Functional characterization of Yersinia pestis aerobic glycerol metabolism.

Yersinia pestis biovar Orientalis isolates have lost the capacity to ferment glycerol. Herein we provide experimental validation that a 93 bp in-frame deletion within the glpD gene encoding the glycerol-3-phosphate dehydrogenase present in all biovar Orientalis strains is sufficient to disrupt aerobic glycerol fermentation. Furthermore, the inability to ferment glycerol is often insured by a variety of additional mutations within the glpFKX operon which prevents glycerol internalization and conversion to glycerol-3-phosphate. The physiological impact of functional glpFKX in the presence of dysfunctional glpD was assessed. Results demonstrate no change in growth kinetics at 26 °C and 37 °C. Mutants deficient in glpD displayed decreased intracellular accumulation of glycerol-3-phosphate, a characterized inhibitor of cAMP receptor protein (CRP) activation. Since CRP is rigorously involved in global regulation Y. pestis virulence, we tested a possible influence of a single glpD mutation on virulence. Nonetheless, subcutaneous and intranasal murine challenge was not impacted by glycerol metabolism. As quantified by crystal violet assay, biofilm formation of the glpD-deficient KIM6+ mutant was mildly repressed; whereas, chromosomal restoration of glpD in CO92 resulted in a significant increase in biofilm formation.

Application of a Broad Range Lytic Phage LPST94 for Biological Control of Salmonella in Foods.

Salmonella, one of the most common food-borne pathogens, is a significant public health and economic burden worldwide. Lytic phages are viable alternatives to conventional technologies for pathogen biocontrol in food products. In this study, 40 Salmonella phages were isolated from environmentally sourced water samples. We characterized the lytic range against Salmonella and among all isolates, phage LPST94 showed the broadest lytic spectrum and the highest lytic activity. Electron microscopy and genome sequencing indicated that LPST94 belongs to the Ackermannviridae family. Further studies showed this phage is robust, tolerating a wide range of pH (4-12) and temperature (30-60 °C) over 60 min. The efficacy of phage LPST94 as a biological control agent was evaluated in various food products (milk, apple juice, chicken breast, and lettuce) inoculated with non-typhoidal Salmonella species at different temperatures. Interestingly, the anti-Salmonella efficacy of phage LPST94 was greater at 4 °C than 25 °C, although the efficacy varied between different food models. Adding phage LPST94 to Salmonella inoculated milk decreased the Salmonella count by 3 log10 CFU/mL at 4 °C and 0.84 to 2.56 log10 CFU/mL at 25 °C using an MOI of 1000 and 10000, respectively. In apple juice, chicken breast, and lettuce, the Salmonella count was decreased by 3 log10 CFU/mL at both 4 °C and 25 °C after applying phage LPST94 at an MOI of 1000 and 10,000, within a timescale of 48 h. The findings demonstrated that phage LPST94 is a promising candidate for biological control agents against pathogenic Salmonella and has the potential to be applied across different food matrices.

Isolation, Characterization, and Application of Bacteriophage LPSE1 Against Salmonella enterica in Ready to Eat (RTE) Foods.

Salmonella infection is an important foodborne consumer health concern that can be mitigated during food processing. Bacteriophage therapy imparts many advantages over conventional chemical preservatives including pathogen specificity, natural derivation, potency, and providing a high degree of safety. The objective of this study aimed to isolate and characterize a phage that effectively control Salmonella food contamination. Out of 35 isolated phages, LPSE1 demonstrated a broad Salmonella host range, robust lytic ability, extensive pH tolerance, and prolonged thermal stability. The capacity for phage LPSE1 to control Salmonella Enteritidis-ATCC13076 in milk, sausage, and lettuce was established. Incubation of LPSE1 at 28°C in milk reduced recoverable Salmonella by approximately 1.44 log10 CFU/mL and 2.37 log10 CFU/mL at MOI of 1 and 100, respectively, as relative to the phage-excluded control. Upon administration of LPSE1 at an MOI of 1 in sausage, Salmonella count decreased 0.52 log10 at 28°C. At MOI of 100, the count decreased 0.49 log10 at 4°C. Incubation of LPSE1 on lettuce reduced recoverable Salmonella by 2.02 log10, 1.71 log10, and 1.45 log10 CFU/mL at an MOI of 1, 10, and 100, respectively, as relative to the negative control. Taken together, these findings establish LPSE1 as an effective weapon against human pathogenic Salmonella in various ready to eat foods.

Immunogenicity and protective capacity of EF-Tu and FtsZ of Streptococcus suis serotype 2 against lethal infection.

Vaccine development efforts against Streptococcus suis serotype 2 (S. suis 2) are often constrained by strain/serotype antigen variability. Bioinformatics analyses revealed two highly conserved S. suis 2 factors, EF-Tu and FtsZ. Murine immunization with recombinant proteins emulsified in white oil adjuvant or eukaryotic DNA vaccine vectors provided significant protection against lethal S. suis 2 challenge. Immune responses elicited by recombinant protein immunization revealed the robust generation of humoral immune responses, with a mixed induction of Th1-type and Th2-type responses. Furthermore, the antiserum from mice immunized with recombinant proteins significantly inhibited the growth of S. suis 2 in healthy pig whole blood, suggesting the triggering of a strong opsonizing response. Histological examination found that immunizing mice with purified recombinant proteins reduced neutrophil and macrophage accumulation in brain and lung tissues after challenge with virulent S. suis. Taken together, these findings reveal that EF-Tu and FtsZ may be promising targets for subunit and DNA vaccine candidates against S. suis 2 infection.

Complete Genome Sequence of Salmonella enterica Lytic Bacteriophage LPST10, Isolated in China.

Bacteriophage LPST10 was isolated from Wuhan, China. Lytic activity was demonstrated against multiple Salmonella enterica serovars, including Salmonella enterica serovar Typhimurium strains. This bacteriophage has a 47,657-bp double-stranded DNA genome encoding 87 putative coding sequences.

CRP-Mediated Carbon Catabolite Regulation of Yersinia pestis Biofilm Formation Is Enhanced by the Carbon Storage Regulator Protein, CsrA.

The natural transmission of Yersinia pestis is reliant upon biofilm blockage of the flea vector. However, the environmentally-responsive adaptive regulators which facilitate Y. pestis biofilm production in accordance with the flea midgut milieu are not well understood. We seek to establish the impact of available carbon source metabolism and storage upon Y. pestis biofilm production. Our findings demonstrate that Y. pestis biofilm production is subject to carbon catabolite regulation in which the presence of glucose impairs biofilm production; whereas, the sole metabolism of alternate carbon sources promotes robust biofilm formation. This observation is facilitated by the cAMP receptor protein, CRP. In accordance with a stark growth defect, deletion of crp in both CO92 and KIM6+ Y. pestis strains significantly impaired biofilm production when solely utilizing alternate carbon sources. Media supplementation with cAMP, a small-molecule activator of CRP, did not significantly alter Y. pestis biofilm production. Furthermore, CRP did not alter mRNA abundance of previously-characterized hms biofilm synthesis and regulation factors. Therefore, our findings indicate CRP does not confer a direct stimulatory effect, but may indirectly promote Y. pestis biofilm production by facilitating the alternate carbon source expression profile. Additionally, we assessed the impact of the carbon storage regulator protein, CsrA, upon Y. pestis biofilm production. Contrary to what has been described for E. coli, Y. pestis biofilm formation was found to be enhanced by CsrA. Regardless of media composition and available carbon source, deletion of csrA significantly impaired Y. pestis biofilm production. CsrA was found to promote Y. pestis biofilm production independent of glycogen regulation. Loss of csrA did not significantly alter relative hmsH, hmsP, or hmsT mRNA abundance. However, deletion of hmsP in the csrA-deficient mutant enabled excessive biofilm production, suggesting CsrA enables potent Y. pestis biofilm production through cyclic diguanylate regulation.