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Nontyphoidal Salmonella strains are the main source of pathogenic bacterial contamination in the poultry industry. Recently, Salmonella enterica serovar Kentucky has been recognized as the most prominent serovar on carcasses in poultry-processing plants. Previous studies showed that flagella are one of the main factors that contribute to bacterial attachment to broiler skin. However, the precise role of flagella and the mechanism of attachment are unknown. There are two different flagellar subunits (fliC and fljB) expressed alternatively in Salmonella enterica serovars using phase variation. Here, by making deletions in genes encoding flagellar structural subunits (flgK, fliC, and fljB), and flagellar motor (motA), we were able to differentiate the role of flagella and their rotary motion in the colonization of broiler skin and cellular attachment. Utilizing a broiler skin assay, we demonstrated that the presence of FliC is necessary for attachment to broiler skin. Expression of the alternative flagellar subunit FljB enables Salmonella motility, but this subunit is unable to mediate tight attachment. Deletion of the flgK gene prevents proper flagellar assembly, making Salmonella significantly less adherent to broiler skin than the wild type. S. Kentucky with deletions in all three structural genes, fliC, fljB, and flgK, as well as a flagellar motor mutant (motA), exhibited less adhesion and invasion of Caco-2 cells, while an fljB mutant was as adherent and invasive as the wild-type strain.IMPORTANCE In this work, we answered clearly the role of flagella in S. Kentucky attachment to the chicken skin and Caco-2 cells. We demonstrated that the presence of FliC is necessary for attachment to broiler skin. Expression of the alternative flagellar subunit FljB enables Salmonella motility, but this subunit is unable to mediate strong attachment. Deletion of the flgK gene prevents proper flagellar assembly, making Salmonella significantly less adherent to broiler skin than the wild type. S. Kentucky with deletions in all three structural genes, fliC, fljB, and flgK, as well as a flagellar motor mutant (motA), exhibited less adhesion and invasion of Caco-2 cells, while an fljB mutant was as adherent and invasive as the wild-type strain. We expect these results will contribute to the understanding of the mechanisms of Salmonella attachment to food products.

Measuring functional opsonic antibodies against group A streptococci is an important component of the clinical development path for effective vaccines. Prior studies have used an assay developed over 60 years ago that relied on whole human blood as the source of phagocytes and complement, both of which are critical components of antibody-mediated killing assays. In this study, we adapted an assay that uses the HL-60 human promyelocytic leukemia cell line as phagocytic cells and baby rabbit serum as a source of complement for detection of opsonic antibodies against group A streptococci. On the basis of some of the known biological characteristics of the bacteria, we modified the assay conditions to support the evaluation of 21 epidemiologically important M types and demonstrated the utility and reproducibility of the assay for measurement of functional opsonic antibody levels.

BackgroundRegardless of sanitation practices implemented to reduce Salmonella prevalence in poultry processing plants, the problem continues to be an issue. To gain an understanding of the attachment mechanism of Salmonella to broiler skin, a bioluminescent-based mutant screening assay was used. A random mutant library of a field-isolated bioluminescent strain of Salmonella enterica serovar Kentucky was constructed. Mutants’ attachment to chicken skin was assessed in 96-well plates containing uniform 6 mm diameter pieces of circular chicken skin. After washing steps, mutants with reduced attachment were selected based on reduced bioluminescence, and transposon insertion sites were identified.ResultsAttachment attenuation was detected in transposon mutants with insertion in genes encoding flagella biosynthesis, lipopolysaccharide core biosynthesis protein, tryptophan biosynthesis, amino acid catabolism pathway, shikimate pathway, tricarboxylic acid (TCA) cycle, conjugative transfer system, multidrug resistant protein, and ATP-binding cassette (ABC) transporter system. In particular, mutations in S. Kentucky flagellar biosynthesis genes (flgA, flgC, flgK, flhB, and flgJ) led to the poorest attachment of the bacterium to skin.ConclusionsThe current study indicates that attachment of Salmonella to broiler skin is a multifactorial process, in which flagella play an important role.

Group A streptococcal infections are a significant cause of global morbidity and mortality. A leading vaccine candidate is the surface M protein, a major virulence determinant and protective Ag. One obstacle to the development of M protein–based vaccines is the >200 different M types defined by the N-terminal sequences that contain protective epitopes. Despite sequence variability, M proteins share coiled-coil structural motifs that bind host proteins required for virulence. In this study, we exploit this potential Achilles heel of conserved structure to predict cross-reactive M peptides that could serve as broadly protective vaccine Ags. Combining sequences with structural predictions, six heterologous M peptides in a sequence-related cluster were predicted to elicit cross-reactive Abs with the remaining five nonvaccine M types in the cluster. The six-valent vaccine elicited Abs in rabbits that reacted with all 11 M peptides in the cluster and functional opsonic Abs against vaccine and nonvaccine M types in the cluster. We next immunized mice with four sequence-unrelated M peptides predicted to contain different coiled-coil propensities and tested the antisera for cross-reactivity against 41 heterologous M peptides. Based on these results, we developed an improved algorithm to select cross-reactive peptide pairs using additional parameters of coiled-coil length and propensity. The revised algorithm accurately predicted cross-reactive Ab binding, improving the Matthews correlation coefficient from 0.42 to 0.74. These results form the basis for selecting the minimum number of N-terminal M peptides to include in potentially broadly efficacious multivalent vaccines that could impact the overall global burden of group A streptococcal diseases.

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BackgroundCritical to the development of Salmonellosis in humans is the interaction of the bacterium with the epithelial lining of the gastrointestinal tract. Traditional scientific reasoning held type III secretion system (T3SS) as the virulence factor responsible for bacterial invasion. In this study, field-isolated Salmonella enterica serovar Kentucky and a known human pathogen Salmonella enterica serovar Typhimurium were mutated and evaluated for the invasion of human colorectal adenocarcinoma epithelial cells.Results S. enterica serovar Kentucky was shown to actively invade a eukaryotic monolayer, though at a rate that was significantly lower than Typhimurium. Additionally, strains mutated for T3SS formation were less invasive than the wild-type strains, but the decrease in invasion was not significant in Kentucky.ConclusionsStrains mutated for T3SS formation were able to initiate invasion of the eukaryotic monolayer to varying degrees based on strain, In the case of Kentucky, the mutated strain initiated invasion at a level that was not significantly different from the wild-type strain. A different result was observed for Typhimurium as the mutation significantly lowered the rate of invasion in comparison to the wild-type strain.