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Introduction: Pseudomonas aeruginosa is a leading cause of canine otitis externa. Enrofloxacin is often applied topically to treat this condition, although recalcitrant and recurring infections are common. There is evidence that exposure to blue light (400-470 nm) has a bactericidal effect on P. aeruginosa and other microorganisms., Methods: In the present study, we tested the biocidal effect of blue light (375-450 nm), alone or in combination with enrofloxacin, against six isolates of P. aeruginosa from dogs with otitis externa (5 of which were resistant to enrofloxacin)., Results: Treatment of planktonic cell cultures with blue light resulted in significant ( p < 0.5) reductions in Colony Forming Units (CFU) for all seven strains tested, in some cases below the limit of detection. The greatest bactericidal effect was observed following exposure to light at 405 nm wavelength ( p < 0.05). Exposure to blue light for 20 min usually resulted in a greater reduction in Pseudomonas aeruginosa than enrofloxacin treatment, and combination treatment typically resulted in the largest reductions in CFU. Analysis of the genome sequences of these strains established that enrofloxacin resistance was likely the result of a S466F substitution in GyrB. However, there was no clear association between genotype and susceptibility to blue light treatment., Discussion: These results suggest that blue light treatment, particularly at 405 nm wavelength, and especially in combination with enrofloxacin therapy, could be an effective treatment for otherwise recalcitrant canine otitis externa caused by Pseudomonas aeruginosa . It may also provide a way of extending the usefulness of enrofloxacin therapy which would otherwise be ineffective as a sole therapeutic agent., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gigante, Hadis, Secker, Shaw, Cooper, Palin, Milward and Atterbury.)

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 CaCO 3 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 ) CaCO 3 in the formulation further reduced phage loss to <0.5 log 10 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-CaCO 3 encapsulation is a promising approach for targeted intestinal delivery of genomically diverse Salmonella bacteriophages.

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N -terminus. The N -terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis , Mycobacterium vaccae , Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis , Escherichia coli , Pseudomonas aeruginosa and Bacillus subtilis , demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N -terminus of Ms6 LysB binds to the PG.

Background: Enteric infections caused by Salmonella spp. remain a major public health burden worldwide. Chickens are known to be a major reservoir for this zoonotic pathogen. The presence of Salmonella in poultry farms and abattoirs is associated with financial costs of treatment and a serious risk to human health. The use of bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization of chickens could be reduced. In a prior study, phages Eϕ151 and Tϕ7 significantly reduced broiler chicken caecal colonization by S. Enteritidis and S. Typhimurium respectively., Methods: Salmonella-free Ross broiler chickens were orally infected with S. Enteritidis P125109 or S. Typhimurium 4/74. After 7 days of infection, the animals were euthanased, and 25cm 2 sections of skin were collected. The skin samples were sprayed with a phage suspension of either Eϕ151 (S. Enteritidis), Tϕ7 phage suspension (S. Typhimurium) or SM buffer (Control). After incubation, the number of surviving Salmonellas was determined by direct plating and Most Probable Number (MPN). To determine the rate of reduction of Salmonella numbers on the skin surface, a bioluminescent S. Typhimurium DT104 strain was cultured, spread on sections of chicken breast skin, and after spraying with a Tϕ11 phage suspension, skin samples were monitored using photon counting for up to 24 h., Results: The median levels of Salmonella reduction following phage treatment were 1.38 log 10 MPN (Enteritidis) and 1.83 log 10 MPN (Typhimurium) per skin section. Treatment reductions were significant when compared with Salmonella recovery from control skin sections treated with buffer (p < 0.0001). Additionally, significant reduction in light intensity was observed within 1 min of phage Tϕ11 spraying onto the skin contaminated with a bioluminescent Salmonella recombinant strain, compared with buffer-treated controls (p < 0.01), implying that some lysis of Salmonella was occurring on the skin surface., Conclusions: The results of this study suggest that phages may be used on the surface of chicken skin as biocontrol agents against Salmonella infected broiler chicken carcasses. The rate of bioluminescence reduction shown by the recombinant Salmonella strain used supported the hypothesis that at least some of the reduction observed was due to lysis occurred on the skin surface.

Aim: Following previous research on improving the cleaning of crates used to transport broiler chickens from the farm to the abattoir, a demonstration project was undertaken to investigate improvements in crate washing on a commercial scale., Methods and Results: The soak tank of a conventional crate washing system was replaced with a high-performance washer fitted with high-volume, high-pressure nozzles. The wash water could be heated, and a greatly improved filtration system ensured that the nozzles did not lose performance or become blocked. Visual cleanliness scores and microbial counts were determined for naturally contaminated crates which had been randomly assigned to different cleaning protocols., Conclusions: When a combination of mechanical energy, heat and chemicals (i.e. detergent and disinfectant) was used, the results showed significant improvements to crate cleaning. Reductions of up to 3·6 and 3·8 log 10 CFU per crate base were achieved for Campylobacter and Enterobacteriaceae, respectively, along with a marked improvement in visual cleanliness., Significance and Impact of the Study: Broiler transport crates may become heavily contaminated with faeces and this may contribute to the spread of disease between farms. The results of this trial may be of use in reducing the spread of zoonotic pathogens in the poultry meat supply chain., (© 2020 The Society for Applied Microbiology.)

All dsDNA phages encode two proteins involved in host lysis, an endolysin and a holin that target the peptidoglycan and cytoplasmic membrane, respectively. Bacteriophages that infect Gram-negative bacteria encode additional proteins, the spanins, involved in disruption of the outer membrane. Recently, a gene located in the lytic cassette was identified in the genomes of mycobacteriophages, which encodes a protein (LysB) with mycolyl-arabinogalactan esterase activity. Taking in consideration the complex mycobacterial cell envelope that mycobacteriophages encounter during their life cycle, it is valuable to evaluate the role of these proteins in lysis. In the present work, we constructed an Ms6 mutant defective on lysB and showed that Ms6 LysB has an important role in lysis. In the absence of LysB, lysis still occurs but the newly synthesized phage particles are deficiently released to the environment. Using cryo-electron microscopy and tomography to register the changes in the lysis phenotype, we show that at 150 min post-adsorption, mycobacteria cells are incompletely lysed and phage particles are retained inside the cell, while cells infected with Ms6 wt are completely lysed. Our results confirm that Ms6 LysB is necessary for an efficient lysis of Mycobacterium smegmatis , acting, similarly to spanins, in the third step of the lysis process., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.