Salmonella pathogenicity island-14 is a critical virulence factor responsible for systemic infection in chickens caused by Salmonella gallinarum.

Salmonella enterica serovar Gallinarum (S. gallinarum) is an important host-specific pathogen that causes fowl typhoid, a severe systemic, septicemic, and fatal infection, in chickens. S. gallinarum causes high morbidity and mortality in chickens and poses a significant burden and economic losses to the poultry industry in many developing countries. However, the virulence factors and mechanisms of S. gallinarum-induced systemic infection in chickens remain poorly understood. In this study, we constructed a Salmonella pathogenicity island-14 (SPI-14) mutant strain (mSPI-14) of S. gallinarum and evaluated the pathogenicity of mSPI-14 in the chicken systemic infection model. The mSPI-14 exhibited the same level of bacterial growth and morphological characteristics but significantly reduced resistance to bile acids compared with the wild-type (WT) strain in vitro. The virulence of mSPI-14 was significantly attenuated in the chicken oral infection model in vivo. Chickens infected with WT showed typical clinical symptoms of fowl typhoid, with all birds succumbing to the infection within 6 to 9 days post-inoculation, and substantial increases in bacterial counts and significant pathological changes in the liver and spleen were observed. In contrast, all mSPI-14-infected chickens survived, the bacterial counts in the organs were significantly lower, and no significant pathological changes were observed in the liver and spleen. The expression of interleukin (IL)-1ß, IL-12, CXCLi1, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the liver of mSPI-14-infected chickens were significantly lower than those in the WT-infected chickens. These results indicate that SPI-14 is a crucial virulence factor in systemic infection of chickens, and avirulent mSPI-14 could be used to develop a new attenuated live vaccine to prevent S. gallinarum infection in chickens.

Synthetic phage-based approach for sensitive and specific detection of Escherichia coli O157.

Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.

Selective bacteriophages reduce the emergence of resistant bacteria in bacteriophage-antibiotic combination therapy.

Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC, the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance.

Harnessing a T1 Phage-Derived Spanin for Developing Phage-Based Antimicrobial Development.

The global increase in the prevalence of drug-resistant bacteria has necessitated the development of alternative treatments that do not rely on conventional antimicrobial agents. Using bacteriophage-derived lytic enzymes in antibacterial therapy shows promise; however, a thorough comparison and evaluation of their bactericidal efficacy are lacking. This study aimed to compare and investigate the bactericidal activity and spectrum of such lytic enzymes, with the goal of harnessing them for antibacterial therapy. First, we examined the bactericidal activity of spanins, endolysins, and holins derived from 2 Escherichia coli model phages, T1 and T7. Among these, T1-spanin exhibited the highest bactericidal activity against E. coli. Subsequently, we expressed T1-spanin within bacterial cells and assessed its bactericidal activity. T1-spanin showed potent bactericidal activity against all clinical isolates tested, including bacterial strains of 111 E. coli, 2 Acinetobacter spp., 3 Klebsiella spp., and 3 Pseudomonas aeruginosa. In contrast, T1 phage-derived endolysin showed bactericidal activity against E. coli and P. aeruginosa, yet its efficacy against other bacteria was inferior to that of T1-spanin. Finally, we developed a phage-based technology to introduce the T1-spanin gene into target bacteria. The synthesized non-proliferative phage exhibited strong antibacterial activity against the targeted bacteria. The potent bactericidal activity exhibited by spanins, combined with the novel phage synthetic technology, holds promise for the development of innovative antimicrobial agents.

Identification of IMP Dehydrogenase as a Potential Target for Anti-Mpox Virus Agents.

Mpox virus (formerly monkeypox virus [MPXV]) is a neglected zoonotic pathogen that caused a worldwide outbreak in May 2022. Given the lack of an established therapy, the development of an anti-MPXV strategy is of vital importance. To identify drug targets for the development of anti-MPXV agents, we screened a chemical library using an MPXV infection cell assay and found that gemcitabine, trifluridine, and mycophenolic acid (MPA) inhibited MPXV propagation. These compounds showed broad-spectrum anti-orthopoxvirus activities and presented lower 90% inhibitory concentrations (0.026 to 0.89 µM) than brincidofovir, an approved anti-smallpox agent. These three compounds have been suggested to target the postentry step to reduce the intracellular production of virions. Knockdown of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanosine biosynthesis and a target of MPA, dramatically reduced MPXV DNA production. Moreover, supplementation with guanosine recovered the anti-MPXV effect of MPA, suggesting that IMPDH and its guanosine biosynthetic pathway regulate MPXV replication. By targeting IMPDH, we identified a series of compounds with stronger anti-MPXV activity than MPA. This evidence shows that IMPDH is a potential target for the development of anti-MPXV agents. IMPORTANCE Mpox is a zoonotic disease caused by infection with the mpox virus, and a worldwide outbreak occurred in May 2022. The smallpox vaccine has recently been approved for clinical use against mpox in the United States. Although brincidofovir and tecovirimat are drugs approved for the treatment of smallpox by the U.S. Food and Drug Administration, their efficacy against mpox has not been established. Moreover, these drugs may present negative side effects. Therefore, new anti-mpox virus agents are needed. This study revealed that gemcitabine, trifluridine, and mycophenolic acid inhibited mpox virus propagation and exhibited broad-spectrum anti-orthopoxvirus activities. We also suggested IMP dehydrogenase as a potential target for the development of anti-mpox virus agents. By targeting this molecule, we identified a series of compounds with stronger anti-mpox virus activity than mycophenolic acid.

Potential Anti-Mpox Virus Activity of Atovaquone, Mefloquine, and Molnupiravir, and Their Potential Use as Treatments.

BACKGROUND: Mpox virus (MPXV) is a zoonotic orthopoxvirus and caused an outbreak in 2022. Although tecovirimat and brincidofovir are approved as anti-smallpox drugs, their effects in mpox patients have not been well documented. In this study, by a drug repurposing approach, we identified potential drug candidates for treating mpox and predicted their clinical impacts by mathematical modeling. METHODS: We screened 132 approved drugs using an MPXV infection cell system. We quantified antiviral activities of potential drug candidates by measuring intracellular viral DNA and analyzed the modes of action by time-of-addition assay and electron microscopic analysis. We further predicted the efficacy of drugs under clinical concentrations by mathematical simulation and examined combination treatment. RESULTS: Atovaquone, mefloquine, and molnupiravir exhibited anti-MPXV activity, with 50% inhibitory concentrations of 0.51-5.2 µM, which was more potent than cidofovir. Whereas mefloquine was suggested to inhibit viral entry, atovaquone and molnupiravir targeted postentry processes. Atovaquone was suggested to exert its activity through inhibiting dihydroorotate dehydrogenase. Combining atovaquone with tecovirimat enhanced the anti-MPXV effect of tecovirimat. Quantitative mathematical simulations predicted that atovaquone can promote viral clearance in patients by 7 days at clinically relevant drug concentrations. CONCLUSIONS: These data suggest that atovaquone would be a potential candidate for treating mpox.

wecB Gene of Salmonella Gallinarum Plays a Critical Role in Systemic Infection of Fowl Typhoid.

Salmonella enterica serovar Gallinarum (S. Gallinarum) is a host-specific pathogen causing fowl typhoid, a severe systemic infection in poultry, which leads to substantial economic losses due to high morbidity and mortality in many developing countries. However, less is known about the pathogenic characteristics and mechanism of S. Gallinarum-induced systemic infection in chickens. In this study, we deleted the S. Gallinarum UDP-N-acetylglucosamine-1-phosphate transferase gene, which contributes to the biosynthesis of enterobacterial common antigen (ECA), and studied the pathogenicity of this wecB::Cm strain in a chicken model of systemic infection. The wecB::Cm mutant strain showed comparable growth but lower resistance to bile acid and nalidixic acid than the wild-type strain in vitro. In the oral infection model of chickens, the virulence of the wecB::Cm strain was significantly attenuated in vivo. Chickens infected with wild-type strain showed typical clinical signs and pathological changes of fowl typhoid and died between 6 and 9 days post-infection, and the bacteria rapidly disseminated to systemic organs and increased in the livers and spleens. In contrast, the wecB::Cm mutant strain did not cause chicken death, there were no significant clinical changes, and the bacterial numbers in the liver and spleen of the chickens were significantly lower than those of the chickens infected with the wild-type strain. In addition, the expression of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and CXCLi1 in the livers of wecB::Cm-infected chickens was significantly lower than that of the chickens infected with the wild-type strain. Furthermore, the attenuated wecB::Cm strain could persistently colonize the liver and spleen at low levels for up to 25 days post-infection and could induce a protective immune response in the chickens. These results indicate that the wecB gene is an important virulence factor of S. Gallinarum in the chicken model of systemic infection, and the avirulent wecB::Cm mutant could possibly be used as a live-attenuated vaccine strain for controlling fowl typhoid.

Characteristics of systemic infection and host responses in chickens experimentally infected with Salmonella enterica serovar Gallinarum biovar Gallinarum.

Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) is a host-specific pathogen causing systemic infection in poultry, which leads to significant economic losses due to high mortality. However, little is known about the dynamic process of systemic infection and pathogenic characteristics of S. Gallinarum in chickens. In the present study, we developed an oral infection model that reproduces the pathology of S. Gallinarum and clarified the host immune response of the infected chickens. Chickens at 20 days of age orally inoculated at a dose of 108 colony forming unit (CFU) showed typical clinical signs of fowl typhoid and died between 6 and 10 days post infection. The inoculated S. Gallinarum rapidly disseminated to multple organs and the bacterial counts increased in the liver and spleen at 3 days post infection. Pathological changes associated wirh inflammation in the liver and spleen became apparent at 4 days post infection, and increased expression of interferon (IFN)-γ and interleuikin (IL)-12 in the liver and spleen did not observed until 3 days post infection. These results indicate that S. Gallinarum rapidly spread to entire body through intestine, and the low-level of inflammatory responses in the liver during the early stage of infection may contribute to rapid, systemic dissemination of the bacteria. Our infection model and findings will contribute to the better understanding of the pathogenic mechanism of S. Gallinarum, and provide new insights into the prevention and control of fowl typhoid.

Differential Distribution of Salmonella Serovars and Campylobacter spp. Isolates in Free-Living Crows and Broiler Chickens in Aomori, Japan.

Salmonella and Campylobacter cause foodborne enteritis mainly via the consumption of raw/undercooked contaminated poultry meat and products. Broiler flocks are primarily colonized with these bacteria; however, the underlying etiology remains unclear. The present study was conducted in order to obtain further information on the prevalence and genotypic distribution of Salmonella and Campylobacter in free-living crows and broiler flocks in a region for 2 years, thereby facilitating estimations of the potential risk of transmission of C. jejuni from crows to broiler flocks. Salmonella serovars Bredeney and Derby were isolated from 8 and 3 out of 123 captured crows, respectively, both of which are not common in broiler chickens. Campylobacter were isolated from all 89 crows tested and C. jejuni was prevalent (85 crows). Pulsed field gel electrophoresis showed broad diversity in the crow isolates of C. jejuni. However, 3 crow isolates and 2 broiler isolates showing similar banding patterns were assigned to different sequence types in multi-locus sequence typing. These results indicate that crows do not share Salmonella serovars with broilers, and harbor various genotypes of C. jejuni that differ from those of broiler flocks. Thus, our results indicate that crows are not a potential vector of these bacteria to broiler flocks in this region.