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Background Salmonella enterica serovar Enteritidis, the most common cause of human gastroenteritis, employs several virulence factors including lipopolysaccharide (LPS) for infection and establishment of disease inside the host. The LPS of S. enterica serovar Enteritidis consists of lipid A, core oligosaccharide and O-antigen (OAg). The OAg consists of repeating units containing different sugars. The sugars of OAg are synthesized and assembled by a set of enzymes encoded by genes organized into clusters. Present study focuses on the effect of deletion of genes involved in biosynthesis of OAg repeating units on resistance to antimicrobial peptides and virulence in mice. Methods In the present study, the OAg biosynthesis was impaired by deleting tyv, prt and wbaV genes involved in tyvelose biosynthesis and its transfer to OAg. The virulence phenotype of resulting mutants was evaluated by assessing resistance to antimicrobial peptides, serum complement, adhesion, invasion and in vivo colonization. Results Deletion of the above three genes resulted in the production of OAg-negative LPS. All the OAg-negative mutants showed phenotype reported for rough strains. Interestingly, [DELA]wbaV mutant showed increased resistance against antimicrobial peptides and normal human serum. In addition, the [DELA]wbaV mutant also showed increased adhesion and invasion as compared to the other two O-Ag negative mutants [DELA]tyv and [DELA]prt. In vivo experiments also confirmed the increased virulent phenotype of [DELA]wbaV mutant as compared to [DELA]prt mutant. Conclusion OAg-negative mutants are known to be avirulent; however, this study demonstrates that certain OAg negative mutants e.g. [DELA]wbaV may also show resistance to antimicrobial peptides and cause colitis in Streptomyces pretreated mouse model. Keywords: S. Enteritidis, Lipopolysaccharide, OAg-negative, wbaV gene, Antimicrobial peptides, Virulence, OAg biosynthesis, Author(s): Sangeeta Jaiswal[sup.1] , Niladri Bhusan Pati[sup.1] , Manupriyam Dubey[sup.1] , Chandrashekhar Padhi[sup.1] , Prakash Kumar Sahoo[sup.1] , Shilpa Ray[sup.1] , Aryashree Arunima[sup.1] , Nirmal Kumar Mohakud[sup.2] and Mrutyunjay Suar[sup.1] [...]

Background Salmonella enterica serovar Enteritidis infections are known to exhibit worldwide prevalence with increased morbidity and mortality. The conventional strategies like antibiotic therapy and vaccination have not only proved to be of sub-optimal efficacy but also led to the development of multidrug resistant strains of Salmonella. Antimicrobial activities of probiotics against various enteropathogens and other health promoting effects have assumed greater significance in recent years. The present study aims to evaluate the efficacy of a Lactobacillus plantarum strain (KSBT 56, isolated from a traditional food product of India), in preventing Salmonella enterica serovar Enteritidis growth and pathogenicity in vitro. Methods and results The cell free culture supernatant (CFCS) of KSBT 56 strain notably inhibited the growth of Salmonella Enteritidis without affecting the growth of other gram-positive lactic acid bacteria. The isolated KSBT 56 strain produces lactic acid similar to other standard probiotic strains like Lactobacillus plantarum MTCC 1407. The free radical production by KSBT 56 strain was studied by using sodC mutant of S. Enteritidis, which exhibited reduced growth in the presence of CFCS of the KSBT 56 strain, indicating the inhibitory activity of free radicals on the growth of S. Enteritidis. Our results also showed a significant reduction in the biofilm forming ability of Salmonella Enteritidis in the presence of the KSBT 56 strain (2 log cfu/ml, p = 0.01). Further, the anti-infective characteristics of KSBT 56 strain was validated by gentamicin protection assay which revealed 80% reduction in the invasion of Salmonella Enteritidis to HCT-116 cell line (Salmonella Enteritidis and KSBT 56 in a 1:1 ratio) and delayed addition of Salmonella Enteritidis by 1 h. Similarly, the reduced adhesion of Salmonella to the HCT-116 cells was observed along with the down regulation of hilA gene of Salmonella Pathogenicity Island 1 (SPI1) indicating that they might have acted synergistically to decrease the invasion of the pathogen into the cell line. Conclusions KSBT 56 strain effectively inhibited the growth, invasion and the biofilm forming ability of Salmonella Enteritidis without inhibiting the growth of other Lactobacillus strains. Overall, our result suggested that KSBT 56 can be used as a potential probiotic strain with considerable beneficial effects on the host. Keywords: Probiotic, Lactobacillus, Salmonella, HCT-116, Cell free culture supernatant, Author(s): Jugal Kishore Das[sup.1] , Debasmita Mishra[sup.1] , Pratikshya Ray[sup.1] , Prangya Tripathy[sup.1] , Tushar K Beuria[sup.2] , Neera Singh[sup.1] and Mrutyunjay Suar[sup.1] Background Salmonella enterica is a major food [...]

Background: Salmonella enterica serovar Enteritidis, the most common cause of human gastroenteritis, employs several virulence factors including lipopolysaccharide (LPS) for infection and establishment of disease inside the host. The LPS of S. enterica serovar Enteritidis consists of lipid A, core oligosaccharide and O-antigen (OAg). The OAg consists of repeating units containing different sugars. The sugars of OAg are synthesized and assembled by a set of enzymes encoded by genes organized into clusters. Present study focuses on the effect of deletion of genes involved in biosynthesis of OAg repeating units on resistance to antimicrobial peptides and virulence in mice. Methods: In the present study, the OAg biosynthesis was impaired by deleting tyv, prt and wbaV genes involved in tyvelose biosynthesis and its transfer to OAg. The virulence phenotype of resulting mutants was evaluated by assessing resistance to antimicrobial peptides, serum complement, adhesion, invasion and in vivo colonization. Results: Deletion of the above three genes resulted in the production of OAg-negative LPS. All the OAg-negative mutants showed phenotype reported for rough strains. Interestingly, ΔwbaV mutant showed increased resistance against antimicrobial peptides and normal human serum. In addition, the ΔwbaV mutant also showed increased adhesion and invasion as compared to the other two O-Ag negative mutants Δtyv and Δprt. In vivo experiments also confirmed the increased virulent phenotype of ΔwbaV mutant as compared to Δprt mutant. Conclusion: OAg-negative mutants are known to be avirulent; however, this study demonstrates that certain OAg negative mutants e.g. ΔwbaV may also show resistance to antimicrobial peptides and cause colitis in Streptomyces pretreated mouse model. [ABSTRACT FROM AUTHOR]

Background: Salmonella enterica serovar Enteritidis infections are known to exhibit worldwide prevalence with increased morbidity and mortality. The conventional strategies like antibiotic therapy and vaccination have not only proved to be of sub-optimal efficacy but also led to the development of multidrug resistant strains of Salmonella. Antimicrobial activities of probiotics against various enteropathogens and other health promoting effects have assumed greater significance in recent years. The present study aims to evaluate the efficacy of a Lactobacillus plantarum strain (KSBT 56, isolated from a traditional food product of India), in preventing Salmonella enterica serovar Enteritidis growth and pathogenicity in vitro. Methods and results: The cell free culture supernatant (CFCS) of KSBT 56 strain notably inhibited the growth of Salmonella Enteritidis without affecting the growth of other gram-positive lactic acid bacteria. The isolated KSBT 56 strain produces lactic acid similar to other standard probiotic strains like Lactobacillus plantarum MTCC 1407. The free radical production by KSBT 56 strain was studied by using sodC mutant of S. Enteritidis, which exhibited reduced growth in the presence of CFCS of the KSBT 56 strain, indicating the inhibitory activity of free radicals on the growth of S. Enteritidis. Our results also showed a significant reduction in the biofilm forming ability of Salmonella Enteritidis in the presence of the KSBT 56 strain (2 log cfu/ml, p = 0.01). Further, the anti-infective characteristics of KSBT 56 strain was validated by gentamicin protection assay which revealed 80% reduction in the invasion of Salmonella Enteritidis to HCT-116 cell line (Salmonella Enteritidis and KSBT 56 in a 1:1 ratio) and delayed addition of Salmonella Enteritidis by 1 h. Similarly, the reduced adhesion of Salmonella to the HCT-116 cells was observed along with the down regulation of hilA gene of Salmonella Pathogenicity Island 1 (SPI1) indicating that they might have acted synergistically to decrease the invasion of the pathogen into the cell line. Conclusions: KSBT 56 strain effectively inhibited the growth, invasion and the biofilm forming ability of Salmonella Enteritidis without inhibiting the growth of other Lactobacillus strains. Overall, our result suggested that KSBT 56 can be used as a potential probiotic strain with considerable beneficial effects on the host. [ABSTRACT FROM AUTHOR]

Background: Non-typhoidal Salmonella (NTS) infections caused primarily by S. Enteritidis and S. Typhimurium particularly in immunocompromised hosts have accounted for a large percentage of fatalities in developed nations. Antibiotics have revolutionized the cure of enteric infections but have also led to the rapid emergence of pathogen resistance. New powerful therapeutics involving metabolic enzymes are expected to be potential targets for combating microbial infections and ensuring effective health management. Therefore, the need for new antimicrobials to fight such health emergencies is paramount. Enteric bacteria successfully evade the gut and colonize their hosts through specialized virulence strategies. An important player, alanine racemase is a key enzyme facilitating bacterial survival. Results: This study aims at understanding the contribution of alanine racemase genes alr, dadX and SEN3897 to Salmonella survival in vitro and in vivo. We have shown SEN3897 to function as a unique alanine racemase in S. Enteritidis which displayed essential alanine racemase activity. Interestingly, the sole presence of this gene in alr dadX double mutant showed a strict dependence on d-alanine supplementation both in vitro and in vivo. However, Alr complementation in d-alanine auxotrophic strain restored the alanine racemase deficiency. The Km and Vmax of SEN3897 was 89.15 ± 10.2 mM, 400 ± 25.6 µmol/(min mg) for l-alanine and 35 ± 6 mM, 132.5 ± 11.3 µmol/(min mg) for d-alanine, respectively. In vitro assays for invasion and survival as well as in vivo virulence assays involving SEN3897 mutant showed attenuated phenotypes. Further, this study also showed attenuation of d-alanine auxotrophic strain in vivo for the development of potential targets against Salmonella that can be investigated further. Conclusion: This study identified a third alanine racemase gene unique in S. Enteritidis which had a potential effect on survival and pathogenesis in vitro and in vivo. Our results also confirmed that SEN3897 by itself wasn't able to rescue d-alanine auxotrophy in S. Enteritidis which further contributed to its virulence properties. [ABSTRACT FROM AUTHOR]