galacto-oligosaccharides during gestation. This study aimed to determine if dietary galacto-oligosaccharide supplementation during gestation could improve immunity, reduce rotavirus infection and modulate the microbiota in sows and neonates in a commercial farm setting with confirmed natural endemic rotavirus challenge. Methods: In a randomized controlled trial, control sows received lactation diet with no galacto-oligosaccharide supplementation and test sows received lactation diet with 30 g/day galacto-oligosaccharide top-dressed into feed daily, seven days before farrowing. Colostrum was collected from sows 24 hours post-partum and tested for rotavirus specific antibodies. Fecal samples were collected from sows and piglets three days post-partum, tested for rotavirus A by qPCR and the microbiome composition assessed by 16s rRNA gene sequencing. Results: Supplementation with galacto-oligosaccharides during gestation significantly increased rotavirus-specific IgG and IgA in sow colostrum and reduced the number of rotavirus positive piglet fecal samples. Abundance of potential pathogens Treponema and Clostridiales were higher in fecal samples from non-galacto-oligosaccharide fed sows, their piglets and rotavirus positive samples. Discussion: This study demonstrates that galacto-oligosaccharide supplementation during gestation significantly increases rotavirus specific IgG and IgA in sow colostrum thereby reducing neonatal rotavirus infection and suppresses potential pathogenic bacteria in nursing sows and neonatal piglets.

Galacto-Oligosaccharides Increase the Abundance of Beneficial Probiotic Bacteria and Improve Gut Architecture and Goblet Cell Expression in Poorly Performing Piglets, but Not Performance.

Poorly performing piglets receiving commercial milk replacers do not benefit from the naturally occurring probiotic galacto-oligosaccharides otherwise found in sow milk. Study objectives were to investigate the effects of complete milk replacer supplemented with galacto-oligosaccharides on the microbiome, gut architecture and immunomodulatory goblet cell expression of poorly performing piglets that could benefit from milk replacement feeding when separated from sows and housed with fit siblings in environmentally controlled pens. The study is novel in that it is one of the first to investigate the effects of supplementing complete milk replacer with galacto-oligosaccharides in poorly performing piglets. Gastrointestinal tract samples were collected from piglets, and the microbiome composition was assessed by 16s ribosomal ribonucleic acid gene sequencing. Gut architectural features, villus/crypt ratio and enumeration of goblet cells in tissues were assessed by histopathological techniques. The most abundant taxa identified at the genus level were Lactobacillus, Streptococcus, Prevotella, Lactococcus and Leuconostoc. Milk replacer plus galacto-oligosaccharides significantly improved gut architectural features and villus/crypt ratio throughout the gastrointestinal tract, increased the number of goblet cells and revealed a differential abundance of beneficial probiotic bacteria, particularly Lactobacillus and Bifidobacterium. In these respects, galacto-oligosaccharide-supplemented milk replacer may be a useful addition to animal husbandry in poorly performing, non-thriving animals when moved to environmentally controlled pens away from sows and fit siblings, thereby modulating the microbiome and gastrointestinal tract performance.

Common colonic community indicators of the suckling pig microbiota where diversity and abundance correlate with performance.

The primary objective of this study was to investigate if common colonic community indicators could be identified from the microbiota of 22-day-old suckling pigs in repeated small-scale trials. A total of three separate trials were conducted at different times in the same year and facility with genetically similar animals. Colonic samples were collected from four pigs in each trial and the microbiome composition assessed by 16s rRNA gene sequencing. Pig weight, average daily gain (ADG), bacterial diversity, and abundance were not significantly different between repeated trials, except for a significant difference in Jaccard Similarity. At genus level, the most abundant taxa identified were Porphyromonadaceae unclassified (15.81%), Ruminococcaceae unclassified, (12.78%), Prevotella (7.26%), Clostridiales unclassified (6.99%), Lactobacillus (6.58%), Phascolarctobacterium (6.52%), and Firmicutes unclassified (5.69%). The secondary objective was to establish if pooled data in terms of microbial diversity and abundance of the colonic microbiota related to weight and ADG. Pig weight at day 22 and ADG positively correlated with α-diversity. Abundance of potential protein digesting and short-chain fatty acid producing operational taxonomic units ascribed to Terrisporobacter, Ruminococcaceae unclassified, Intestinimonas, and Dorea correlated with weight and ADG, suggesting a nutritional role for these common colonic community microbiota members in suckling pigs.

Venatorbacter cucullus gen. nov sp. nov a novel bacterial predator.

A novel Gram-stain negative, aerobic, halotolerant, motile, rod-shaped, predatory bacterium ASxL5T, was isolated from a bovine slurry tank in Nottinghamshire, UK using Campylobacter hyointestinalis as prey. Other Campylobacter species and members of the Enterobacteriaceae were subsequently found to serve as prey. Weak axenic growth on Brain Heart Infusion agar was achieved upon subculture without host cells. The optimal growth conditions were 37 °C, at pH 7. Transmission electron microscopy revealed some highly unusual morphological characteristics related to prey availability. Phylogenetic analyses using 16S rRNA gene sequences showed that the isolate was related to members of the Oceanospirillaceae family but could not be classified clearly as a member of any known genus. Whole genome sequencing of ASxL5T confirmed the relationship to members the Oceanospirillaceae. Database searches revealed that several ASxL5T share 16S rRNA gene sequences with several uncultured bacteria from marine, and terrestrial surface and subsurface water. We propose that strain ASxL5T represents a novel species in a new genus. We propose the name Venatorbacter cucullus gen. nov., sp. nov. with ASxL5T as the type strain.

Application of a novel phage vB_SalS-LPSTLL for the biological control of Salmonella in foods.

Salmonella is one of the most common foodborne pathogens around the world. Phages are envisioned as a new strategy to control foodborne pathogenic bacteria and food safety. A Salmonella specific lytic phage vB_SalS-LPSTLL (LPSTLL) was selected for food applications on the basis of lytic range, lytic efficiency, functional stability and characteristics. Phage LPSTLL was able to lyse 11 Salmonella serotypes, which represents the broadest range reported Salmonella phages, and was able to suppress the growth of Salmonella enterica in liquid culture over nine hours. LPSTLL exhibited rapid reproductive activity with a short latent period and a large burst size in one-step growth experiment. LPSTLL remained active over a pH range of 3.0 to 12.0, and at incubation temperatures up to 60 °C for 60 min, indicating wide applicability for food processing and storage. Significant reductions of viable Salmonella were observed in diverse foods (milk, apple juice, chicken and lettuce) with reductions up to 2.8 log CFU/mL recorded for milk. Sensory evaluation indicated that treatment with phage LPSTLL did not alter the visual or tactile quality of food matrices. Genome analysis of LPSTLL indicated the absence of any virulence or antimicrobial resistance genes. Genomic comparisons suggest phage LPSTLL constitutes a novel member of a new genus, the LPSTLLvirus with the potential for Salmonella biocontrol in the food industry.

Bacteriophages to Control Multi-Drug Resistant Enterococcus faecalis Infection of Dental Root Canals.

Phage therapy is an alternative treatment to antibiotics that can overcome multi-drug resistant bacteria. In this study, we aimed to isolate and characterize lytic bacteriophages targeted against Enterococcus faecalis isolated from root canal infections obtained from clinics at the Faculty of Dentistry, Ismalia, Egypt. Bacteriophage, vB_ZEFP, was isolated from concentrated wastewater collected from hospital sewage. Morphological and genomic analysis revealed that the phage belongs to the Podoviridae family with a linear double-stranded DNA genome, consisting of 18,454, with a G + C content of 32.8%. Host range analysis revealed the phage could infect 10 of 13 E. faecalis isolates exhibiting a range of antibiotic resistances recovered from infected root canals with efficiency of plating values above 0.5. One-step growth curves of this phage showed that it has a burst size of 110 PFU per infected cell, with a latent period of 10 min. The lytic activity of this phage against E. faecalis biofilms showed that the phage was able to control the growth of E. faecalis in vitro. Phage vB_ZEFP could also prevent ex-vivo E. faecalis root canal infection. These results suggest that phage vB_ZEFP has potential for application in phage therapy and specifically in the prevention of infection after root canal treatment.

Acetate and auto-inducing peptide are independent triggers of quorum sensing in Lactobacillus plantarum.

The synthesis of plantaricin in Lactobacillus plantarum is regulated by quorum sensing. However, the nature of the extra-cytoplasmic (EC) sensing domain of the histidine kinase (PlnB1) and the ability to recognize the auto-inducing peptide PlnA1 is not known. We demonstrate the key motif Ile-Ser-Met-Leu of auto-inducing peptide PlnA1 binds to the hydrophobic region Phe-Ala-Ser-Gln-Phe of EC loop 2 of PlnB1 via hydrophobic interactions and hydrogen bonding. Moreover, we identify a new inducer, acetate, that regulates the synthesis of plantaricin by binding to a positively charged region (Arg-Arg-Tyr-Ser-His-Lys) in loop 4 of PlnB1 via electrostatic interaction. The side chain of Phe143 on loop 4 determined the specificity and affinity of PlnB1 to recognize acetate. PlnA1 activates quorum sensing in log phase growth and acetate in stationary phase to maintain the synthesis of plantaricin under conditions of reduced growth. Acetate activation of PlnB was also evident in four types of PlnB present in different Lb. plantarum strains. Finally, we proposed a model to explain the developmental regulation of plantaricin synthesis by PlnA and acetate. These results have potential applications in improving food fermentation and bacteriocin production.

Campylobacter bacteriophage DA10: an excised temperate bacteriophage targeted by CRISPR-cas.

BACKGROUND: Lytic bacteriophages that infect Campylobacter spp. have been utilized to develop therapeutic/decontamination techniques. However, the association of Campylobacter spp. and bacteriophages has been the focus of several strands of research aimed at understanding the complex relationships that have developed between predators and prey over evolutionary time. The activities of endogenous temperate bacteriophages have been used to evaluate genomic rearrangements and differential protein expression in host cells, and mechanisms of resistance to bacteriophage infection in campylobacters such as phase variation and CRISPR-mediated immunity. RESULTS: Temperate bacteriophage DA10 represents a novel excised and infective virus capable of replication in a restricted set of C. jejuni and C. coli hosts. Whole genome sequencing reveals that DA10 (35,379 bp) forms part of a novel group of temperate bacteriophages that have limited distribution among database host genome sequences. Analysis of potential host genomes reveals a robust response against DA10 and DA10-like bacteriophages is driven by CRISPR-mediated immunity with 75% of DA10 ORFs represented as ~ 30 bp spacer sequences in numerous Campylobacter Type II-C CRISPR arrays. Several DA10-like homologues have been identified in a small sub-set of C. jejuni and C. coli genome sequences (ranging from near complete integrated prophage sequences to fragments recognisable in the sequence read archive). CONCLUSIONS: A complete intact DA10-like prophage in C. jejuni CJ677CC520 provides evidence that the associations between host and DA10-like bacteriophages are long-standing in evolutionary timescales. Extensive nucleotide substitution and loss can be observed in the integrated DA10-like prophage of CJ677CC520 compared to other relatives as observed through pairwise genome comparisons. Examining factors that have limited the population expansion of the prophage, while others appear to have thrived and prospered (Mu-like, CJIE-like, and lytic Campylobacter bacteriophages) will assist in identifying the underlying evolutionary processes in the natural environment.

In Vitro Evaluation of the Effects of Commercial Prebiotic GOS and FOS Products on Human Colonic Caco-2 Cells.

Prebiotic oligosaccharides are widely used as human and animal feed additives for their beneficial effects on the gut microbiota. However, there are limited data to assess the direct effect of such functional foods on the transcriptome of intestinal epithelial cells. The purpose of this study is to describe the differential transcriptomes and cellular pathways of colonic cells directly exposed to galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS). We have examined the differential gene expression of polarized Caco-2 cells treated with GOS or FOS products and their respective mock-treated cells using mRNA sequencing (RNA-seq). A total of 89 significant differentially expressed genes were identified between GOS and mock-treated groups. For FOS treatment, a reduced number of 12 significant genes were observed to be differentially expressed relative to the control group. KEGG and gene ontology functional analysis revealed that genes up-regulated in the presence of GOS were involved in digestion and absorption processes, fatty acids and steroids metabolism, potential antimicrobial proteins, energy-dependent and -independent transmembrane trafficking of solutes and amino acids. Using our data, we have established complementary non-prebiotic modes of action for these frequently used dietary fibers.

Bacteriophages to Control Campylobacter in Commercially Farmed Broiler Chickens, in Australia.

This study describes the development and use of bacteriophage cocktails to control Campylobacter in broiler chickens, in a commercial setting, in Queensland Australia, following the birds from farm to the processing plant. The components of the bacteriophage cocktails were selected to be effective against the maximum number of Campylobacter jejuni and Campylobacter coli isolates encountered on SE Queensland farms. Farms were identified that had suitable Campylobacter target populations and phage were undetectable 1 week prior to the intended treatment. Cocktails of phages were administered at 47 days of age. Groups of study birds were slaughtered the following day, on-farm, at the end of flock transport to the plant, and at processing (approximately 28 h post-treatment). On Farm A, the phage treatment significantly reduced Campylobacter levels in the ceca at the farm in the range of 1-3 log10 CFU/g (p = 0.007), compared to mock treated controls. However, individual birds sampled on farm (1/10) or following transport (2/10) exhibited high cecal Campylobacter counts with low phage titers, suggesting that treatment periods > 24 h may be required to ensure phage replication for effective biocontrol in vivo. At the time of the trial the control birds in Farm B were phage positive despite having been negative one week earlier. There was no significant difference in the cecal Campylobacter counts between the treatment and control groups following treatment but a fall of 1.7 log10 CFU/g was observed from that determined from birds collected the previous week (p = 0.0004). Campylobacter isolates from both farms retained sensitivity to the treatment phages. These trials demonstrated bacteriophages sourced from Queensland farms have the potential to reduce intestinal Campylobacter levels in market ready broiler chickens.

Bacteriophage ZCSE2 is a Potent Antimicrobial Against Salmonella enterica Serovars: Ultrastructure, Genomics and Efficacy.

Developing novel antimicrobials capable of controlling multidrug-resistant bacterial pathogens is essential to restrict the use of antibiotics. Bacteriophages (phages) constitute a major resource that can be harnessed as an alternative to traditional antimicrobial therapies. Phage ZCSE2 was isolated among several others from raw sewage but was distinguished by broad-spectrum activity against Salmonella serovars considered pathogenic to humans and animals. Lytic profiles of ZCSE2 against a panel of Salmonella were determined together with low temperature activity and pH stability. The morphological features of the phage and host infection processes were characterized using a combination of transmission electron and atomic force microscopies. Whole genome sequencing of ZCSE2 produced a complete DNA sequence of 53,965 bp. No known virulence genes were identified in the sequence data, making ZCSE2 a good candidate for phage-mediated biological control purposes. ZCSE2 was further tested against S. Enteritidis in liquid culture and was observed to reduce the target bacterium to below the limits of detection from initial concentrations of 107-108 Colony Forming Units (CFU)/mL. With a broad host-range against pathogenic Salmonella serovars, phage ZCSE2 constitutes a potential tool against a major cause of human and animal disease.

Application of a Novel Phage LPSEYT for Biological Control of Salmonella in Foods.

Salmonella is a leading cause of foodborne diseases, and in recent years, many isolates have exhibited a high level of antibiotic resistance, which has led to huge pressures on public health. Phages are a promising strategy to control food-borne pathogens. In this study, one of our environmental phage isolates, LPSEYT, was to be able to restrict the growth of zoonotic Salmonella enterica in vitro over a range of multiplicity of infections. Phage LPSEYT exhibited wide-ranging pH and thermal stability and rapid reproductive activity with a short latent period and a large burst size. Phage LPSEYT demonstrated potential efficiency as a biological control agent against Salmonella in a variety of food matrices, including milk and lettuce. Morphological observation, comparative genomic, and phylogenetic analysis revealed that LPSEYT does not belong to any of the currently identified genera within the Myoviridae family, and we suggest that LPSEYT represents a new genus, the LPSEYTvirus. This study contributes a phage database, develops beneficial phage resources, and sheds light on the potential application value of phages LPSEYT on food safety.

Development of a Lyophilization Process for Campylobacter Bacteriophage Storage and Transport.

Bacteriophages are a sustainable alternative to control pathogenic bacteria in the post-antibiotic era. Despite promising reports, there are still obstacles to phage use, notably titer stability and transport-associated expenses for applications in food and agriculture. In this study, we have developed a lyophilization approach to maintain phage titers, ensure efficacy and reduce transport costs of Campylobacter bacteriophages. Lyophilization methods were adopted with various excipients to enhance stabilization in combination with packaging options for international transport. Lyophilization of Eucampyvirinae CP30A using tryptone formed a cake that limited processing titer reduction to 0.35 ± 0.09 log10 PFU mL-1. Transmission electron microscopy revealed the initial titer reduction was associated with capsid collapse of a subpopulation. Freeze-dried phages were generally stable under refrigerated vacuum conditions and showed no significant titer changes over 3 months incubation at 4 °C (p = 0.29). Reduced stability was observed for lyophilized phages that were incubated either at 30 °C under vacuum or at 4 °C at 70% or 90% relative humidity. Refrigerated international transport and rehydration of the cake resulted in a total phage titer reduction of 0.81 ± 0.44 log10 PFU mL-1. A significantly higher titer loss was observed for phages that were not refrigerated during transport (2.03 ± 0.32 log10 PFU mL-1). We propose that lyophilization offers a convenient method to preserve and transport Campylobacter phages, with minimal titer reduction after the drying process.

Application of a Broad Range Lytic Phage LPST94 for Biological Control of Salmonella in Foods.

Salmonella, one of the most common food-borne pathogens, is a significant public health and economic burden worldwide. Lytic phages are viable alternatives to conventional technologies for pathogen biocontrol in food products. In this study, 40 Salmonella phages were isolated from environmentally sourced water samples. We characterized the lytic range against Salmonella and among all isolates, phage LPST94 showed the broadest lytic spectrum and the highest lytic activity. Electron microscopy and genome sequencing indicated that LPST94 belongs to the Ackermannviridae family. Further studies showed this phage is robust, tolerating a wide range of pH (4-12) and temperature (30-60 °C) over 60 min. The efficacy of phage LPST94 as a biological control agent was evaluated in various food products (milk, apple juice, chicken breast, and lettuce) inoculated with non-typhoidal Salmonella species at different temperatures. Interestingly, the anti-Salmonella efficacy of phage LPST94 was greater at 4 °C than 25 °C, although the efficacy varied between different food models. Adding phage LPST94 to Salmonella inoculated milk decreased the Salmonella count by 3 log10 CFU/mL at 4 °C and 0.84 to 2.56 log10 CFU/mL at 25 °C using an MOI of 1000 and 10000, respectively. In apple juice, chicken breast, and lettuce, the Salmonella count was decreased by 3 log10 CFU/mL at both 4 °C and 25 °C after applying phage LPST94 at an MOI of 1000 and 10,000, within a timescale of 48 h. The findings demonstrated that phage LPST94 is a promising candidate for biological control agents against pathogenic Salmonella and has the potential to be applied across different food matrices.

Galacto-Oligosaccharides Modulate the Juvenile Gut Microbiome and Innate Immunity To Improve Broiler Chicken Performance.

Improvements in growth performance and health are key goals in broiler chicken production. Inclusion of prebiotic galacto-oligosaccharides (GOS) in broiler feed enhanced the growth rate and feed conversion of chickens relative to those obtained with a calorie-matched control diet. Comparison of the cecal microbiota identified key differences in abundances of Lactobacillus spp. Increased levels of Lactobacillus johnsonii in GOS-fed juvenile birds at the expense of Lactobacillus crispatus were linked to improved performance (growth rate and market weight). Investigation of the innate immune responses highlighted increases of ileal and cecal interleukin-17A (IL-17A) gene expression counterposed to a decrease in IL-10. Quantification of the autochthonous Lactobacillus spp. revealed a correlation between bird performance and L. johnsonii abundance. Shifts in the cecal populations of key Lactobacillus spp. of juvenile birds primed intestinal innate immunity without harmful pathogen challenge.IMPORTANCE Improvements in the growth rate of broiler chickens can be achieved through dietary manipulation of the naturally occurring bacterial populations while mitigating the withdrawal of antibiotic growth promoters. Prebiotic galacto-oligosaccharides (GOS) are manufactured as a by-product of dairy cheese production and can be incorporated into the diets of juvenile chickens to improve their health and performance. This study investigated the key mechanisms behind this progression and pinpointed L. johnsonii as a key species that facilitates the enhancements in growth rate and gut health. The study identified the relationships between the GOS diet, L. johnsonii intestinal populations, and cytokine immune effectors to improve growth.

Trileucine and Pullulan Improve Anti-Campylobacter Bacteriophage Stability in Engineered Spray-Dried Microparticles.

Spray drying biologics into a powder can increase thermal stability and shelf-life relative to liquid formulations, potentially eliminating the need for cold chain infrastructure for distribution in developing countries. In this study, process modelling, microparticle engineering, and a supplemented phase diagram were used to design physically stable fully amorphous spray-dried powder capable of stabilizing biological material. A greater proportion of anti-Campylobacter bacteriophage CP30A remained biologically active after spray drying using excipient formulations containing trehalose and a high glass transition temperature amorphous shell former, either trileucine or pullulan, as compared to the commonly used crystalline shell former, leucine, or a low glass transition temperature amorphous shell former, pluronic F-68. Particle formation models suggest that the stabilization was achieved by protecting the bacteriophages against the main inactivating stress, desiccation, at the surface. The most promising formulation contained a combination of trileucine and trehalose for which the combined effects of feedstock preparation, spray drying, and 1-month dry room temperature storage resulted in a titer reduction of only 0.6 ± 0.1 log10(PFU mL-1). The proposed high glass transition temperature amorphous formulation platform may be advantageous for stabilizing biologics in other spray drying applications in the biomedical engineering industry.

Resistance mechanisms adopted by a Salmonella Typhimurium mutant against bacteriophage.

Bacteriophages have key roles in regulating bacterial populations in most habitats. A Salmonella Typhimurium mutant (N18) with impaired sensitivity to phage fmb-p1 was obtained and examined, the adsorption efficiency of fmb-p1 to N18 was reduced to 6%, compared to more than 97% for wild type S. Typhimurium CMCC50115. Reduced adsorption was accompanied by a reduction of 90% in the LPS content compared to wild type. Electron microscopy showed phage scattered around N18 with minimal engagement, while the phage were efficiently adsorbed to the wild type with tails oriented towards the bacterial surface. Evidence suggests fmb-p1 can slightly infect N18 and this does not give rise to an increase of phage titer. RT-qPCR data show that several Salmonella genes involved in lipopolysaccharide synthesis and five virulence related genes were down-regulated upon exposure of N18 to phage fmb-p1. In contrast, phage resistance related genes such as the SOS response, restriction-modification (RM), and Cas1 gene were up-regulated in N18. These data suggest that although inefficient adsorption and entry is the primary mechanism of resistance, transcriptional responses to phage exposure indicate that alternative resistance mechanisms against phage infection are also brought to bear, including digestion of phage nucleic acids and activation of the SOS. These findings may help develop strategies for biocontrol of Salmonella where multi-resistant bacteria are encountered or emerge in applications for food production, bioremediation or wastewater treatment.

Spray-dried anti-Campylobacter bacteriophage CP30A powder suitable for global distribution without cold chain infrastructure.

Campylobacter jejuni is a leading cause of foodborne illness globally. In this study, a spray drying and packaging process was developed to produce a thermally-stable dry powder containing bacteriophages that retains biological activity against C. jejuni after long distance shipping at ambient temperature. Spray drying using a twin-fluid atomizer resulted in significantly less (p < 0.05) titer reduction than spray drying using a vibrating mesh nebulizer. The use of centrifugation and dilution of filtered bacteriophage lysate in the formulation step resulted in a significantly greater (p < 0.05) proportion of bacteriophages remaining active relative to use of no centrifugation and dilution. The spray-dried bacteriophage powder generated using leucine and trehalose as excipients was flowable, non-cohesive, and exhibited a high manufacturing yield. The powder retained its titer with no significant differences (p > 0.05) in biological activity after storage in suitable packaging for at least 3 weeks at room temperature and after ambient temperature shipping a total distance of approximately 19,800 km, including with a 38 °C temperature excursion. The bacteriophage powder therefore appears suitable for global distribution without the need for cold chain infrastructure.

Encapsulation of E. coli phage ZCEC5 in chitosan-alginate beads as a delivery system in phage therapy.

Bacteriophages can be used successfully to treat pathogenic bacteria in the food chain including zoonotic pathogens that colonize the intestines of farm animals. However, harsh gastric conditions of low pH and digestive enzyme activities affect phage viability, and accordingly reduce their effectiveness. We report the development of a natural protective barrier suitable for oral administration to farm animals that confers acid stability before functional release of bead-encapsulated phages. Escherichia coli bacteriophage ZSEC5 is rendered inactive at pH 2.0 but encapsulation in chitosan-alginate bead with a honey and gelatin matrix limited titer reductions to 1 log10 PFU mL-1. The encapsulated phage titers were stable upon storage in water but achieved near complete release over 4-5 h in a simulated intestinal solution (0.1% bile salt, 0.4% pancreatin, 50 mM KH2PO4 pH 7.5) at 37 °C. Exposure of E. coli O157:H7 to the bead-encapsulated phage preparations produced a delayed response, reaching a maximal reductions of 4.2 to 4.8 log10 CFU mL-1 after 10 h at 37 °C under simulated intestinal conditions compared to a maximal reduction of 5.1 log10 CFU mL-1 at 3 h for free phage applied at MOI = 1. Bead-encapsulation is a promising reliable and cost-effective method for the functional delivery of bacteriophage targeting intestinal bacteria of farm animals.