Pseudomonas spp. in Canine Otitis Externa.

Canine otitis externa (OE) is a commonly diagnosed condition seen in veterinary practice worldwide. In this review, we discuss the mechanisms of the disease, with a particular focus on the biological characteristics of Pseudomonas aeruginosa and the impact that antibiotic resistance has on successful recovery from OE. We also consider potential alternatives to antimicrobial chemotherapy for the treatment of recalcitrant infections. P. aeruginosa is not a typical constituent of the canine ear microbiota, but is frequently isolated from cases of chronic OE, and the nature of this pathogen often makes treatment difficult. Biofilm formation is identified in 40-95% of P. aeruginosa from cases of OE and intrinsic and acquired antibiotic resistance, especially resistance to clinically important antibiotics, highlights the need for alternative treatments. The role of other virulence factors in OE remains relatively unexplored and further work is needed. The studies described in this work highlight several potential alternative treatments, including the use of bacteriophages. This review provides a summary of the aetiology of OE with particular reference to the dysbiosis that leads to colonisation by P. aeruginosa and highlights the need for novel treatments for the future management of P. aeruginosa otitis.

Efficacy of Different Encapsulation Techniques on the Viability and Stability of Diverse Phage under Simulated Gastric Conditions.

Salmonella causes a range of diseases in humans and livestock of considerable public health and economic importance. Widespread antimicrobial use, particularly in intensively produced livestock (e.g., poultry and pigs) may contribute to the rise of multidrug-resistant Salmonella strains. Alternative treatments such as bacteriophages have shown promise when used to reduce the intestinal carriage of Salmonella in livestock. However, the digestive enzymes and low pH encountered in the monogastric GI tract can significantly reduce phage viability and impact therapeutic outcomes. This study deployed alginate-carrageenan microcapsules with and without CaCO3 to protect a genomically diverse set of five Salmonella bacteriophages from simulated gastrointestinal conditions. None of the unprotected phage could be recovered following exposure to pH < 3 for 10 min. Alginate-carrageenan encapsulation improved phage viability at pH 2-2.5 after exposure for 10 min, but not at pH 2 after 1 h. Including 1% (w/v) CaCO3 in the formulation further reduced phage loss to <0.5 log10 PFU/mL, even after 1 h at pH 2. In all cases, phage were efficiently released from the microcapsules following a shift to a neutral pH (7.5), simulating passage to the duodenum. In summary, alginate-carrageenan-CaCO3 encapsulation is a promising approach for targeted intestinal delivery of genomically diverse Salmonella bacteriophages.

Prophylactic Delivery of a Bacteriophage Cocktail in Feed Significantly Reduces Salmonella Colonization in Pigs.

Nontyphoidal Salmonella spp. are a leading cause of human food poisoning and can be transmitted to humans via consuming contaminated pork. To reduce Salmonella spread to the human food chain, bacteriophage (phage) therapy could be used to reduce bacteria from animals' preslaughter. We aimed to determine if adding a two-phage cocktail to feed reduces Salmonella colonization in piglets. This first required spray drying phages to allow them to be added as a powder to feed, and phages were spray dried in different excipients to establish maximum recovery. Although laboratory phage yields were not maintained during scale up in a commercial spray dryer (titers fell from 3 × 108 to 2.4 × 106 PFU/g respectively), the phage titers were high enough to progress. Spray dried phages survived mixing and pelleting in a commercial feed mill, and sustained no further loss in titer when stored at 4°C or barn conditions over 6 months. Salmonella-challenged piglets that were prophylactically fed the phage-feed diet had significantly reduced Salmonella colonization in different gut compartments (P < 0.01). 16S rRNA gene sequencing of fecal and gut samples showed phages did not negatively impact microbial communities as they were similar between healthy control piglets and those treated with phage. Our study shows delivering dried phages via feed effectively reduces Salmonella colonization in pigs. IMPORTANCE Infections caused by Salmonella spp. cause 93.8 million cases of human food poisoning worldwide, each year of which 11.7% are due to consumption of contaminated pork products. An increasing number of swine infections are caused by multidrug-resistant (MDR) Salmonella strains, many of which have entered, and continue to enter the human food chain. Antibiotics are losing their efficacy against these MDR strains, and thus antimicrobial alternatives are needed. Phages could be developed as an alternative approach, but research is required to determine the optimal method to deliver phages to pigs and to determine if phage treatment is effective at reducing Salmonella colonization in pigs. The results presented in this study address these two aspects of phage development and show that phages delivered via feed prophylactically to pigs reduces Salmonella colonization in challenged pigs.

Predatory bacteria as living antibiotics - where are we now?

Antimicrobial resistance (AMR) is a global health and economic crisis. With too few antibiotics in development to meet current and anticipated needs, there is a critical need for new therapies to treat Gram-negative infections. One potential approach is the use of living predatory bacteria, such as Bdellovibrio bacteriovorus (small Gram-negative bacteria that naturally invade and kill Gram-negative pathogens of humans, animals and plants). Moving toward the use of Bdellovibrio as a 'living antibiotic' demands the investigation and characterization of these bacterial predators in biologically relevant systems. We review the fundamental science supporting the feasibility of predatory bacteria as alternatives to antibiotics.

Antibacterial and Antivirulence Activity of Manuka Honey against Genetically Diverse Staphylococcus pseudintermedius Strains.

Staphylococcus pseudintermedius causes opportunistic infections in dogs. It also has significant zoonotic potential, with the emergence of multidrug resistance leading to difficulty treating both animal and human infections. Manuka honey has previously been reported to inhibit many bacterial pathogens, including methicillin-resistant Staphylococcus aureus, and is successfully utilized in both clinical and veterinary practice. Here, we evaluated the ability of manuka honey to inhibit strains of S. pseudintermedius grown alone and in combination with antibiotics, as well as its capacity to modulate virulence within multiple S. pseudintermedius isolates. All 18 of the genetically diverse S. pseudintermedius strains sequenced and tested were inhibited by ≤12% (wt/vol) medical-grade manuka honey, although tolerance to five clinically relevant antibiotics was observed. The susceptibility of the isolates to four of these antibiotics was significantly increased (P ≤ 0.05) when combined with sublethal concentrations of honey, although sensitivity to oxacillin was decreased. Virulence factor (DNase, protease, and hemolysin) activity was also significantly reduced (P ≤ 0.05) in over half of isolates when cultured with sublethal concentrations of honey (13, 9, and 10 isolates, respectively). These findings highlight the potential for manuka honey to be utilized against S. pseudintermedius infections.IMPORTANCEStaphylococcus pseudintermedius is an important member of the skin microbial community in animals and can cause opportunistic infections in both pets and their owners. The high incidence of antimicrobial resistance in S. pseudintermedius highlights that this opportunistic zoonotic pathogen can cause infections which require prolonged and intensive treatment to resolve. Manuka honey has proven efficacy against many bacterial pathogens and is an accepted topical treatment for infections in both veterinary and clinical practice, and so it is a particularly appropriate antimicrobial for use with zoonotic pathogens such as S. pseudintermedius Here, we demonstrate that not only is manuka honey highly potent against novel multidrug-resistant S. pseudintermedius isolates, it also acts synergistically with clinically relevant antibiotics. In addition, manuka honey modulates S. pseudintermedius virulence activity, even at subinhibitory concentrations. In a clinical setting, these attributes may assist in controlling infection, allowing a more rapid resolution and reducing antibiotic use.

Dual Predation by Bacteriophage and Bdellovibrio bacteriovorus Can Eradicate Escherichia coli Prey in Situations where Single Predation Cannot.

Bacteria are preyed upon by diverse microbial predators, including bacteriophage and predatory bacteria, such as Bdellovibrio bacteriovorus While bacteriophage are used as antimicrobial therapies in Eastern Europe and are being applied for compassionate use in the United States, predatory bacteria are only just beginning to reveal their potential therapeutic uses. However, predation by either predator type can falter due to different adaptations arising in the prey bacteria. When testing poultry farm wastewater for novel Bdellovibrio isolates on Escherichia coli prey lawns, individual composite plaques were isolated containing both an RTP (rosette-tailed-phage)-like-phage and a B. bacteriovorus strain and showing central prey lysis and halos of extra lysis. Combining the purified phage with a lab strain of B. bacteriovorus HD100 recapitulated haloed plaques and increased killing of the E. coli prey in liquid culture, showing an effective side-by-side action of these predators compared to their actions alone. Using approximate Bayesian computation to select the best fitting from a variety of different mathematical models demonstrated that the experimental data could be explained only by assuming the existence of three prey phenotypes: (i) sensitive to both predators, (ii) genetically resistant to phage only, and (iii) plastic resistant to B. bacteriovorus only. Although each predator reduces prey availability for the other, high phage numbers did not abolish B. bacteriovorus predation, so both predators are competent to coexist and are causing different selective pressures on the bacterial surface while, in tandem, controlling prey bacterial numbers efficiently. This suggests that combinatorial predator therapy could overcome problems of phage resistance.IMPORTANCE With increasing levels of antibiotic resistance, the development of alternative antibacterial therapies is urgently needed. Two potential alternatives are bacteriophage and predatory bacteria. Bacteriophage therapy has been used, but prey/host specificity and the rapid acquisition of bacterial resistance to bacteriophage are practical considerations. Predatory bacteria are of interest due to their broad Gram-negative bacterial prey range and the lack of simple resistance mechanisms. Here, a bacteriophage and a strain of Bdellovibrio bacteriovorus, preyed side by side on a population of E. coli, causing a significantly greater decrease in prey numbers than either alone. Such combinatorial predator therapy may have greater potential than individual predators since prey surface changes selected for by each predator do not protect prey against the other predator.

Veterinary use of bacteriophage therapy in intensively-reared livestock.

Zoonoses are infectious diseases transmitted directly or indirectly between animals and humans. Several important zoonotic pathogens colonize farm animals asymptomatically, which may lead to contamination of the food chain and public health hazards. Moreover, routine sampling of carcasses at retail by government authorities over the past 20 years suggests the prevalence of antibiotic resistance in foodborne pathogens has increased. If this continues, antibiotics may be ineffective against such pathogens in the future and alternative approaches, such as phage therapy, may be necessary. Intensive livestock farming is the only realistic way of meeting the demand for meat from an increasing global population and growth in middle class consumers in developing countries, particularly in Asia. This review elaborates on the use of phages to control zoonotic pathogens in intensively-reared livestock (poultry and pigs).

Reviving Phage Therapy for the Treatment of Cholera.

Cholera remains a major risk in developing countries, particularly after natural or man-made disasters. Vibrio cholerae El Tor is the most important cause of these outbreaks, and is becoming increasingly resistant to antibiotics, so alternative therapies are urgently needed. In this study, a single bacteriophage, Phi_1, was used to control cholera prophylactically and therapeutically in an infant rabbit model. In both cases, phage-treated animals showed no clinical signs of disease, compared with 69% of untreated control animals. Bacterial counts in the intestines of phage-treated animals were reduced by up to 4 log10 colony-forming units/g. There was evidence of phage multiplication only in animals that received a V. cholerae challenge. No phage-resistant bacterial mutants were isolated from the animals, despite extensive searching. This is the first evidence that a single phage could be effective in the treatment of cholera, without detectable levels of resistance. Clinical trials in human patients should be considered.

Complete Genome Sequences of Seven Vibrio cholerae Phages Isolated in China.

The complete genome sequences of seven closely related Vibrio cholerae phages isolated from environmental sites in southeastern China are reported here. Phages QH, CJY, H1, H2, H3, J2, and J3 are members of the Podoviridae family and are highly similar to the previously sequenced Vibrio phages VP2, VP5, and phiVC8.

Complete Genome Sequences of Vibrio cholerae-Specific Bacteriophages 24 and X29.

The complete genomes of two Vibrio cholerae bacteriophages of potential interest for cholera bacteriophage (phage) therapy were sequenced and annotated. The genome size of phage 24 is 44,395 bp encoding 71 putative proteins, and that of phage X29 is 41,569 bp encoding 68 putative proteins.

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.

BACKGROUND: Evolution 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 ~1x10(7) 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. RESULTS: To 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. CONCLUSIONS: The 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.

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.

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

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 host-specific bacteriophages to the surface of chicken skin leads to a reduction in recovery of Campylobacter jejuni.

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.

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 degrees C.