Robert J. Atterbury

Bacteriophage Therapy To Reduce Campylobacter jejuni Colonization of Broiler Chickens

ABSTRACT Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log10 CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens.

Bacteriophage Therapy To Reduce Salmonella Colonization of Broiler Chickens

ABSTRACT Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by ≥4.2 log10 CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by ≥2.19 log10 CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.

Application of Host-Specific Bacteriophages to the Surface of Chicken Skin Leads to a Reduction in Recovery of Campylobacter jejuni

ABSTRACT Retail poultry products are widely purported as the major infection vehicle for human campylobacteriosis. Numerous intervention strategies have sought to reduce Campylobacter contamination on broiler carcasses in the abattoir. This study reports the efficacy of bacteriophage in reducing the number of recoverable Campylobacter jejuni cells on artificially contaminated chicken skin.

Isolation and Characterization of Campylobacter Bacteriophages from Retail Poultry

ABSTRACT 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.

Effects of orally administered Bdellovibrio bacteriovorus on the well-being and Salmonella colonization of young chicks

ABSTRACT Bdellovibrio bacteriovorus is a bacterium which preys upon and kills Gram-negative bacteria, including the zoonotic pathogens Escherichia coli and Salmonella. Bdellovibrio has potential as a biocontrol agent, but no reports of it being tested in living animals have been published, and no data on whether Bdellovibrio might spread between animals are available. In this study, we tried to fill this knowledge gap, using B. bacteriovorus HD100 doses in poultry with a normal gut microbiota or predosed with a colonizing Salmonella strain. In both cases, Bdellovibrio was dosed orally along with antacids. After dosing non-Salmonella-infected birds with Bdellovibrio, we measured the health and well-being of the birds and any changes in their gut pathology and culturable microbiota, finding that although a Bdellovibrio dose at 2 days of age altered the overall diversity of the natural gut microbiota in 28-day-old birds, there were no adverse effects on their growth and well-being. Drinking water and fecal matter from the pens in which the birds were housed as groups showed no contamination by Bdellovibrio after dosing. Predatory Bdellovibrio orally administered to birds that had been predosed with a gut-colonizing Salmonella enterica serovar Enteritidis phage type 4 strain (an important zoonotic pathogen) significantly reduced Salmonella numbers in bird gut cecal contents and reduced abnormal cecal morphology, indicating reduced cecal inflammation, compared to the ceca of the untreated controls or a nonpredatory ΔpilA strain, suggesting that these effects were due to predatory action. This work is a first step to applying Bdellovibrio therapeutically for other animal, and possibly human, infections.

Application of a bacteriophage cocktail to reduce Salmonella Typhimurium U288 contamination on pig skin

Multidrug-resistant Salmonella Typhimurium U288 is a significant pathogen of pigs, accounting for over half of all outbreaks on UK pig production premises. The potential of this serovar, and other salmonellae, to enter the food chain during the slaughtering process requires that efforts be made to reduce the prevalence of these bacteria at both the pre- and post-harvest stages of production. A bacteriophage cocktail (PC1) capable of lysing various Salmonella enterica serovars was designed using the broad host-range phage Felix 01, and three phages isolated from sewage. PC1 applied to pig skin experimentally-contaminated with U288 achieved significant reductions (P<0.05) in Salmonella counts when stored at 4 °C over 96 h. Reductions of >1 log₁₀ unit were observed when the ratio of phage applied was in excess of the bacterial concentration. The treatment was found to be effective at a multiplicity of infection (MOI) of 10 or above, with no significant reductions taking place when the MOI was less than 10. Under these conditions U288 counts of log₁₀ 4.1-4.3 CFU were reduced to undetectable levels following the application of PC1 to pig skin (>99% reduction). These data suggest phage cocktails could be employed post-slaughter as a means to reduce Salmonella contamination of pig carcasses.

Correlation of Campylobacter Bacteriophage with Reduced Presence of Hosts in Broiler Chicken Ceca

ABSTRACT Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log10 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log10 6.9 CFU/g).

Bacteriophage biocontrol in animals and meat products

Since their discovery almost a century ago, bacterial viruses (bacteriophages or ‘phages’) have been used to prevent and treat a multitude of bacterial infections (phage therapy: PT). In addition, they have been the basis for many advances in genetics and biochemistry. Phage therapy was performed on human subjects in the United States, Europe and Asia in the few decades following their discovery. However, Western countries largely abandoned PT in favour of antibiotics in the 1940s. The relatively recent renaissance of PT in the West can be attributed partly to the increasing prevalence of antibiotic resistance in human and animal pathogens. However, the stringent controls on human trials now required in the United States and Europe have led to a greater number of domestic animal and agricultural applications as an alternative to PT in man. This trend is set to continue, at least in the short term, with recent approval from the Food and Drug Administration allowing commercial phage treatments to be used in human food in the USA. Nevertheless, despite these significant milestones and the growing number of successful PT trials, significant obstacles remain to their widespread use in animals, food and ultimately medicine in many parts of the world. This review will provide a brief overview of the history of PT in the West and will summarize some of the key findings of phage biocontrol studies in animals and meat products.

Genome analysis of a simultaneously predatory and prey-independent, novel Bdellovibrio bacteriovorus from the River Tiber, supports in silico predictions of both ancient and recent lateral gene transfer from diverse bacteria

BackgroundEvolution equipped Bdellovibrio bacteriovorus predatory bacteria to invade other bacteria, digesting and replicating, sealed within them thus preventing nutrient-sharing with organisms in the surrounding environment. Bdellovibrio were previously described as “obligate predators” because only by mutations, often in gene bd0108, are 1 in ~1x107 of predatory lab strains of Bdellovibrio converted to prey-independent growth. A previous genomic analysis of B. bacteriovorus strain HD100 suggested that predatory consumption of prey DNA by lytic enzymes made Bdellovibrio less likely than other bacteria to acquire DNA by lateral gene transfer (LGT). However the Doolittle and Pan groups predicted, in silico, both ancient and recent lateral gene transfer into the B. bacteriovorus HD100 genome.ResultsTo test these predictions, we isolated a predatory bacterium from the River Tiber- a good potential source of LGT as it is rich in diverse bacteria and organic pollutants- by enrichment culturing with E. coli prey cells. The isolate was identified as B. bacteriovorus and named as strain Tiberius. Unusually, this Tiberius strain showed simultaneous prey-independent growth on organic nutrients and predatory growth on live prey. Despite the prey-independent growth, the homolog of bd0108 did not have typical prey-independent-type mutations. The dual growth mode may reflect the high carbon content of the river, and gives B. bacteriovorus Tiberius extended non-predatory contact with the other bacteria present. The HD100 and Tiberius genomes were extensively syntenic despite their different cultured-terrestrial/freshly-isolated aquatic histories; but there were significant differences in gene content indicative of genomic flux and LGT. Gene content comparisons support previously published in silico predictions for LGT in strain HD100 with substantial conservation of genes predicted to have ancient LGT origins but little conservation of AT-rich genes predicted to be recently acquired.ConclusionsThe natural niche and dual predatory, and prey-independent growth of the B. bacteriovorus Tiberius strain afforded it extensive non-predatory contact with other marine and freshwater bacteria from which LGT is evident in its genome. Thus despite their arsenal of DNA-lytic enzymes; Bdellovibrio are not always predatory in natural niches and their genomes are shaped by acquiring whole genes from other bacteria.

Investigation into the animal species contents of popular wet pet foods

BackgroundThe use of the generic term “meat and animal derivatives” in declared ingredient lists of pet foods in the European Union is virtually universal. In the wake of the 2013 “horse meat scandal” in the human food chain, we examined the presence and authenticity of animal sources (cow, chicken, pig and horse) of proteins in a range of popular wet pet foods in the United Kingdom.FindingsSeventeen leading dog and cat foods were sampled for the relative presence of DNA from each of the four animal species by quantitative real-time polymerase chain reaction. No horse DNA was detected. However, there was detection at substantial levels of unspecified animal species in most products tested. In 14 out of 17 samples, bovine, porcine and chicken DNA were found in various proportions and combinations but were not explicitly identified on the product labels. Of the 7 products with prominent headline descriptions containing the term “with beef”, only 2 were found to contain more bovine DNA (>50%) than pig and chicken DNA combined.ConclusionsThere is a need for the pet food industry to show greater transparency to customers in the disclosure of the types of animal proteins (animal species and tissue types) in their products. Full disclosure of animal contents will (a) allow more informed choices to be made on purchases which are particularly important for pets with food allergies, (b) reduce the risk of product misinterpretation by shoppers, and (c) avoid potential religious concerns.