Influence of corn starch based bio-active edible coating containing fumaric acid on the lipid quality and microbial shelf life of silver pomfret fish steaks stored at 4 °C.

The present study aimed at assessing the impact of addition of fumaric acid (0.5%), as an active agent, in a corn starch (2%) based edible coating, on the lipid quality and microbial shelf life of silver pomfret (Pampus argenteus) fish steaks stored at 4 °C. Treating fish steaks with FA resulted in a bacteriostatic effect leading to reduced counts of total mesophilic and psychrotrophic bacteria, H2S producing bacteria and Pseudomonas spp. The total mesophilic bacterial count of uncoated control sample exceeded the permissible limit of 7 log cfu g-1 on 6th day and had the lowest microbial shelf life. FA incorporation in the CS coating improved the microbial stability of fish steaks resulting in a shelf life of 15 days. The outcomes of the study suggest that CS based coating is beneficial in delaying lipid oxidation as displayed by the lower TBA and PV values while FA is an effective agent for further increasing the preservative action of CS coating by significantly inhibiting microbial growth as well as lipid quality deterioration, which could be exploited by the seafood industry as an active packaging component.

Pufferfish poisoning from Arothron stellatus: The first confirmed case in India with exact DNA sequencing-based species identification.

A puffer fish poisoning case was reported from the coastal city of Veraval in the Gujarat state of India with patient reporting symptoms of giddiness, vertigo, aphasia and heaviness of head following consumption of cooked fish. Treatment was purely symptomatic and supportive. The patient was discharged from the hospital in a stable condition after 4 days. The suspected fish species was later identified using DNA (Deoxyribonucleic Acid) sequencing as Arothron stellatus with 100% identity.

Performance evaluation of a selective-enrichment-isolation protocol for Salmonella enterica from seafood.

In this study using 57 finfish samples of marine origin, selective enrichment in Rappaport-Vassiliadis (RV) broth followed by isolation on the Hektoen enteric agar (HEA) yielded 50 (53.2%) of 94 isolates. The results suggest RV-HEA as the most suitable media combination for the recovery of Salmonella from tropical seafood.

Assessment of structural chromosomal instability phenotypes as biomarkers of carboplatin response in triple negative breast cancer: the TNT trial.

BACKGROUND: In the TNT trial of triple negative breast cancer (NCT00532727), germline BRCA1/2 mutations were present in 28% of carboplatin responders. We assessed quantitative measures of structural chromosomal instability (CIN) to identify a wider patient subgroup within TNT with preferential benefit from carboplatin over docetaxel. PATIENTS AND METHODS: Copy number aberrations (CNAs) were established from 135 formalin-fixed paraffin-embedded primary carcinomas using Illumina OmniExpress SNP-arrays. Seven published [allelic imbalanced CNA (AiCNA); allelic balanced CNA (AbCNA); copy number neutral loss of heterozygosity (CnLOH); number of telomeric allelic imbalances (NtAI); BRCA1-like status; percentage of genome altered (PGA); homologous recombination deficiency (HRD) scores] and two novel [Shannon diversity index (SI); high-level amplifications (HLAMP)] CIN-measurements were derived. HLAMP was defined based on the presence of at least one of the top 5% amplified cytobands located on 1q, 8q and 10p. Continuous CIN-measurements were divided into tertiles. All nine CIN-measurements were used to analyse objective response rate (ORR) and progression-free survival (PFS). RESULTS: Patients with tumours without HLAMP had a numerically higher ORR and significantly longer PFS in the carboplatin (C) than in the docetaxel (D) arm [56% (C) versus 29% (D), PHLAMP,quiet = 0.085; PFS 6.1 months (C) versus 4.1 months (D), Pinteraction/HLAMP = 0.047]. In the carboplatin arm, patients with tumours showing intermediate telomeric NtAI and AiCNA had higher ORR [54% (C) versus 20% (D), PNtAI,intermediate = 0.03; 62% (C) versus 33% (D), PAiCNA,intermediate = 0.076]. Patients with high AiCNA and PGA had shorter PFS in the carboplatin arm [3.4 months (high) versus 5.7 months (low/intermediate); and 3.8 months (high) versus 5.6 months (low/intermediate), respectively; Pinteraction/AiCNA = 0.027, Padj.interaction/AiCNA = 0.125 and Pinteraction/PGA = 0.053, Padj.interaction/PGA = 0.176], whilst no difference was observed in the docetaxel arm. CONCLUSIONS: Patients with tumours lacking HLAMP and demonstrating intermediate CIN-measurements formed a subgroup benefitting from carboplatin relative to docetaxel treatment within the TNT trial. This suggests a complex and paradoxical relationship between the extent of genomic instability in primary tumours and treatment response in the metastatic setting.

Tropical shrimp aquaculture farms harbour pathogenic Vibrio parahaemolyticus with high genetic diversity and Carbapenam resistance.

In characterization of food borne pathogens from the environment, assessment of virulence, genetic diversity and AMR are essential preludes to formulate preventive strategies and to combat the spread. This study aimed to identify and characterize pathogenic Vibrio parahaemolyticus in the coastal aquaculture farms of Kerala, India. Twenty-seven ß-haemolytic V. parahaemolyticus were isolated from 7 out of 40 farms studied. Among the 27 isolates, 15 possessed the tdh gene and 4 had trh. ERIC PCR and PFGE illustrated the presence of pathogenic isolates that shared genetic similarity with clinical strains. One pathogenic isolate was identified to be multidrug resistant (MDR) and 59% exhibited a MAR index of 0.2 or above. Seventy four percent of the pathogenic isolates were ESBL producers and 3.7% of them were carbapenemase producers phenotypically. This asks for adoption of control measures during farming to prevent the transmission of pathogenic V. parahaemolyticus to the environment and food chain.

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon.

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

Prevalence, Virulence Characterization, AMR Pattern and Genetic Relatedness of Vibrio parahaemolyticus Isolates From Retail Seafood of Kerala, India.

Vibrio parahaemolyticus, a halophilic bacterium often found in the marine or estuarine environment is a well-known enteropathogen responsible for foodborne outbreaks associated with seafood. The pathogenic strains of V. parahaemolyticus are marked by the presence of thermostable direct hemoylsin (tdh) and/or TDH related hemolysin (trh) genes. This study aimed to investigate the prevalence and characteristics of potentially pathogenic V. parahaemolyticus in selected retail markets of Cochin, Kerala, along the south-western coast of the Indian subcontinent. One hundred samples collected from 10 retail markets were analyzed for the presence of pathogenic isolates of V. parahaemolyticus. Out of the 721 presumptive isolates, 648 were confirmed to be V. parahaemolyticus by toxR gene amplification, among which 29 were Kanagawa phenomenon (KP) positive. Among these potentially pathogenic isolates, 17 possessed the tdh gene whereas none of them had the trh gene. The faint amplification bands produced during the amplification of tdh gene from two isolates was confirmed by sequencing. Multiplex O serotyping identified O1 serotype as the most prevalent serotype among the 29 potentially pathogenic isolates. Further, studies on the pandemic nature of these isolates revealed that 14 of the 29 were positive for the PGS-PCR, whereas all the isolates were negative for GS-PCR and HU-α PCR. The antibiogram of the isolates revealed that three isolates had significant Multiple Antibiotic Resistance (MAR) index of 0.2 or above. Pathogenic isolates resistant to second, third and fourth generation Cephalosporins were found to be present in the seafood studied. The molecular fingerprinting studies using ERIC-PCR, and PFGE revealed that three of these isolates shared close genetic similarities with the clinical strains. The environmental and seafood isolates that produced faint amplification bands during the amplification of tdh gene suggests that the tdh gene often goes undetected in environmental isolates. The conventional methods used to identify the pathogenic V. parahaemolyticus would be good for clinical isolates, but a more elaborate method is recommended for the detection of tdh gene in environmental isolates. This is the first comprehensive study on pathogenic V. parahaemolyticus in Kerala, India and demonstrates for the first time, the isolation of potentially pathogenic V. parahaemolyticus, carrying tdh gene from seafood collected from retail markets in Kerala.

Prevalence of malnutrition in medical and surgical gastrointestinal outpatients.

BACKGROUND: UK NICE guidelines, state that patients attending an outpatient clinic for the first time, should be screened for malnutrition. AIMS: To determine the prevalence of malnutrition in the medical and surgical gastroenterology outpatient department (OPD) using body mass index (BMI) and % weight loss (%WL) and to assess the physicians'/surgeons' response to malnutrition being detected. METHODS: The BMI and the %WL were determined for every patient over a 2 week period before the clinician saw the patient. The BMI and %WL were scored as in the Malnutrition Universal Screening Tool (MUST). RESULTS: 605 patients (316 females) of mean age 54 years were included. 150 (25%) were new patients. 519 (86%) had a normal BMI and %WL. 86 (14%) had a BMI <20 kg/m2 or had 5% WL. 61 (10%) were in MUST "medium risk" and 25 (4%) were in MUST "high risk" of malnutrition. 15 (60%) of the "high risk" patients were under the care of or had been referred to a dietitian compared to 19 (28%) of "medium risk" patients. The prevalence of malnutrition was independent of sex, age, history of previous surgery or underlying comorbidities. There was no difference in the prevalence of malnutrition between new and follow up patients. Malnutrition was more common in patients with IBD (38, 18%) vs non-IBD (48, 12%) and patients with cancer (11, 25%) vs non cancer (75, 13%) (p < 0.05). CONCLUSIONS: The prevalence of malnutrition in medical and surgical gastrointestinal outpatients was 14%. IBD and cancer patients had the highest prevalence. Most patients with malnutrition (52, 61%) were not being seen by a dietitian.

Multilocus Sequence Typing and Staphylococcal Protein A Typing Revealed Novel and Diverse Clones of Methicillin-Resistant Staphylococcus aureus in Seafood and the Aquatic Environment.

Methicillin-resistant Staphylococcus aureus (MRSA) has been a global health concern since the 1960s, and isolation of this pathogen from food-producing animals has been increasing. However, little information is available on the prevalence of MRSA and its clonal characteristics in seafood and the aquatic environment. In this study, 267 seafood and aquatic environment samples were collected from three districts of Kerala, India. Staphylococcal protein A (spa) typing and multilocus sequence typing (MLST) was performed for 65 MRSA strains isolated from 20 seafood and aquatic environment samples. The MRSA clonal profiles were t657-ST772, t002-ST5, t334-ST5, t311-ST5, t121-ST8, t186-ST88, t127-ST1, and two non-spa assignable strains. Whole spa gene sequence analysis along with MLST confirmed one strain as t711-ST6 and another as a novel MRSA clone identified for the first time in seafood and the aquatic environment with a t15669 spa type and a new MLST profile of ST420-256-236-66-82-411-477. The MRSA strains were clustered into five clonal complexes based on the goeBURST algorithm, indicating high diversity among MRSA strains in seafood and the aquatic environment. The novel clone formed a separate clonal complex with matches to three loci. This study recommends large-scale spa typing and MLST of MRSA isolates from seafood and the aquatic environment to determine the prevalence of new MRSA clones. This monitoring process can be useful for tracing local spread of MRSA isolates into the seafood production chain in a defined geographical area.

Occurrence of viral pathogens in Penaeus monodon post-larvae from aquaculture hatcheries.

Viral pathogens appear to exert the most significant constraints on the growth and survival of crustaceans under culture conditions. The prevalence of viral pathogens White Spot Syndrome Virus (WSSV), Hepatopancreatic Parvo Virus (HPV), Monodon Baculo Virus (MBV) and Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) in Penaeus monodon post-larvae was studied. Samples collected from different hatcheries and also samples submitted by farmers from Kerala were analyzed. Out of 104 samples collected, WSSV was detected in 12.5% of the post-larvae samples. Prevalence of concurrent infections by HPV, MBV and WSSV (either dual or triple infection) was present in 60.6% of the total post-larvae tested. Out of the 51 double positives, 98% showed either HPV or IHHNV infection. HPV or IHHNV was detected in 11 post-larval samples showing triple viral infection. This is the first report of IHHNV from India. Result of this study reveals the lack of efficient screening strategies to eradicate viruses in hatchery reared post-larvae.

White spot syndrome virus infection: Threat to crustacean biodiversity in Vembanad Lake, India.

The Vembanad Lake located on the south-west coast of India, an ecological hotspot is the nursing ground of many economically important crustaceans. The prevalence of white spot syndrome virus (WSSV) among crustaceans from farmed, estuarine and marine environments surrounding the Vembanad Lake, India was detected using PCR. A total of 308 samples from aquaculture ponds consisting of six species of crustaceans collected from five different farms were tested for the presence of WSSV. Of these, 67% were found to carry the virus. A total of 258 samples of crustaceans from the Cochin backwater system that forms a part of the Vembanad lake viz., Metapenaeus dobsoni, Metapenaeus monoceros, Penaeus monodon and Penaeus indicus were found to contain WSSV in 62% of the samples. Fifteen species of crustaceans caught from the seas off Cochin were also screened for the presence of WSSV. Out of these, twelve species had WSSV incidence levels ranging from 6-23%. WSSV was not detected from three species of deep sea crustaceans tested. The black tiger shrimp, Penaeus monodon had the highest incidence of WSSV among the species screened in farmed, estuarine and marine environments.

Draft Genome Sequence of the Halophilic and Highly Halotolerant Gammaproteobacteria Strain MFB021.

We report the 4.25-Mbp first draft sequence of Gammaproteobacteria strain MFB021, a moderate halophile isolated from petroleum-contaminated soil in Cochin, India. The genome of the strain MFB021 was sequenced to understand the mechanism of hydrocarbon degradation and the halophilicity of the bacterium.

First draft genome sequence of a member of the genus mangrovibacter, isolated from an aquaculture farm in India.

Mangrovibacter sp. MFB070, a Gram-negative, facultatively anaerobic, nitrogen-fixing bacterium, was isolated from an aquaculture farm in Cochin, India. Here, we report the first draft genome sequence of a member of the genus Mangrovibacter, which may help us to elucidate the evolutionary status of this genus. The draft genome sequence of the Mangrovibacter sp. consists of 5,361,682 bp, encoding 4,971 predicted coding sequences in 57 contigs.

Dynamic changes in dna methylation status in peripheral blood Mononuclear cells following an acute bout of exercise: Potential impact of exercise-induced elevations in interleukin-6 concentration.

The aim of the present study was to examine the relationship between interleukin (IL)-6 concentrations and DNA methylation in the peripheral blood mononuclear cells (PBMCs) of trained runners after a bout of prolonged, strenuous exercise. Eight healthy trained males completed a treadmill run at 60% vVO(2max) for 120 min followed by a 5-km time trial in a fasted condition. Whole blood samples were taken prior to, immediately before and 24 h following exercise. From these samples, PBMCs were isolated for analysis and plasma IL-6 concentrations were measured. The methylation status of DNA extracted from PBMCs was analysed using the Illumina 27k methylation beadchip platform. Global DNA methylation status was unaltered immediately and up to 24 hours following a bout of prolonged exercise in comparison to pre-exercise. Despite no change in global DNA methylation, plasma IL-6 concentrations were significantly related to the DNA methylation status of 11 genes. Our study demonstrates that the methylome is stable, while discovering a novel link between exercise-induced increases in circulating IL-6 and the DNA methylation status of 11 individual genes. Based on our preliminary findings, the mechanisms by which changes in plasma IL-6 concentrations and DNA methylation in response to exercise interact require further study.

Differential gene expression profile of the hepatopancreas of white spot syndrome virus infected Fenneropenaeus indicus by suppression subtractive hybridization.

Suppression Subtractive Hybridization was employed in order to identify the differentially expressed genes in the hepatopancreas of white spot syndrome virus infected Fenneropenaeus indicus. A forward subtracted cDNA library generated 356 clones following a white spot syndrome virus infection. A total of 345 clones with more than 100 nucleotides were selected for further analysis using bioinformatics tools after vector screening. Twenty-three contigs and 111 singletons were generated from a total of 134 consensuses. The consensuses, on a sequence homology search using BLASTX (NCBI), revealed that 74 (55%) of them had no significant match to reported sequences in the database, suggesting that they were found for the first time and are probably associated with shrimp immune function. Out of the remaining 60 (45%) consensuses, 43 had significant homology to known protein sequences in the database while 17 consensuses are homologous to unknown proteins in the database which are considered novel. The most abundant genes in the subtracted library were antimicrobial peptides accounting for 56 clones; among which one is a member of SNF2 family of proteins and another belonged to PfP1 family of proteins on analysis using Antimicrobial peptide predictor software. The other predicted genes in the subtracted library include signal transduction molecules (GTPase, Serine threonine kinase, Armadillo repeats etc), antioxidant enzymes (Cytochrome oxidase, Monomeric sarcosine oxidase and Catalase), active transporters (Nuclear Localization Signal [NLS], calcium ATPase, sodium glutamate symporter, Store-Operated Calcium Entry [SOCE] and ribonucleoprotein [RNP]) contributing to 19, 14 and 5 clones respectively. Three clones are homologous to reverse transcriptase; a first time report in shrimp and one each belong to cell adhesion molecule and Proteinase. InterProScan at EMBL, when used for an integrated search at PROSITE predicted; signal sequences and transmembrane regions for 13 clones. This is the first report on the differential gene expression in WSSV-infected F. indicus. The high expression of immune related genes in response to virus infection in shrimp will provide a new insight into the crustacean innate immunity. Further work on the functionality of the unknown genes in shrimps will give an overview on the role of the differentially expressed genes during viral infection and increase our understanding for developing antiviral measures by making use of the shrimp defense mechanism.

Functional characterization of trehalose biosynthesis genes from E. coli: an osmolyte involved in stress tolerance.

Trehalose (1-alpha-D-glucopyranosyl-1-alpha-D-glucopyranoside), a non-reducing disaccharide is a major compatible solute, which maintains fluidity of membranes and protects the biological structure of organisms under stress. In this study, trehalose-6-phosphate synthase (otsA) and trehalose-6-phosphate phosphatase (otsB) genes encoding for trehalose biosynthesis from Escherichia coli was cloned as an operon and expressed in E. coli M15(pREP4). The recombinant E. coli strain showed a threefold increase in the activity of otsBA pathway enzymes, compared to the control strain. The transgenic E. coli accumulated up to 0.86 mg/l of trehalose. The sequence of otsA and otsB genes reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in the altered amino acid sequences of the translated proteins.

Molecular screening, isolation, and characterization of enterohemorrhagic Escherichia coli O157:H7 from retail shrimp.

Foodborne outbreaks attributed to the contamination of foods with enterohemorrhagic Escherichia coli (EHEC) O157:H7 are a growing global concern. Fish and shrimp samples obtained from different retail fish markets in Cochin, India, were screened by direct PCR assays targeting three important virulence markers of EHEC, the intimin protein (eaeA gene), enterohemolysin (hlyA gene), and Shiga toxin (stx gene). One shrimp (Fenneropenaeus indicus) sample was positive for all these virulence markers, and seven typical E. coli O157:H7 isolates were recovered from the marker-positive shrimp sample. This is the first report of recovery of typical E. coli O157:H7 from fish or shellfish in India. All the typical EHEC isolates had a characteristic reaction in eosin methylene blue agar and belonged to IMViC (indole, methyl red, Voges Proskauer, Simmons citrate reactions) biotype I. These isolates also were negative for sorbitol and methylumbelliferyl-beta-glucuronide and exhibited beta-hemolytic activity. One isolate showed self-agglutination for E. coli O157 antisera and produced a false-positive reaction with CHROMagar O157. These typical EHEC isolates belonged to a restricted biotype group and had a very low multiple antibiotic resistance index. Isolation of E. coli O157:H7 in fish and shellfish indicates that strict adherence to hygienic handling methods and proper cooking or processing is needed before consumption of these products.

Characterization and phylogenetic analysis of ectoine biosynthesis genes from Bacillus halodurans.

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), is a natural compound, which serves as a protective substance in many bacterial cells. In this study, the putative ectABC gene cluster from Bacillus halodurans was heterologously expressed in E. coli and the production of ectoine was confirmed by HPLC analysis. The activity of the enzymes coded by the ectA, B and C genes were found to be higher in induced transgenic cells compared to the uninduced cells. Phylogenetic analysis revealed sequence identities ranging from 36-73% for ectA gene, 55-81% for ectB gene and 55-80% for ectC gene indicating that the enzymes are evolutionarily well conserved.

Cloning and heterologous expression of ectoine biosynthesis genes from Bacillus halodurans in Escherichia coli.

The genes involved in the biosynthetic pathway of ectoine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) from Bacillus halodurans were cloned as an operon and expressed in E. coli. Analysis of the deduced ectoine biosynthesis cluster amino acid sequence revealed that the ectoine operon contain 2,389 bp, encoded by three genes; ectA, ectB and ectC that encode proteins of 189, 427 and 129 amino acids with deduced molecular masses of 21,048, 47,120 and 14,797 Da respectively. Extracts of induced cells showed two bands at 41 kDa and 17 kDa, possibly corresponding to the products of the later two genes. However the expression of ectA gene could not be ascertained by SDS-PAGE. The activity of the ectA protein was confirmed by an acylation assay. The transgenic E. coli accumulated upto 4.6 mg ectoine/l culture. This is the first report of an engineered E. coli strain carrying the ectoine genes of the alkaliphilic bacterium, B. halodurans.

Control of intestinal inflammation by regulatory T cells.

Regulatory T(Treg)-cell populations have been identified in a number of disease models. In this review we focus on the role of naturally occurring Treg cells in the control of intestinal inflammation. Specifically, we discuss their mechanism of action with particular emphasis on the role of anti-inflammatory cytokines and cell surface molecules.