Inhibition of growth and virulence of Vibrio cholerae by carvacrol, an essential oil component of Origanum spp.

AIMS: In the age where bacterial resistance to conventional antibiotics is increasing at an alarming rate, the use of the traditional plant, herb extracts or other bioactive constituents is gradually becoming popular as an anti-virulence agent to treat pathogenic diseases. Carvacrol, a major essential oil fraction of Oregano, possesses a wide range of bioactivities. Therefore, we aimed to study the effect of sub-inhibitory concentrations of carvacrol on major virulence traits of Vibrio cholerae. METHODS AND RESULTS: We have used in vitro as well as ex vivo models to access the anti-pathogenic role of carvacrol. We found that the sub-inhibitory concentration of carvacrol significantly repressed bacterial mucin penetrating ability. Carvacrol also reduced the adherence and fluid accumulation in the rabbit ileal loop model. Reduction in virulence is associated with the downregulated expression of tcpA, ctxB, hlyA and toxT. Furthermore, carvacrol inhibits flagellar synthesis by downregulating the expression of flrC and most of the class III genes. CONCLUSIONS: Carvacrol exhibited anti-virulence activity against V. cholerae, which involved many events including the inhibition of mucin penetration, adhesion, reduced expression of virulence-associated genes culminating in reduced fluid accumulation. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate that carvacrol possesses inhibitory activity against V. cholerae pathogenesis and might be considered as a potential bio-active therapeutic alternative to combat cholera.

Exploring the physical and quality attributes of muffins incorporated with microencapsulated squalene as a functional food additive.

Squalene, a triterpenoid compound is proven to possess immense bioactivities by virtue of its high antioxidant activity. The present study was designed to investigate the quality attributes of muffins as influenced by addition of encapsulated squalene. Nutritional analysis showed that calorific value of prepared muffins has ranged from 480.78 ± 0.10 to 501.61 ± 0.38 kcal. Baking loss was lowest in case of muffins prepared with encapsulated squalene with its crumb region recorded higher moisture content. Color kinetics study indicated that browning index (BI) was higher in crust portion of encapsulated squalene enriched muffins. Scanning electron micrographs showing that muffins with encapsulated squalene had stronger structural organization. This was further supported by the textural studies showed that the muffins with encapsulated squalene was cohesive, springier and chewy with less gumminess and stiffness indicating their efficacy in improving the textural quality. Oxidative stability and microbiological quality were also high in squalene enriched foods suggesting that squalene might have some antimicrobial effects. Outcome of the study indicated that encapsulated squalene can be very well utilised as a functional food ingredient in ready -to-eat functional foods. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s13197-020-04955-9).

Identification of common virulence factors present in enterotoxigenic Escherichia coli isolated from diarrhoeal patients in Kolkata, India.

AIMS: Development of an effective vaccine against enterotoxigenic Escherichia coli (ETEC) is largely dependent on the conscientious understanding of different virulence associated factors from diverse geographical areas. So, the objective of this study is to elucidate the distribution of enterotoxins, CF and NCVF in clinical ETEC strains isolated between 2008 and 2014 from two hospitals in Kolkata, India. METHODS AND RESULTS: Multiplex PCR method was used for detection of two enterotoxin genes, 11 common CFs and five common NCVFs. Among the 350 tested ETEC strains, 61% strains possessed est+elt genes, 25% est and 14% elt. Among 56% CF positive ETEC strains, CS21 was the prevalent one (37%) followed by CS6 (36%). NCVF genes were present in 59% of the ETEC strains; eatA was the most prevalent (65%) followed by etpA (51%). There were 29% strains negative for any CFs or NCVFs. CONCLUSIONS: We conclude that a pattern exists between CS6, eatA and toxins. We observed est with or without elt, CS6 with or without CS5 and with or without eatA were present in 24% of clinical ETEC strains (59/250) analysed. CS21 has emerged as another predominant CF but it had diverse CFs and NCVFs. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence of ETEC virulence factors would help in tracking ETEC globally and suggests the need of a multivalent ETEC vaccine.

Antioxidant, functional properties and amino acid composition of pepsin-derived protein hydrolysates from whole tilapia waste as influenced by pre-processing ice storage.

In recent years, hygienic handling of fishery waste is demanded owing to the fact that the fishery waste is an ideal raw material for the preparation of bioactive compounds. In the present study, the effect of pre-processing storage (at 4 ± 2 °C) of whole tilapia waste (WTW) on the properties of its protein hydrolysate derived using pepsin was evaluated. Fish protein hydrolysates (FPH) were prepared from 0, 24 and 48 h old ice stored WTW and designated as FPH-0, FPH-1, and FPH-2, respectively. Total amino acids, total essential amino acids and total hydrophobic amino acids of FPH samples increased with the storage period of raw material (WTW). Antioxidant activities such as DPPH (2, 2 diphynyl-1-picrylhydrazyl) free radical scavenging activity and ferric reducing power of FPH samples were dose dependent. FPH-0 had better antioxidant properties including linoleic acid peroxidation inhibition activity than FPH-1 and FPH-2. The DNA nicking assay revealed the protective effect of FPH preparations against Fenton's reaction mediated oxidative damage. FPH-2 had better emulsifying properties and foaming stability whereas the FPH-0 had relatively good foaming capacity. SDS-PAGE indicated the presence of peptides ranging from 116 to < 14.4 kDa in FPH-0 and less than 18 kDa in FPH-1 and FPH-2. The present study, clearly demonstrated that whole tilapia waste can effectively be converted to FPH and could be a potential ingredient in functional food and as a rich source of high-quality protein in animal feed formulations.

Phlorotannins-bioactivity and extraction perspectives.

Phlorotannins, a seaweed based class of polyphenolic compounds, have proven to possess potential bioactivities such as antioxidant, antimicrobial, anti-allergic, anti-diabetic, anti-inflammatory, anti-cancerous, neuroprotection etc. These bioactivities have further increased demand globally and sustainable techniques such as supercritical fluid extraction, microwave assisted extraction, enzyme assisted extraction, extraction using deep eutectic solvents etc. are being explored currently for production of phlorotannin-rich extracts. In spite of such well documented bioactivities, very few phlorotannin-based nutraceuticals are available commercially which highlights the significance of generating consumer awareness about their physiological benefits. However, for industry level commercialization accurate quantification of phlorotannins with respect to the different classes is vital requiring sophisticated analytical techniques such as mass spectrometry, 1H-NMR spectroscopy etc. owing to the wide structural diversity. This review summarizes the extraction and bioactivities of phlorotannins based on the findings of in vivo and in vitro studies.

A low-GC Gram-positive Thermoanaerobacter-like bacterium isolated from an Indian hot spring contains Cr(VI) reduction activity both in the membrane and cytoplasm.

AIM: Characterization of an anaerobic thermophilic bacterium and subcellular localization of its Cr(VI)-reducing activity for potential bioremediation applications. METHODS AND RESULTS: 16S rRNA gene sequence-based analyses of bacterial strains isolated from sediment samples of a Bakreshwar (India) hot spring, enriched anaerobically in iron-reducing medium, found them to be 86-96% similar to reported Thermoanaerobacter strains. The most efficient iron reducer among these, BSB-33, could also reduce Cr(VI) at an optimum temperature of 60 degrees C and pH 6.5. Filtered culture medium could reduce Cr(VI) but not Fe(III). Cell-free extracts reduced Cr(VI) inefficiently under aerobic conditions but efficiently anaerobically. Fractionation of the cell-free extracts showed that chromium reduction activity was present in both the cytoplasm and membrane. CONCLUSIONS: BSB-33 reduced Fe(III) and Cr(VI) anaerobically at 60 degrees C optimally. After fractionation, the reducing activity of Cr(VI) was found in both cytoplasmic and membrane fractions. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first systematic study of anaerobic Cr(VI) reduction by a gram-positive thermophilic micro-organism and, in contrast to our results, none of the earlier reports has mentioned Cr(VI)-reducing activity to be present both in the cytoplasm and membrane of an organism. The strain may offer itself as a potential candidate for bioremediation.

Chitosan - Whey protein as efficient delivery system for squalene: Characterization and functional food application.

Squalene, a triterpenoid compound possessing excellent bioactivities, is not being utilized as a functional food ingredient due to its high susceptibility to oxidation. In the present study, the feasibility of chitosan-whey protein as an efficient wall material for squalene encapsulation using spray drying technique was attempted for functional food applications. The encapsulation efficiency of the squalene powder was found to be 75.4 ±â€¯0.22% whereas other physico-chemical properties such as moisture content, flowability, solubility, peroxide value, etc. have shown satisfactory results. The thermogravimetric analysis showed that chitosan-whey protein was able to retain the thermal stability of squalene up to a temperature of 422 °C. Furthermore, the functional food application of the encapsulated squalene in a bakery product (cake) exhibited significantly (p < 0.05) better properties in terms of oxidative stability, sensory attributes than that of cake with pure squalene and control treatment. Hence, it can be concluded that emulsification of squalene in chitosan-whey protein and its subsequent encapsulation by spray drying can be a potential process to produce oxidatively stable encapsulates for the development of functional foods.

Evaluation of chitosan as a wall material for microencapsulation of squalene by spray drying: Characterization and oxidative stability studies.

The present study was aimed at investigating the efficacy of chitosan as a wall material for microencapsulation of squalene by spray drying for functional food applications. Based on different core to wall material ratio (1:1, 0.5:1 and 0.3:1 on w/w basis), emulsions were prepared and evaluated in terms of emulsion stability, particle size, zeta potential, polydispersity Index (PDI), rheology and microstructure. The optimized emulsion combination was spray dried and characterized, physically and chemically. The encapsulation efficiency of the powder was found to be 26±0.6% whereas other properties such as particle size, zeta potential, water activity, hygroscopicity, Carr Index, Hausner ratio have shown satisfactory results. SEM analysis showed that the squalene microcapsules were smooth spherical particles free from dents and fissures. FTIR data further confirmed the encapsulation of squalene with chitosan. However, TGA, oxidative stability and accelerated Rancimat results showed that chitosan was not able to protect squalene from oxidation during storage. The results suggest that chitosan is not an appropriate wall material for microencapsulation of squalene and hence a combination of wall materials could be attempted for the encapsulation of squalene.

Dietary supplementation of thiamine and pyridoxine-loaded vanillic acid-grafted chitosan microspheres enhances growth performance, metabolic and immune responses in experimental rats.

In the present investigation, the effect of dietary supplementation of thiamine and pyridoxine loaded vanillic acid-grafted chitosan microspheres (TPVGC) on growth, metabolic and immune responses in Wistar strain albino rats was studied. Eight experimental groups, namely four groups each for male and female rats were fed with 0, 0.4, 0.8 and 1.6% of TPVGC in the diet. At the end of 45days feeding trials, both male and female rats supplemented with TPVGC had higher weight gain% and specific growth rate than the control groups. Significantly (p<0.05) lower blood glucose level and higher respiratory burst activity were recorded in the treatment groups than the control groups of both male and female rats. Activity of metabolic enzymes (aspartate amino transferase, alanine aminotransferase, alkaline phosphatase and acid phosphatase) and antioxidant enzymes (superoxide dismutase, catalase and glutathione S-transferase) were significantly higher (p<0.05) in the control groups and a decreasing trend in the same was observed with a gradual increase in the inclusion level of TPVGC in the diet of the treatment groups. However, a reverse trend was observed for acetylcholine esterase. It was inferred that dietary supplementation of thiamine and pyridoxine loaded vanillic acid-grafted chitosan enhanced the growth performance, metabolic and immune responses in the animal-model.

A prospective study of phage types & biotypes of Salmonella enterica serotype Typhi isolated from hospitalized children in Kolkata, India.

BACKGROUND & OBJECTIVES: Kolkata and its suburbs in eastern India faced an epidemic of typhoid fever in 1990. A prospective, hospital and laboratory based study over a period of 12 yr (1990-2001), on the phage typing and biotyping pattern of Salmonella enterica serotype Typhi was carried out, to see if there has been a change. METHODS: A total of 338 S. enterica serotype Typhi isolates from 1491 blood samples were phage typed and biotyped. The mean age of isolation was calculated. RESULTS: The age distribution of subjects (neonates to 12 yr) has been analysed. Of the 338 (22.7%) isolates obtained, eight different S. enterica serotype Typhi phage types were detected. Biotype I (95.8%) was more prevalent as compared to biotype II (4.1%). Phage type E1 was the commonest phage type in Kolkata and its suburbs. INTERPRETATION & CONCLUSION: The mean age at isolation was found to be 6.7 +/- 3.3 yr. Biotype I was predominant and it was of interest that all strains of phage type E1 belonged to biotype I.

Effect of environmental factors on expression and activity of chitinase genes of vibrios with special reference to Vibrio cholerae.

AIMS: The aim of this study was to investigate the distribution and inducibility of chitinase genes in vibrios and the effect of environmental factors on the expression level and activity of chitinase genes in Vibrio cholerae strains. METHODS AND RESULTS: Chitin agar plate assays showed that V. cholerae strains were more chitinolytic than non-cholerae vibrios. All of the identified or putative chitinase genes were expressed in V. cholerae (four strains) but not in non-cholerae vibrios (seven species/strains) under standard laboratory growth conditions. In non-cholerae vibrios, these genes were induced by chitin, its monomer N-acetyl-d-glucosamine and on exposure to rabbit intestine, while in V. cholerae strains, these genes showed significant variation in expression levels. To study the effects of environmental factors on the expression and activity of chitinase genes in V. cholerae, bacteria were cultured in different pH, temperature, sodium chloride and nutrients. RT-PCR analysis showed that lower temperatures and higher pH, salinity and nutrition favoured expression of these genes, while their activity increased under higher nutrition content and salinity. CONCLUSIONS: Chitinase genes are distributed in all the relatively small number of strains studied here, and biotic and abiotic factors have significant role in the induction, expression level and activity of this gene family in vibrios. SIGNIFICANCE AND IMPACT OF THE STUDY: Chitinases have important applications especially in recycling of chitin. Vibrios can be used as chitinolytic agents, using suitable culture conditions that maximize the expression and activity of these genes.

A community level syphilis prevention programme: outcome data from a controlled trial.

OBJECTIVES: This study investigated the impact of a small media campaign to reduce syphilis through testing, treatment, and condom use in two urban predominantly African-American communities with high syphilis rates. METHODS: Data were collected from intervention and comparison zip codes using cross sectional street intercept interviews at baseline and 2 years later (n = 1630) following a small media syphilis prevention campaign with role model story posters, billboards, and other merchandise. Community businesses and a community based organisation served as partners, distributing condoms and small media. RESULTS: Comparing intervention with comparison zip codes, there were significant increases in condom use in last sexual act, and some aspects of knowledge of syphilis. However, there was significant cross contamination of media impact, with respondents in the comparison zip code seeing an average of two media items compared with three in the intervention zip code. Media exposure was associated with significant increases in knowledge of syphilis, testing, and condom use. CONCLUSIONS: Targeted community based small media interventions using community partners for distribution are effective in increasing syphilis knowledge, testing, and condom use.

Molecular characterization of the 5' regulatory region of rat sodium-dependent multivitamin transporter gene.

Previous studies have demonstrated the involvement of a specialized, Na(+)-dependent carrier-mediated system for biotin uptake in mammalian intestine. The molecular identity of the carrier protein, the Na(+)-dependent multivitamin transporter (SMVT), has recently been identified. Upon characterization of transcript expression in the rat intestine, four distinct transcript variants (I-IV) due to heterogeneity at the 5'-untranslated region were found (Chatterjee NS, Kumar CK, Ortiz A, Rubin SA, and Said HM. Am J Physiol Cell Physiol 277: C605-C613, 1999). This finding raised the possibility that multiple promoters may be involved in driving the transcription of the SMVT gene. To test this possibility, we cloned the 5' regulatory region of the SMVT gene by genome walking. A 6.5-kb genomic DNA fragment was identified and sequenced. Three putative promoters (P1, P2, and P3) that were separated by exons of the four previously identified transcript variants were, indeed, found. P1 was found to contain multiple putative regulatory regions like GATA-1, AP-1, AP-2, and C/EBP, including several repeats of purine-rich regions and two TATA-like elements. P2 and P3 were GC rich and also revealed the presence of many putative regulatory elements including several SP-1 consensus sequences. The functional identity of each promoter and the minimal regions required for its function were established by the luciferase assay following transfection of rat-derived cultured intestinal epithelial IEC-6 cells. The highest functional activity of the cloned promoters was found to be in the order of P1 > P2 > P3. These findings represent the first characterization of the 5' regulatory region of any mammalian SMVT gene and should assist in the understanding of transcriptional regulation of this important gene.

Molecular mechanism of the intestinal biotin transport process.

Previous studies have characterized different aspects of the cellular/membrane mechanism and regulation of the intestinal uptake process of the water-soluble vitamin biotin. Little, however, is known about the molecular mechanisms of the uptake process. In this study, we have identified a cDNA from rat small intestine that appears to be involved in biotin transport. The open reading frame of this cloned cDNA consisted of 1,905 bases and was identical to that identified for the vitamin transporter in placental tissue. Significant heterogeneity, however, was found in the 5' untranslated region of this clone, with three distinct variants (II, III, IV) being identified in the small intestine; the placental variant (variant I), however, was not present in the small gut. Variant II was found to be the predominant form expressed in the rat small and large intestines. Functional identity of the cloned intestinal cDNA was confirmed by stable expression in COS-7 cells, which showed a four- to fivefold increase in biotin uptake in transfected COS-7 cells compared with controls. The induced biotin uptake in transfected COS-7 cells was found to be 1) Na(+) dependent, 2) saturable as a function of concentration with an apparent K(m) of 8. 77 microM and a V(max) of 779.7 pmol. mg protein(-1). 3 min(-1), and 3) inhibited by unlabeled biotin and pantothenic acid and their structural analogs. The distribution of complementary mRNA transcripts of the cloned cDNA along the vertical and longitudinal axes of the intestinal tract was also determined. Results of this study describe the molecular characteristics of the intestinal biotin absorption process and report the identification of a cDNA that encodes a Na(+)-dependent biotin uptake carrier that appears to exist in the form of multiple variants.