Anti-virulence activity of polyphenolic fraction isolated from Kombucha against Vibrio cholerae.

The use of traditional foods and beverages or their bioactive compounds as anti-virulence agents is a new alternative method to overcome the increased global emergence of antimicrobial resistance in enteric pathogens. In the present study, we investigated the anti-virulence activity of a polyphenolic fraction previously isolated from Kombucha, a 14-day fermented beverage of sugared black tea, against Vibrio cholerae O1. The isolated fraction was mainly composed of the polyphenols catechin and isorhamnetin. The fraction, the individual polyphenols and the combination of the individual polyphenols significantly inhibited bacterial swarming motility and expression of flagellar regulatory genes motY and flaC, even at sub-inhibitory concentrations. The polyphenolic compounds also decreased bacterial protease secretion and mucin penetration in vitro. In vivo study revealed that the polyphenolic fraction significantly inhibited V. cholerae induced fluid accumulation in the rabbit ileal loop model and intestinal colonization in suckling mice model. Therefore, the anti-virulence activity of the Kombucha polyphenolic fraction involved inhibition of motility and protease secretion of V. cholerae, thus preventing bacterial penetration through the mucin layer as well as fluid accumulation and bacterial colonization in the intestinal epithelial cells. The overall results implied that Kombucha might be considered as a potential alternative source of anti-virulence polyphenols against V. cholerae. To the best of our knowledge, this is the first report on the anti-virulence activity of Kombucha, mostly attributed to its polyphenolic content.

A tetravalent Shigella outer membrane vesicles based candidate vaccine offered cross-protection against all the serogroups of Shigella in adult mice.

In today's world and mostly in low and middle income countries, Shigella flexneri and Shigella sonnei remains the major causative agent of clinical bacillary dysentery. Based on contemporary epidemiology, a tetravalent Outer Membrane Vesicle (OMVs) based immunogen was formulated using the most commonly circulating Shigella strains, namely, S. flexneri 2a, S. flexneri 3a, S. flexneri 6 and S. sonnei I, in a 1:1:1:1 ratio. Adult BALB/c mice were orally immunized in a prime-boost-boost manner. Tetravalent Shigella OMVs immunogen induced significant and persistent serum and mucosal antibodies against OMVs, Outer Membrane Proteins (OMPs) and lipopolysaccharides (LPS). Tetravalent OMVs also primed cell mediated immune response effectively. Protective efficacy against six heterologous Shigella strains was checked in an intra-peritoneal mouse model. Immunized mice survived lethal infection better than the non-immunized mice cohort with fewer replicating bacteria isolated from their gut. This study establishes the possibilities of tetravalent OMVs immunogen to become a potent vaccine candidate against human shigellosis, overcoming the limitations of sero-specific cross-protection of Shigella species.

Bivalent non-typhoidal Salmonella outer membrane vesicles immunized mice sera confer passive protection against gastroenteritis in a suckling mice model.

Invasive non-typhoidal Salmonella (iNTS) serovars, especially Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE), cause gastroenteritis worldwide. Due to the emergence of multi-drug resistance in iNTS, a broad-spectrum vaccine is urgently needed for the prevention of iNTS infection. Currently, there is no effective licensed vaccine against iNTS available in the market. We have formulated an outer membrane vesicles (OMVs) based bivalent immunogen as a vaccine candidate to generate broad-spectrum protective immunity against both recently circulating prevalent ST and SE. We have isolated OMVs from ST and SE and formulated the immunogen by mixing both OMVs (1:1 ratio). Three doses of bivalent immunogen significantly induced humoral immune responses against lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) as well as a cell-mediated immune response in adult mice. We also observed that proteins of OMVs act as an adjuvant for generation of high levels of anti-LPS antibodies through T cell activation. We then characterized the one-day old suckling mice model for both ST and SE mediated gastroenteritis and used the model for a passive protection study. In the passive protection study, we found the passive transfer of bivalent OMVs immunized sera significantly reduced ST and SE mediated colonization and gastroenteritis symptoms in the colon of suckling mice compared to non-immunized sera recipients. The overall study demonstrated that OMVs based bivalent vaccine could generate broad-spectrum immunity against prevalent iNTS mediated gastroenteritis. This study also established the suckling mice model as a suitable animal model for vaccine study against iNTS mediated gastroenteritis.

Studies on formulation of a combination heat killed immunogen from diarrheagenic Escherichia coli and Vibrio cholerae in RITARD model.

Multiple diarrheagenic enteric bacterial infections cause global morbidity and mortality. A combination vaccine is needed to combat different diarrhea-causing organisms. In our present work, we formulated a combination of antigens from three different diarrheagenic Escherichia coli strains and three different Vibrio cholerae strains. We demonstrated that our newly formulated combination immunogen was able to raise species-specific immunogenicity. This formulation also gave protection against different diarrheagenic E. coli strains in the removable intestinal tie-adult rabbit diarrhea model. However, protective efficacy was not found against the V. cholerae El Tor Ogawa Haitian variant, but challenged with V. cholerae El Tor Inaba or O139 showed protection in rabbits. This is the first report of a single formulated nonliving heat-killed combination immunogen from different diarrheagenic E. coli and V. cholerae that could bestow protection against different bacteria in an animal model.

A brief review on the immunological scenario and recent developmental status of vaccines against enteric fever.

Enteric fever has been one of the leading causes of severe illness and deaths worldwide. S. Typhi and S. Paratyphi A, B and C are important enteric fever-causing organisms globally. This infection causes about 21 million cases among which 222,000 typhoid related deaths occurred in 2015. These estimates do not reflect the ultimate and real status of the disease due to the lack of unified diagnostic and proper reporting system from typhoid endemic and other regions. Current control strategies have become increasingly ineffective due to the emergence of multi-drug resistance among the strains. This situation worsens the disease-burden in developing as well as in developed countries. Moreover the emergence of S. Paratyphi A as a major enteric fever-causing organism in several Asian countries, demands a prophylactic measure at this hour. Other than two licensed vaccines of S. Typhi, there are no exsisting vaccines for S. Paratyphi A. Moreover, travelers returning from endemic regions are becoming more susceptible to have these infections. In this situation, a need for bivalent approach is required where a single immunogen (consisting from each organism) will be effective against the disease. In this review, we have focused on the general information about typhoidal fever, its spread and epidemiology in brief and the present status of typhoidal vaccines and its future. This review highlights existing gaps in the typhoidal salmonellae research with a special emphasis on the status of present typhoidal salmonellae vaccine research.

Retinoic acid pre-treatment down regulates V. cholerae outer membrane vesicles induced acute inflammation and enhances mucosal immunity.

Bacterial outer membrane vesicles have been extensively investigated and considered as a next generation vaccine. Recently, we have demonstrated that the cholera pentavalent outer membrane vesicles (CPMVs) immunogen induced adaptive immunity and had a strong protective efficacy against the circulating V. cholerae strains in a mouse model. In this present study, we are mainly focusing on reducing outer membrane vesicle (OMV) -mediated toxicity without altering its antigenic property. Therefore, we have selected All-trans Retinoic Acid (ATRA), active metabolites of vitamin A, which have both anti-inflammatory and mucosal adjuvant properties. Pre-treatment of ATRA significantly reduced CPMVs induced TLR2 mediated pro-inflammatory responses in vitro and in vivo. Furthermore, we also found ATRA pre-treatment significantly induced mucosal immune response and protective efficacy after two doses of oral immunization with CPMVs (75µg). This study can help to reduce OMV based vaccine toxicity and induce better protective immunity where children and men suffered from malnutrition mainly in developing countries.

Mice with Streptozotocin-Induced Hyperglycemia are Susceptible to Invasive Enteric Bacterial Infection.

Diabetes mellitus and diarrhea are becoming increasingly burdensome worldwide, particularly in developing countries such as India. Diabetic patients are susceptible to infection with pathogenic bacteria, particularly those causing invasive enteric infections. In this study, we observed changes in the pathophysiological features of mice with streptozotocin-induced hyperglycemia. In our experiments, both hyperglycemic and control mice were infected with pathogenic enteric bacteria-non-typhoidal Salmonella, Shigella flexneri, or Vibrio parahaemolyticus. Morbidity, mortality, and bacterial load were all higher in the diabetic mice than in the control mice, and the phagocytic and bactericidal activities of peritoneal macrophages isolated from hyperglycemic mice were lower than they were in the controls. We hypothesize that hyperglycemia leads to a downregulation of the innate immune response, which in turn increases vulnerability to enteric bacterial infection.

Evaluation of immunogenicity and protective efficacy of combination heat-killed immunogens from three entero-invasive bacteria in rabbit model.

Diarrhea is a very common health problem in both developing and developed countries. Among the major entero-invasive bacteria, Shigella, Salmonella and Campylobacter cause serious problems in different geographic regions. Recently we have shown immunogenicity and protective efficacy of heat killed multi-serotype Shigella immunogen in different animal models. In our present study, we have advanced our research by preparing a combination heat-killed immunogen of three different entero-invasive bacteria, Shigella, Salmonella and Campylobacter. After three doses on 0th, 14th and 28th day of oral immunization with tri-valent heat-killed (TVHK) immunogen in rabbit model, the immunogenicity was determined by differential count of white blood cells and immunoglobulin assay at various time points. During oral immunization differential count of lymphocytes increased where as polymorphonuclear leucocytes (PMN) count decreased. Serum IgG and IgA showed significant elevation during oral immunization and remained at a detectable value upto 120 days. Protection study was performed in both, in vitro and in vivo conditions, using bacteriocidal assay and rabbit ligated ileal loop model, respectively, which conferred protection against homologous bacteria. Moreover, immunoblot assay against whole cell lysate and lipopolysaccharide exhibited significant amount of antigen-specific immunoglobulins raised against three different bacteria which proved that proteins along with lipopolysaccharides played a pivotal role in immunogenicity and protective efficacy. This trivalent heat-killed immunogen could be a low-cost, simple, oral, non-living vaccine candidate for future use against invasive diarrhea.

Retinoic acid decreases the severity of Salmonella enterica serovar Typhimurium mediated gastroenteritis in a mouse model.

Gastroenteritis is a global burden; it's the major cause of morbidity and mortality both in adults and children of developing countries. Salmonella is one of the leading causes of bacteria-mediated gastroenteritis and due to its increasing multidrug antibiotic resistance; Salmonella-mediated gastroenteritis is difficult to control. Retinoic acid, the biologically active agent of vitamin A has an anti-inflammatory effect on experimental colitis. In this study we have shown All trans retinoic acid (ATRA) treatment down regulates Salmonella-mediated colitis in a murine model. Macroscopic signs of inflammation such as decrease in body weight and cecum weight, shorter length of proximal colon and pathological score of colitis were observed less in ATRA treated mice than in a vehicle control group. ATRA treatment not only reduced pro-inflammatory cytokine responses, such as TNF-α, IL-6, IL-1ß, IFN-γ and IL-17 production but also increased IL-10 response in the supernatant of intestinal tissue. Results also suggested that ATRA treatment enhances the number of FoxP3-expressing T regulatory cells in MLN and also decreases bacterial load in systemic organs. We concluded that ATRA treatment indeed reduces Salmonella Typhimurium-mediated gastroenteritis in mice, suggesting it could be an important part of an alternative therapeutic approach to combat the disease.

Zebrafish as a novel model for non-typhoidal Salmonella pathogenesis, transmission and vaccine efficacy.

Salmonella-induced gastroenteritis causes massive morbidity and mortality in both adults and children of developing countries. However, it is difficult to study the mode of infection and vaccine efficacy due to inadequacies of current animal models. For this reason, we have explored using zebrafish as an improved model for non-typhoidal Salmonella (NTS) infection, including Salmonella enterica Typhimurium, Salmonella enterica Enteritidis and Salmonella enterica Weltevreden. In this study, we found that after infection of zebrafish with NTS, severe diarrhea like symptoms were observed and NTS significantly colonized the zebrafish intestine without any manipulation of the normal intestinal microbiota of the fish. Furthermore, these strains can colonize for longer than 72h and induce severe inflammation in the intestine, which may induce fish death. We also found that infected fish can transmit the pathogen into naïve fish. Moreover, we have established that zebrafish is an excellent model for vaccine study. Successive triple bath vaccination with heat-killed single serotype S. Typhimurium and S. Enteritidis immunogen induced protective efficacy against a high dose (10(8)CFU/ml) of infection with these pathogens. This study provides a natural infection model for the study of NTS infection, transmission and vaccine efficacy.