Filamentation of Campylobacter in broth cultures

The transition from rod to filamentous cell morphology has been identified as a response to stressful conditions in many bacterial species and has been ascribed to confer certain survival advantages. Filamentation of Campylobacter jejuni was demonstrated to occur spontaneously on entry in to stationary phase distinguishing it from many other bacteria where a reduction in size is more common. The aim of this study was to investigate the cues that give rise to filamentation of C. jejuni and C. coli and gain insights into the process. Using minimal medium, augmentation of filamentation occurred and it was observed that this morphological change was wide spread amongst C. jejuni strains tested but was not universal in C. coli strains. Filamentation did not appear to be due to release of diffusible molecules, toxic metabolites, or be in response to oxidative stress in the medium. Separated filaments exhibited greater intracellular ATP contents (2.66 to 17.4 fg) than spiral forms (0.99 to 1.7 fg) and showed enhanced survival in water at 4oC and 37oC compared to spiral cells. These observations support the conclusion that the filaments are adapted to survive extra-intestinal environments. Differences in cell morphology and physiology need to be considered in the context of the design of experimental studies and the methods adopted for the isolation of campylobacters from food, clinical and environmental sources

Campylobacters and their bacteriophages from chicken liver: the prospect for phage biocontrol

Consumption of foods containing chicken liver has been associated with Campylobacter enteritis. Campylobacters can contaminate the surface of livers post-mortem but can also arise through systemic infection of colonising bacteria in live birds. The use of bacteriophage to reduce levels of Campylobacter entering the food chain is a promising intervention approach but most phages have been isolated from chicken excreta. This study examined the incidence and contamination levels of Campylobacter and their bacteriophage in UK retail chicken liver. Using enrichment procedures, 87% of 109 chicken livers were surface contaminated with Campylobacter and 83% contaminated within internal tissues. Direct plating on selective agar allowed enumeration of viable bacteria from 43 % of liver samples with counts ranging from 1.8 - > 3.8 log10 CFU/cm2 for surface samples, and 3.0 – > 3.8 log10 CFU/g for internal tissue samples. Three C. jejuni isolates recovered from internal liver tissues were assessed for their ability to colonise the intestines and extra-intestinal organs of broiler chickens following oral infection. All isolates efficiently colonised the chicken intestines but were variable in their abilities to colonise extra-intestinal organs. One isolate, CLB104, could be recovered by enrichment from the livers and kidneys of three of seven chickens. Campylobacter isolates remained viable within fresh livers stored at 4oC over 72 h and frozen livers stored at -20oC over 7 d in atmospheric oxygen, and therefore constitute a risk to human health. Only three Campylobacter-specific bacteriophages were isolated, and these exhibited a limited host range against the Camplylobacter chicken liver isolates. All were identified as group III virulent bacteriophage based on their genome size of 140 kb. The application of broad host range group II virulent phages (8 log10 PFU/g) to liver homogenates containing C. jejuni strains of diverse origin at 4oC resulted in modest but significant reductions in the viable counts ranging from 0.2 to 0.7 log10 CFU/g

Phage Biocontrol of Campylobacter jejuni in Chickens Does Not Produce Collateral Effects on the Gut Microbiota

Bacteriophage biocontrol to reduce Campylobacter jejuni levels in chickens can reduce human exposure and disease acquired through the consumption of contaminated poultry products. Investigating changes in the chicken microbiota during phage treatment has not previously been undertaken but is crucial to understanding the system-wide effects of such treatments to establish a sustainable application. A phage cocktail containing two virulent Campylobacter phages was used to treat broiler chickens colonized with C. jejuni HPC5. Campylobacter counts from cecal contents were significantly reduced throughout the experimental period but were most effective 2 days post-treatment showing a reduction of 2.4 log10 CFU g-1 relative to mock-treated Campylobacter colonized controls. The administered phages replicated in vivo to establish stable populations. Bacteriophage predation of C. jejuni was not found to affect the microbiota structure but selectively reduced the relative abundance of C. jejuni without affecting other bacteria

Isolation and characterization of Campylobacter bacteriophages from retail poultry

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4°C

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

Copyright © 2020 Richards et al 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 galactooligosaccharides (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

Genome Dynamics of Campylobacter jejuni in Response to Bacteriophage Predation

Campylobacter jejuni is a leading cause of food-borne illness. Although a natural reservoir of the pathogen is domestic poultry, the degree of genomic diversity exhibited by the species limits the application of epidemiological methods to trace specific infection sources. Bacteriophage predation is a common burden placed upon C. jejuni populations in the avian gut, and we show that amongst C. jejuni that survive bacteriophage predation in broiler chickens are bacteriophage-resistant types that display clear evidence of genomic rearrangements. These rearrangements were identified as intra-genomic inversions between Mu-like prophage DNA sequences to invert genomic segments up to 590 kb in size, the equivalent of one-third of the genome. The resulting strains exhibit three clear phenotypes: resistance to infection by virulent bacteriophage, inefficient colonisation of the broiler chicken intestine, and the production of infectious bacteriophage CampMu. These genotypes were recovered from chickens in the presence of virulent bacteriophage but not in vitro. Reintroduction of these strains into chickens in the absence of bacteriophage results in further genomic rearrangements at the same locations, leading to reversion to bacteriophage sensitivity and colonisation proficiency. These findings indicate a previously unsuspected method by which C. jejuni can generate genomic diversity associated with selective phenotypes. Genomic instability of C. jejuni in the avian gut has been adopted as a mechanism to temporarily survive bacteriophage predation and subsequent competition for resources, and would suggest that C. jejuni exists in vivo as families of related meta-genomes generated to survive local environmental pressures

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

© Copyright © 2020 The State of Queensland (through the Department Agriculture and Fisheries). 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

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

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

Development of a lyophilization process for Campylobacter bacteriophage storage and transport

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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