Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Isolation, whole genome sequencing and application of a broad-spectrum Salmonella phage.

Salmonella is considered as one of the most common zoonotic /foodborne pathogens in the world. The application of bacteriophages as novel antibacterial agents in food substrates has become an emerging strategy. Bacteriophages have the potential to control Salmonella contamination.We have isolated and characterized a broad-spectrum Salmonella phage, SP154, which can lyse 9 serotypes, including S. Enteritidis, S. Typhimurium, S. Pullorum, S. Arizonae, S. Dublin, S. Cholerasuis, S. Chester, S. 1, 4, [5], 12: i: -, and S. Derby, accounting for 81.9% of 144 isolates. SP154 showed a short latent period (40 min) and a high burst size (with the first rapid burst size at 107 PFUs/cell and the second rapid burst size at approximately 40 PFUs/cell). Furthermore, SP154 activity has higher survival rates across various environmental conditions, including pH 4.0-12.0 and temperatures ranging from 4 to 50 °C for 60 min, making it suitable for diverse food processing and storage applications. Significant reductions in live Salmonella were observed in different foods matrices such as milk and chicken meat, with a decrease of up to 1.9 log10 CFU/mL in milk contamination and a 1 log10 CFU/mL reduction in chicken meat. Whole genome sequencing analysis revealed that SP154 belongs to the genus Ithacavirus, subfamily Humphriesvirinae, within the family Schitoviridae. Phylogenetic analysis based on the terminase large subunit supported this classification, although an alternate tree using the tail spike protein gene suggested affiliation with the genus Kuttervirus, underscoring the limitations of relying on a single gene for phylogenetic inference. Importantly, no virulence or antibiotic resistance genes were detected in SP154. Our research highlights the potential of using SP154 for biocontrol of Salmonella in the food industry.

Salmonella enteritidis acquires phage resistance through a point mutation in rfbD but loses some of its environmental adaptability.

Phage therapy holds promise as an alternative to antibiotics for combating multidrug-resistant bacteria. However, host bacteria can quickly produce progeny that are resistant to phage infection. In this study, we investigated the mechanisms of bacterial resistance to phage infection. We found that Rsm1, a mutant strain of Salmonella enteritidis (S. enteritidis) sm140, exhibited resistance to phage Psm140, which was originally capable of lysing its host at sm140. Whole genome sequencing analysis revealed a single nucleotide mutation at position 520 (C → T) in the rfbD gene of Rsm1, resulting in broken lipopolysaccharides (LPS), which is caused by the replacement of CAG coding glutamine with a stop codon TAG. The knockout of rfbD in the sm140ΔrfbD strain caused a subsequent loss of sensitivity toward phages. Furthermore, the reintroduction of rfbD in Rsm1 restored phage sensitivity. Moreover, polymerase chain reaction (PCR) amplification of rfbD in 25 resistant strains revealed a high percentage mutation rate of 64% within the rfbD locus. We assessed the fitness of four bacteria strains and found that the acquisition of phage resistance resulted in slower bacterial growth, faster sedimentation velocity, and increased environmental sensitivity (pH, temperature, and antibiotic sensitivity). In short, bacteria mutants lose some of their abilities while gaining resistance to phage infection, which may be a general survival strategy of bacteria against phages. This study is the first to report phage resistance caused by rfbD mutation, providing a new perspective for the research on phage therapy and drug-resistant mechanisms.

Salmonella Typhimurium DT 104 response to Lytic bacteriophage and Lactobionic acid on raw chicken breast.

Bacterial food poisoning cases due to Salmonella have been linked with a variety of poultry products. This study evaluated the effects of a Salmonella-specific Lytic bacteriophage and Lactobionic acid (LBA) on Salmonella Typhimurium DT 104 growth on raw chicken breast meat. Each chicken breast was randomly assigned to a treatment group (Control, DI water, phage 1%, phage 5%, LBA 10 mg/mL, LBA 20 mg/mL, and phage 5% + LBA 20 mg/mL) with four chicken breasts per group. Samples were inoculated with 106 CFU/mL of Salmonella and stored at 4 °C for 30 min. The inoculated chicken breasts were randomly assigned to different storage time (0 h, 1 h, 24 h, or 48 h). Both time and treatment showed significance reduction (P < 0.0001) of microbial growth. The weight loss was significantly different (P < 0.0001) between treatments. The LBA treatments were not effective when compared to the control group, but Lytic bacteriophage significantly reduced the amount of microbial growth.

Characterization of Salmonella bacteriophages and their potential use in dishwashing materials.

AIMS: The aims of this study were to isolate and characterize novel Salmonella phages and to evaluate the effectiveness of phage cocktails used as antibacterial agents in dishwashing materials. METHODS AND RESULTS: The effective phages, vB_STy-RN5i1 and vB_STy-RN29, were isolated from drain water samples collected from open markets using Salmonella Typhimurium as the host strain. These phages were identified as members of Podoviridae and Siphoviridae, respectively. Both phages infected at least six Salmonella serovars and rapidly lysed their host within one hour. They were stable at 4-45°C and at pH 6-9 for at least an hour while being evaluated in this study. The phage application results indicated that bacterial cells were reduced by 3⋅1 and 2⋅7 log CFU per ml at room temperature when they encountered the phage cocktail on scouring pads (SPs) and dishwashing sponges (DSs), respectively. CONCLUSIONS: The isolated Salmonella phages, vB_STy-RN5i1 and vB_STy-RN29, had potential against Salm. Typhimurium and could reduce the occurrence of bacterial-cross-contamination from dishwashing materials, which have been reported to be a source of bacteria, to other kitchen utensils and food. SIGNIFICANCE AND IMPACT OF THE STUDY: The successful reduction of bacterial contamination in dishwashing materials by the phage cocktail consisting of vB_STy-RN5i1 and vB_STy-RN29 reveals its potential to be an alternative antimicrobial agent for SPs and DSs.

Development of a broad-spectrum Salmonella phage cocktail containing Viunalike and Jerseylike viruses isolated from Thailand.

Salmonella is one of the most common agents of foodborne disease worldwide. As natural alternatives to traditional antimicrobial agents, bacteriophages (phages) are emerging as highly effective biocontrol agents against Salmonella and other foodborne bacteria. Due to the high diversity within the Salmonella genus and emergence of drug resistant strains, improved efforts are necessary to find broad range and strictly lytic Salmonella phages for use in food biocontrol. Here, we describe the isolation and characterization of two Salmonella phages: ST-W77 isolated on S. Typhimurium and SE-W109 isolated on S. Enteritidis with extraordinary Salmonella specificity. Whole genome sequencing identified ST-W77 as a Myovirus within the Viunalikevirus genus and SE-W109 as a Siphovirus within the Jerseylikevirus genus. Infectivity studies using a panel of S. Typhimurium cell wall mutants revealed both phages require the lipopolysaccharide O-antigen, with SE-W109 also recognizing the flagella, during infection of Salmonella. A combination of both phages was capable of prolonged (one-week) antibacterial activity when added to milk or chicken meat contaminated with Salmonella. Due to their broad host ranges, strictly lytic lifestyles and lack of lysogeny-related genes or virulence genes in their genomes, ST-W77 and SE-W109 are ideal phages for further development as Salmonella biocontrol agents for food production.

Morphologic and genomic characterization of a broad host range Salmonella enterica serovar Pullorum lytic phage vB_SPuM_SP116.

For effective use of phages as antimicrobial agents for controlling multidrug resistant S. Pullorum, it is important to understand phage biology. A lytic S. Pullorum phage was isolated and characterized from chicken feces, and its whole genome was sequenced and analyzed. A new lytic phage-vB_SPuM_SP116 (in brief SP116)- isolated and characterized using S. Pullorum SPu-116 as its host belongs to Myoviridae A1 group. Phage SP116 had a lytic effect on 27 of 37 (72.9%) different serotypes of clinical Salmonella strains. It showed a high bactericidal activity in killing all pathogens in cultures containing 5 × 105 cfu/mL and achieved more than 6.58 and 5.97 log unit reductions in cultures containing 5 × 106 cfu/mL and 5 × 107 cfu/mL, respectively. The one-step growth curve showed that the burst size was up to 118 pfu/bacterial cell. Complete genome sequence analysis revealed a linear, double-stranded DNA genome of 87,510 bp with an average G + C content of 38.84%, including 128 predicted open reading frames (ORFs) and 22 tRNA genes. SP116 was classified as a Felix O1 virus based upon the general phage characterization and the genomic information. Regarding its high efficacy in preventing especially S. Pullorum infection and its lack of any bacterial virulence, antimicrobial resistance, and lysogenesis genes, it could be a potential alternative candidate for the treatment of S. Pullorum infections.

Bioinformatic analyses of a potential Salmonella-virus-FelixO1 biocontrol phage BPS15S6 and the characterisation and anti-Enterobacteriaceae-pathogen activity of its endolysin LyS15S6.

Foodborne Enterobacteriaceae pathogens, especially Salmonella, still seriously threaten food safety. To establish a foundation for further developing phage- and endolysin-based methods combating these pathogens, in this study, the newly isolated Salmonella-virus-FelixO1 phage BPS15S6 for biocontrol purposes was characterised by genomic bioinformatic analysis, and then its endolysin LyS15S6 was obtained using a prokaryotic expression system, characterised in vitro and evaluated in the antibacterial efficacy. It was shown that BPS15S6 had an 87,609-bp genome with 130 open reading frames and does not appear to carry known lysogeny-associated genes and other damaging genetic determinants and is unlikely to perform generalised transduction. Furthermore, LyS15S6 was determined to possess the high enzymatic activity of 1,001,000 U mg-1 and the broad spectrum of lysing 56 tested Gram-negative strains. The assays of thermostability and optimum pH revealed that LyS15S6 was stable up to 40 °C and more active at pH 7. Notably, we demonstrate that edible ε-poly-L-lysine (EPL) can be used as an outer-membrane permeabiliser to improve the antibacterial performance of endolysins. When combined with 1 µg ml-1 EPL, 2 µM LyS15S6 could cause 3-4 log viable cell reductions of the three tested Enterobacteriaceae pathogens in vitro after 2 h of reaction at 25 °C and 2.56 and 3.14 log reductions of Salmonella ATCC13076 after 15 min of reaction at 25 °C and 2 h of reaction at 8 °C respectively. A new strategy, the combined application of endolysins and edible EPL for combating Enterobacteriaceae pathogens in food, is thus presented in this work.

Phages for biocontrol in foods: What opportunities for Salmonella sp. control along the dairy food chain?

Controlling the presence of pathogenic bacteria, such as Salmonella sp., in dairy products production is a burning issue since contamination with Salmonella can occur at any stage of the production chain. The use of Salmonella-phages applied as control agents has gained considerable interest. Nonetheless, Salmonella-phage applications specifically intended for ensuring the safety of dairy products are scarce. This review identifies recent advances in the use of Salmonella-phages that are or could be applied along the dairy food chain, in a farm-to-fork approach. Salmonella-phages can be promising tools to reduce the shedding of Salmonella in cattle, and to reduce and control Salmonella occurrence in postharvest food (such as food additives), and in food processing facilities (such as biosanitizing agents). These control measures, combined with existing methods and other biocontrol agents, constitute new opportunities to reduce Salmonella occurrence along the dairy food production, and consequently to alleviate the risk of Salmonella contamination in dairy products.

Evaluation of storage conditions and efficiency of a novel microencapsulated Salmonella phage cocktail for controlling S. enteritidis and S. typhimurium in-vitro and in fresh foods.

S. Enteritidis and S. Typhimurium are typically linked to foodborne outbreaks. Phages have continued to expand in various food applications. In this study, microencapsulation is applied for enhancing the stability and efficacy of phages as bio-control agent. Microencapsulated phage cocktail kept in aluminium laminated foil bag (LF) at 4 °C showed the highest survivability with a titer loss of 0.5 log PFU/g after 12 weeks of storage. Titer loss of phage cocktail lysate >4 log PFU/mL was observed after 12 weeks, at 4 °C. Color change of microencapsulated phage cocktail kept in LF at 4 °C did not show any significant difference during storage, and water activity (free water content) at 0.13 was found in these conditions. In-vitro study, S. Enteritidis and S. Typhimurium were decreased 1.79 and 3.63 log CFU/mL, respectively at 37 °C. Whereas, 0.43 and 0.76 log CFU/mL, respectively were observed at 10 °C. In foods, S. Enteritidis and S. Typhimurium were decreased 0.57 and 1.78 log CFU/cm2, respectively in meat. Whereas, 0.86 and 1.2 log CFU/g, respectively were observed in sprout. Foods with/without microencapsulated phage cocktail showed non-significant differences in liking scores after 2 days of storage. Overall, microencapsulated phage cocktail suggests another alternative for phage-based biocontrol with improved stability and efficacy for food application.

Effective inhibition of Salmonella Typhimurium in fresh produce by a phage cocktail targeting multiple host receptors.

Salmonella contamination of fresh produce is the primary bacterial cause of a significant number of foodborne outbreaks and infections. Bacteriophages can be used as natural antibacterial agents to control foodborne pathogens. However, the rapid development of bacterial resistance to phage infection is a significant barrier to practical phage application. To overcome this problem, we developed a novel phage cocktail consisting of the three phages (BSPM4, BSP101 and BSP22A) that target different host receptors, including flagella, O-antigen and BtuB, respectively. Whole genome sequence analysis of the phages revealed that three phages do not harbor genes involved in lysogen formation or toxin production, suggesting they are safe for use as biocontrol agents in foods. In vitro treatment of the phage cocktail resulted in a significant reduction in the development of bacterial resistance. Phage cocktail treatments achieved 4.7-5.5 log CFU/cm2 reduction of viable cell number in iceberg lettuce and 4.8-5.8 log CFU/cm2 reduction in cucumber after 12 h at room temperature (25 °C). The phage cocktail exhibited good antimicrobial efficiency, suggesting that it could reduce S. Typhimurium contamination of fresh produce. The strategy of developing cocktails of phages that target multiple host receptors can be used to develop novel biocontrol agents of S. Typhimurium.

Characterization of SE-P3, P16, P37, and P47 bacteriophages infecting Salmonella Enteritidis.

The aim of this study was to identify and characterize the SE-P3, P16, P37, and P47 phages infecting Salmonella Enteritidis. Transmission electron microscopy analysis showed that the SE phages belonged to the Myoviridae or Siphoviridae family and had plaque sizes between 0.622 ± 0.027 and 1.630 ± 0.036 mm in diameter. sefC, pefA, spvC, sopE, and gipA virulent gene regions were absent in their genome and their calculated genome sizes were between 35.9 and 37.8 kbp. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the protein profiles of each phage were different. The SE phages had a short latent period (10-20 min), large burst size (76-356 PFU/cell), and a short burst time (25-35 min). The multiplicity of infection values and mutant frequency of the phages were 0.01-0.0001 and 10-7 , respectively. They were very infective against their host bacteria when applied at 20°C, 30°C, or 37°C and adsorbed to their host cells by 96.20-97.65% in the first 5 min of incubation, and also Ca2+ ions did not have a significant effect on their adsorption. The SE phages were resistant to wide pH ranges and high temperatures. These results indicate that the SE phages are good candidates as therapeutic and biocontrol agents against foodborne pathogenic S. Enteritidis.

Role of phage ϕ1 in two strains of Salmonella Rissen, sensitive and resistant to phage ϕ1.

BACKGROUND: The study describes the Salmonella Rissen phage ϕ1 isolated from the ϕ1-sensitive Salmonella Rissen strain RW. The same phage was then used to select the resistant strain RRϕ1+, which can harbour or not ϕ1. RESULTS: Following this approach, we found that ϕ1, upon excision from RW cells with mitomycin, behaves as a temperate phage: lyses host cells and generates phage particles; instead, upon spontaneous excision from RRϕ1+ cells, it does not generate phage particles; causes loss of phage resistance; switches the O-antigen from the smooth to the rough phenotype, and favors the transition of Salmonella Rissen from the planktonic to the biofilm growth. The RW and RRϕ1+ strains differ by 10 genes; of these, only two (phosphomannomutase_1 and phosphomannomutase_2; both involved in the mannose synthesis pathway) display significant differences at the expression levels. This result suggests that phage resistance is associated with these two genes. CONCLUSIONS: Phage ϕ1 displays the unusual property of behaving as template as well as lytic phage. This feature was used by the phage to modulate several phases of Salmonella Rissen lifestyle.

Isolation and identification of Salmonella pullorum bacteriophage YSP2 and its use as a therapy for chicken diarrhea.

Salmonella pullorum is the major pathogen that is harmful to the poultry industry in developing countries, and the treatment of chicken diarrhea caused by S. pullorum has become increasingly difficult. In this study, a virulent bacteriophage YSP2, which was able to specifically infect Salmonella, was isolated and characterized. Phage YSP2 was classified in the Siphoviridae family and had a short latent period of 10 min. No bacterial virulence- or lysogenesis-related ORF is present in the YSP2 genome, making it eligible for use in phage therapy. Experiments in vivo investigated the potential use of phages as a therapy against diarrhea in chickens caused by S. pullorum in a chicken diarrhea model, demonstrating that a single oral administration of YSP2 (1 × 1010 PFU/mL, 80 µL/chicken) 2 h after S. pullorum oral administration at a double median lethal dose was sufficient to protect chickens against diarrhea. Gross inspection showed that YSP2 can effectively reduce organ damage and significantly relieve hemorrhage in the intestine and liver tissue. Moreover, YSP2 can maintain a high curative effect when diluted to 108 PFU/mL. In light of its therapeutic effect on chicken diarrhea, YSP2 may serve as an alternative treatment strategy for infections caused by S. pullorum.

Three Salmonella enterica serovar Enteritidis bacteriophages from the Siphoviridae family are promising candidates for phage therapy.

Salmonella is a common and widely distributed foodborne pathogen that is frequently implicated in gastrointestinal infections. The emergence and spread of Salmonella strains resistant to multiple antibiotics poses a significant health threat, highlighting the urgent need for early and effective therapeutic strategies. We isolated a total of 32 phages from water samples and anal swabs from pigs. Of these, three phages that produced large, clear plaques were selected for further study using the following methods: electron microscopy, analysis of the life cycle parameters, genetic analysis, inhibition of bacterial growth, and activity against biofilms. The three Salmonella phages (vB_SenS_CSP01, vB_SenS_PHB06, and vB_SenS_PHB07) were assigned to the family Siphoviridae on the basis of their morphology. All showed polyvalent infectivity, and individual phages or phage cocktails could inhibit the growth of host Salmonella enterica serovar Enteritidis strains or reduce biofilm formation by Salmonella enterica serovar Typhimurium. In summary, these three phages merit further research as biocontrol agents for Salmonella infection.

Isolation of lytic bacteriophages infecting Salmonella Typhimurium and Salmonella Enteritidis.

The objectives of this study were to isolate, purify and determine host range of lytic bacteriophages infecting foodborne the pathogen Salmonella Typhimurium and S. Enteritidis. River/stream water, sewage, raw foods, wastewater from food processing plants, slaughterhouse and fish farms and water from troughs were used for the screening of bacteriophages. The richest sources in terms of phages infecting S. Typhimurium and Enteritidis were found to be sewage, wastewaters of slaughterhouse, food processing and fisheries and streams. A total of 33 S. Typhimurium and 56 S. Enteritidis phages were isolated and purified from the samples. It was demostrated that host ranges of the isolated phages were quite wide. The numbers of bacteria types inhibited by S. Typhimurium or Enteritidis phages were changed among 1-15 and 1-19, respectively. It was found that 75.8% (25 out of 33) and 83.93% (47 out of 56) of isolated S. Typhimurium or Enteritidis phages formed clear plaques and were capable of lysing at least six or two Salmonella serovars. Beside Salmonella serovars, some S. Typhimurium (15 out of 33, 45.5%) and S. Enteritidis phages (5 out of 56, 8.93%) were also infective against E. coli strains. The host ranges of S. Typhimurium phages were wider than those of S. Enteritidis.

Comparative Characteristics of Discrimination of S. enterica Isolates by Phagotyping Test and Dienes Test.

We propose an original methodological approach to discrimination of newly isolated Salmonella enterica strains with the use of Dienes test. Dienes test is used for identification of P. vulgaris and P. mirabilis strains. It consists in growth suppression by mobile bacterial strain cultures and the formation of a demarcation line (Dienes line) between the strains growing towards each other. Similarities and differences between salmonella phagotyping method and Dienes test-based discrimination of the strains are detected. The studied sample of salmonellas was divided into 12 phagotypes. Cluster analysis has shown that most of the salmonella strains could not be clusterized by both methods. Discrimination by different methods has shown that the largest clusters contain the same strains. Clusterization of salmonella strains by different methods shows moderate congruency. Rand index used for comparison of the results of the sample clusterization by different methods is 0.88. High heterogeneity of salmonella strains is presumably explained by heterogeneity of antagonism factors within the S. enterica species. Intraspecies antagonism is essential for limitation of the horizontal gene transfer in closely related strains and for increase of the genetic heterogeneity of salmonella population in the host.

Salmonella Typhimurium, the major causative agent of foodborne illness inactivated by a phage lysis system provides effective protection against lethal challenge by induction of robust cell-mediated immune responses and activation of dendritic cells.

Salmonella Typhimurium infection via foodborne transmission remains a major public health threat even in developed countries. Vaccines have been developed to reduce the disease burden at the pre-harvest stage, but the cell-mediated immune response against intracellular invasion of the pathogen is not sufficiently elicited by conventional killed Salmonella vaccines, which are safer than live vaccines. In this study, we developed a genetically inactivated vaccine candidate by introducing lysis plasmid pJHL454 harboring the λ phage holin-endolysin system into S. Typhimurium; we designated this vaccine JOL1950. In vitro expression of endolysin was validated by immunoblotting, and complete inactivation of JOL1950 cells was observed following 36 h of the lysis. Electron microscopic examinations by scanning electron microscopy and immunogold labeling transmission EM revealed conserved surface antigenic traits of the JOL1950 cells after lysis. An in vivo immunogenicity study in mice immunized with lysed cells showed significantly increased serum IgG, IgG1, and IgG2a levels. Further, we observed markedly increased in vitro cell proliferation and upregulation of Th1, Th2, and Th17 cytokines in the repulsed splenic T-cells of immunized mice. In dendritic cells (DCs) treated with lysed JOL1950, we observed a significant increase in dendritic cell activation, co-stimulatory molecule production, and levels of immunomodulatory cytokines. In addition, Th1 and Th17 cytokines were also released by naïve CD4+ T-cells pulsed with primed DCs. Lysed JOL1950 also protected against lethal challenge in immunized mice. Together, these results indicate that our vaccine candidate has great potential to induce cell-mediated immunity against S. Typhimurium by facilitating the activation of DCs.

Isolation, Characterization, and Bioinformatic Analyses of Lytic Salmonella Enteritidis Phages and Tests of Their Antibacterial Activity in Food.

Salmonella Enteritidis remains a major threat for food safety. To take efforts to develop phage-based biocontrol for S. Enteritidis contamination in food, in this study, the phages against S. Enteritidis were isolated from sewage samples, characterized by host range assays, DNA restriction enzyme pattern analyses, and transmission electron microscope observations, and tested for antibacterial activity in food; some potent phages were further characterized by bioinformatic analyses. Results showed that based on the plaque quality and host range, seven lytic phages targeting S. Enteritidis were selected, considered as seven distinct phages through DNA physical maps, and classified as Myoviridae or Siphoviridae family by morphologic observations; the combined use of such seven strain phages as a "food additive" could succeed in controlling the artificial S. Enteritidis contamination in the different physical forms of food at a range of temperatures; by bioinformatic analyses, both selected phage BPS11Q3 and BPS15Q2 seemed to be newfound obligate lytic phage strains with no indications for any potentially harmful genes in their genomes. In conclusion, our results showed a potential of isolated phages as food additives for controlling S. Enteritidis contamination in some salmonellosis outbreak-associated food vehicles, and there could be minimized potential risk associated with using BPS11Q3 and BPS15Q2 in food.

Treatment of Salmonella-Contaminated Eggs and Pork with a Broad-Spectrum, Single Bacteriophage: Assessment of Efficacy and Resistance Development.

Numerous studies have assessed the efficacy of phage-based methods to inhibit Salmonella contamination in food products. As with most antibacterials, bacteria can develop resistance to phage in vitro. Here, we applied a single broad-spectrum Salmonella phage, vB_SalS_SJ_2 (SJ2; 108 PFU; MOI = 10), to Salmonella-contaminated meat and eggs to quantify the development of resistance in actual food matrices. Treatment with a single phage significantly reduced Salmonella Typhimurium contamination in both ground pork and liquid egg at various time points. Similarly, the same phage significantly reduced Salmonella Enteritidis in both food matrices. Efficacy was temperature dependent as larger reductions were seen at higher temperatures (21°C) versus lower temperatures (4°C) at 24 h. Following phage treatment, over 10,000 Salmonella isolates were examined for resistance to the treatment phage. The percentages of phage-resistant Salmonella (either serovar) recovered from phage-treated versus untreated pork did not differ. Conversely, significantly (p < 0.05) higher percentages of phage-resistant Salmonella Typhimurium (92.50% vs. 0.56% of control) and Salmonella Enteritidis (50.83% vs. 0.56% of control) isolates were observed in phage-treated versus untreated egg samples after incubation at room temperature for 48 h. Taken together, these data indicate that the food matrix may influence the emergence of phage resistance with resistance developing more rapidly in foods with less complex microbial communities. Future studies will focus on the impact the development of resistance in production and processing settings may have on the efficacy of phage treatments for longer term biocontrol of pathogens.