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Marine fish species were analyzed for culturable and total metagenomic microbial diversity, antibiotic resistance (AR) pattern, and horizontal gene transfer in culturable microorganisms. We observed a high AR microbial load of 3 to 4 log CFU g -1 . Many fish pathogens like Providencia, Staphylococcus, Klebsiella pneumoniae, Enterobacter, Vagococcus, and Aeromonas veronii were isolated. Photobacterium and Vibrio were two major fish and human pathogens which were identified in the fish metagenome. Other pathogens that were identified were Shewanella, Acinetobacter, Psychrobacter, and Flavobacterium. Most of these pathogens were resistant to multiple antibiotics such as erythromycin, kanamycin, neomycin, streptomycin, penicillin, cefotaxime, bacitracin, rifampicin, trimethoprim, ciprofloxacin, and doxycycline with a high multiple antibiotic resistance index of 0.54-0.77. The fish microflora showed high prevalence of AR genes like bla TEM , Class I integron, tetA, aph(3')-IIIa, ermB, aadA, and sul1. Nineteen of 26 AR isolates harbored Class I integrons showing high co-resistance to trimethoprim, kanamycin, doxycycline, and cefotaxime. Mobile R-plasmids from 6 of the 12 AR pathogens were transferred to recipient E. coli after conjugation. The transconjugants harbored the same R-plasmid carrying bla CTX-M , dfr1, tetA, bla TEM , and cat genes. This study confirms that fish is a potential carrier of AR pathogens which can enter the human gut via food chain. To the best of our knowledge, this is the first study in the Indian subcontinent reporting a direct evidence of spread of AR pathogens to humans from specific marine fish consumption.

Aeromonas hydrophila has emerged as an important human pathogen as it causes gastroenteritis and extra-intestinal infections. Information regarding the influence of environmental stresses on gene expression profile of A. hydrophila is lacking. The impact of nutrient replenishment, nutrient deprivation, acid stress, and cold shock on housekeeping, general stress-response, and virulence genes was studied using quantitative real-time PCR in two A. hydrophila strains, CECT 839 T , and A331. These sub-lethal stresses invoked significant changes in the expression of these genes in a strain-dependent manner. Overall, nutrient replenishment and deprivation significantly induced the expression of housekeeping (rpoD), general stress regulators (uspA and rpoS), and virulence (aer) genes, indicating their importance in regulating the survival and virulence of A. hydrophila under these stress conditions. rpoS gene was significantly induced under cold shock; whereas, acid stress significantly induced the expression of uspA gene. This is the first study to investigate the effect of environmental parameters on the expression of stress-response and virulence genes in A. hydrophila strains., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Cancer stem cells (CSCs) are increasingly being realized to play a significant role in the mechanism of chemo-, radio-resistance, and metastasis of cancer. However, studies for spectral markers of CSCs using Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy are limited in the literature., Materials and Methods: In the present study, CSCs obtained from single cell assay of human lung adenocarcinoma (A549) cells were characterized using CD44+/CD24-/low phenotype expression, Hoechst 33342 dye efflux assay, and expression of stemness genes. Spectral changes in cancer cells and clones enriched with CSCs were studied by FT-IR and CD spectroscopy., Results: The changes in FT-IR spectra of clones enriched with CSCs showed the difference in the secondary protein structure as compared to nonstem cancer cells. Moreover, A549 clone cells showed higher C-O band of carbohydrates and deoxyribose ring vibrations of Z-form of DNA. These results were further corroborated with CD spectroscopy that showed increased alpha helix proteins and difference in DNA conformation in clones enriched with CSCs. FT-IR studies also showed higher imidazole-metal interactions in clones enriched with CSCs. These results are in agreement with higher activity of one of the metalloproteins that is, superoxide dismutase in clones enriched with CSCs and their increased radioresistance., Conclusions and General Significance: Overall, these observations provide novel FT-IR and CD spectroscopy signatures in A549 clones enriched with CSCs, which may have implications in the quantifying magnitude of CSCs as prognostic markers in cancer therapy.

Aeromonas are regarded as opportunistic as well as primary pathogens of humans and fish, and are associated with gastroenteritis and septicemia in humans. Production of N-acyl-homoserine lactone (AHL) signal molecules and biofilm was determined in 22 Aeromonas isolates, from different food products in India, using thin-layer chromatography (TLC) analysis and microtiter-plate assay, respectively. Overall, highly heterogeneous patterns of AHL production were observed, with the production of N-butanoyl homoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL) by the majority (81.8%) of Aeromonas food isolates. Moreover, putative N-pentanoyl homoserine lactone (C5-HSL), N-heptanoyl homoserine lactone (C7-HSL), and N-octanoyl homoserine lactone (C8-HSL) were produced by 72.7%, 27.3%, and 9.1% of isolates, respectively. This is the 1st report of production of C7-HSL by Aeromonas species. Aeromonas food isolates were highly variable in their biofilm forming abilities with majority of them as weak biofilm producers in 2 different media, TSB and M9 minimal medium supplemented with 0.4% glucose. The genes encoding for putative virulence factors, glycerophospholipid cholesterol acyltransferase (gcat), heat-labile cytotonic enterotoxin (alt), heat-stable cytotonic enterotoxin (ast), serine protease (ser), polar flagella (fla), and lateral flagella (lafA) were present in 95.5%, 59.1%, 22.7%, 81.8%, 77.3%, and 22.7% of the strains, respectively. Class 1 integrons (100 to 3000 bp) were found in 68.2% of food isolates; whereas, 50% isolates contained class 2 integrons (150 to 1600 bp). This study provides a baseline data on the diversity of AHLs, biofilm forming ability and presence of virulence genes and integrons in Aeromonas food isolates from India., (© 2015 Institute of Food Technologists®)

Vibrio vulnificus, an important food-borne pathogen, is known to enter viable but nonculturable (VBNC) state under low temperature and low nutrition stress conditions. Present study examined the time required for induction of VBNC state and temperature which induces resuscitation of V. vulnificus YJ016. The change in cell morphology and gene expression during VBNC state and in resuscitated cells was also examined. V. vulnificus incubated in artificial sea water at 4 °C entered VBNC state after considerably extended time (70 days). An increase in temperature by 6 °C from the VBNC induction temperature (4 °C) resulted in resuscitation of VBNC cells; however, maximum resuscitation was observed when VBNC cells were held at 23 °C for 24 h. VBNC cells changed their morphology from comma shape to coccoid shape. Two rounds of induction of VBNC and resuscitation were possible with V. vulnificus cells; however, there was progressive reduction in number of resuscitated cells and after 190 days cells failed to resuscitate. Significant up-regulation of genes related to membrane proteins [porinH (10.4-fold), ompU (2.9-fold)], regulatory proteins [envZ (5.6-fold), toxR (4.5-fold), toxS (4.8-fold)], oxidative stress related protein katG (2.3-fold), cell division/maintenance proteins [ftsZ (4.3), mreB (6.5-fold)] and resuscitating promoter factor yeaZ (fourfold) was observed during resuscitation with respect to VBNC state indicating that these genes play a role during resuscitation. Gene expression data presented here would enhance our understanding of resuscitation of V. vulnificus from VBNC state. The results also highlight the importance of maintenance of low temperature during storage of seafood.

Vibrio vulnificus, a halophilic pathogenic bacterium of marine environments, encounters changes in salinity in its natural habitat and in the food-processing environment. The comparative response of V. vulnificus to hyperosmotic and hypoosmotic stress in terms of gene expression was investigated. Genes belonging to the proU operon for transport of compatible solutes and compatible solute synthesis were significantly upregulated (3- to 4.7-fold) under hyperosmotic stress. Under hypoosmotic stress, upregulation of genes coding for mechanosensitive channels of small conductance (mscS) was not observed. In hyperosmotic conditions a 2.3-fold decrease in the expression of aqpZ was observed. A 2-fold induction in gyrA was observed in V. vulnificus cells on exposure to hyperosmotic stress. groEL genes, VVA1659 (1.6-fold), and VV3106 (1-fold) were induced in hypoosmotic condition. Results of this study indicate that to manage hyperosmotic stress, V. vulnificus accumulated osmoprotectants through uptake or through endogenous synthesis of compatible solutes. Expression of mscS may not be necessary for immediate protection in cells exposed to hyper- and hypo-osmotic stress. Comparative analysis of important osmotic-stress-related genes showed up- or down-regulation of 14 genes in hyperosmotic stress as compared with up- or down-regulation of only 7 genes in hypoosmotic stress, indicating that the cells respond asymmetrically to hyper- and hypo-osmotic stress.

Aeromonas are responsible for causing gastroenteritis and extra-intestinal infections in humans. Twenty-two Aeromonas strains isolated from different food sources were re-identified up to species level using rpoD gene sequence analysis. Biochemical tests and 16S rRNA gene sequencing were insufficient to identify Aeromonas till species level. However, incorporation of additional biochemical tests lead to correct identification of 95.5 % strains up to species level. The 16S rRNA gene sequencing was useful to identify Aeromonas isolates at the genus level only. Sequences of the rpoD gene showed greater discriminatory power than 16S rRNA gene and provided conclusive discrimination of the strains for which the phenotypic species identification was uncertain. All these 22 strains were accurately identified up to species level by rpoD gene as A. salmonicida (6), A. veronii bv. veronii (4), A. caviae (3), A. hydrophila (2), A. veronii bv. sobria (2), A. jandaei (1), A. trota (1), A. sobria (1), A. allosaccharophila (1) and A. bivalvium (1). All these strains were also characterized using whole cell protein (WCP) analysis by gradient SDS-PAGE and showed different whole cell protein (WCP) profile [22-28 polypeptide bands (~10 to >97 kDa)], indicating high genetic diversity. The present work emphasizes the use of molecular methods such as rpoD gene sequencing along with comprehensive biochemical tests for the rapid and accurate identification of Aeromonas isolates till species level. The WCP profile can be subsequently used to characterize Aeromonas isolates below species level.

Salmonella is one of the most important food-borne pathogens. The MINI-MSRV MPN method was modified by replacing the isolation and confirmation of Salmonella on selective chromogenic agar with PCR/RT-PCR. This modification reduced the time for quantification by the MINI-MSRV MPN method by 24 h. Ninety-seven different food samples, comprising chicken, mutton, fish, and sprouts from different markets in Mumbai, India, were analyzed for quantification of Salmonella species by the MINI-MSRV MPN and modified MINI-MSRV MPN methods. Seventy-four percent of the chicken samples were found positive for Salmonella. However, Salmonella was found to be absent in fish, mutton, and sprouts samples. Salmonella load in the chicken sample was in the range of 1.30 to 120 MPN/g. This genotypic confirmation has advantage over variable phenotypic confirmation of pathogens., (© 2011 Institute of Food Technologists®)

A total of 154 food samples (chicken, fish, and ready-to-eat sprouts) from various retail outlets in Mumbai, India, were analyzed for the presence of Aeromonas spp. over a period of 2 y (January 2006 to March 2008). Twenty-two Aeromonas isolates belonging to 7 different species were isolated from 18 (11.7%) food samples. The highest percentages of isolation were from chicken (28.6%) followed by fish (20%) and sprout (2.5%) samples. Aeromonas caviae, A. veronii bv. sobria, and A. salmonicida were the most frequently isolated species from sprouts, chicken, and fish samples, respectively. The genes encoding for putative virulence factors, cytotoxic enterotoxin (act), hemolysin (hly), aerolysin (aer), elastase (ahyB), and lipase (lip) were detected using polymerase chain reaction method in 59.1%, 40.9%, 22.7%, 54.5%, and 31.8% of the strains, respectively. The isolated Aeromonas strains were found to be positive for virulence factors, that is, amylase, DNase, gelatinase, protease, and lipase production. More than 60% isolates were also positive for β-hemolytic activity. All these food isolates were found to be resistant to ampicillin and bacitracin, and sensitive to gentamicin, 3rd-generation cephalosporins (ceftazidime, cephotaxime, ceftriaxone), and chloramphenicol. Seventeen (77.2%) isolates harbored single and/or multiple plasmids (approximately 5 to >16 kb). The XbaI digestion patterns of chromosomal DNA of these isolates, using pulsed field gel electrophoresis, showed high genetic diversity among these isolates. Our results demonstrate the presence of various Aeromonas spp. with virulence potential and antimicrobial resistance in different food products marketed in Mumbai, India. The potential health risks posed by consumption of these raw or undercooked food products should not be underestimated., (© 2011 Bhabha Atomic Research Centre, India Journal of Food Science © 2011 Institute of Food Technologists®)

Antioxidant enzymes and antioxidant metabolites appear to have different roles in the oxidative stress resistance responses of radiation-resistant bacteria belonging to the Deinococcus-Thermus group. Twelve distinct strains belonging to 7 Deinococcus species were characterized for their responses to hydrogen peroxide, ciprofloxacin, and ionizing radiation. The levels of catalase and peroxidase activities in these strains showed a positive correlation with resistance to hydrogen peroxide and ciprofloxacin. However, the levels of these enzymes and carotenoids did not appear to contribute significantly to radiation resistance. Our findings support the idea that enzymatic defense systems are not sufficient to account for the extreme radiation resistance of Deinococcus species. Consistent with previously published reports, the Deinococcus strains had high intracellular manganese/iron ratios. No significant correlation was found between intracellular manganese/iron ratios and radiation resistance within different Deinococcus species, suggesting that other components are involved in conferring radiation resistance.

A radiation-resistant, Gram-stain-positive, non-motile, non-sporulating, aerobic, coccoid bacterium, strain 3ax(T), was isolated from a marine fish (black pomfret, Parastromateus niger), with radiation as a selective pressure. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 3ax(T) exhibited highest similarity (97.9 %) with Deinococcus proteolyticus DSM 20540(T). The DeltaT(m) for DNA-DNA hybridization of D. proteolyticus DSM 20540(T) and strain 3ax(T) was 15.3 degrees C, indicating that the novel strain was distinct from D. proteolyticus DSM 20540(T). The predominant respiratory quinone was MK-8. Strain 3ax(T) could grow at 20-42 degrees C; the optimum temperature for growth was 35 degrees C. The strain grew well at pH 6-9, with optimum growth at pH 7. The cell wall contained ornithine. The major fatty acids were 16 : 0, 16 : 1omega7c, 16 : 1omega9c and 18 : 1omega9c. Three phosphoglycolipids and one aminophospholipid were the major polar lipids. Based on the genotypic, phenotypic and chemotaxonomic characteristics, strain 3ax(T) was significantly different from D. proteolyticus DSM 20540(T) and, therefore, it was identified as representing a novel species of the genus Deinococcus, for which the name Deinococcus piscis sp. nov. is proposed. The type strain is 3ax(T) (=MTCC9123(T) =DSM 19767(T)).

Deinococcus species exhibit an extraordinary ability to withstand ionizing radiation (IR). Most of the studies on radiation resistance have been carried out with exponential phase cells. The studies on radiation resistance of Deinococcus radiodurans R1 with respect to different phases of growth showed that late stationary phase cells of D. radiodurans R1 were fourfold more sensitive to IR and heat as compared with exponential or early stationary phase cells. The increased sensitivity of D. radiodurans R1 to IR in the late stationary phase was not due to a decrease in the intracellular Mn/Fe ratio or an increase in the level of oxidative protein damage. The resistance to IR was restored when late stationary phase cells were incubated for 15 min in fresh medium before irradiation, indicating that replenishment of exhausted nutrients restored the metabolic capability of the cells to repair DNA damage. These observations suggest that stress tolerance mechanisms in D. radiodurans R1 differ from established paradigms.

A rapid nested PCR assay for detection of Salmonella from food was developed. The sensitivity of the assay developed was comparable to the traditional culture based methods with an advantage in reduction of assay time. The assay procedure with artificially contaminated samples was able to detect as low as 4CFU Salmonella/25g of food samples (sprout, carrot, cucumber and poultry meat). With two synthetic primers of 26 mer TS11 and 25 mer TS4, a 1.2kb fragment was amplified which served as a template for amplification of final 375bp product using TS11 and TS5 primers. No non-specific amplification from the native microbial flora of food samples was observed. The reaction generates a single band specific to Salmonella which allows the analyst to interpret data at ease and without any confusion. Enriched broth serves as template for the reaction which removes labour intensive DNA isolation procedures. In case of artificially contaminated samples, 6h enriched lactose broth can serve as template. However, for market samples where the organisms are under environmental stress, it is desirable to use template from Rappaport Vasiliadis medium. The assay also employes internal amplification control, which is amplified into a 300bp fragment and thus serves as positive control for the reaction and any possibility of false negative due to inhibitory action of food components on PCR reaction can be ruled out.

Many virulence phenotypes of Salmonella enterica are encoded by genes located on pathogenicity islands. Based on genome analysis, it is predicted that Salmonella pathogenicity island (SPI)-8 is restricted to Salmonella serovars Typhi and Paratyphi A, and SPI-10 to Salmonella serovars Typhi, Paratyphi, Enteritidis, Dublin and Gallinarum. This study was conducted to investigate the distribution of SPI-8 and SPI-10 among Salmonella isolates from sprouts, fish, water and blood. A total of 110 Salmonella isolates and 6 Salmonella serovars from the Microbial Type Culture Collection, Chandigarh, India, were screened. All isolates belonging to Salmonella serovars Washington, Enteritidis and Paratyphi A had both SPI-8 and SPI-10. All Salmonella serovar Typhi isolates from water and blood had both SPI-8 and SPI-10, whereas isolates from fish contained only SPI-8. SPI-8 and SPI-10 were also detected in only 3 out of 42 isolates belonging to Salmonella serovar Typhimurium. Both SPI-8 and SPI-10 were absent in Salmonella serovars Worthington, Dublin, Paratyphi B and Paratyphi C. These results contradict the predictions from Salmonella genome sequences available in GenBank and indicate that SPI-8 and SPI-10 are widely distributed among Salmonella serovars and that virulence factors other than those on SPI-8 and SPI-10 may be responsible for host specificity. This is the first report on the distribution of SPIs in Salmonella isolates from India.

The effect of radiation processing on the germination of the sprout seeds mung (Phaseolus aureus), matki (Phaseolus aconitifolius), chana (Cicer arietinum), and vatana (Pisum sativum) in terms of percent germination, germination yield, sprout length, vitamin C content, and texture was investigated. Gradual decreases in the percent germination, germination yield, and sprout length with increases in radiation dose (0.5 to 2.0 kGy) were observed. Vitamin C content and texture remained unaffected for the seeds treated with doses of up to 2 kGy. To determine the efficacy of radiation treatment in elimination of foodborne pathogens, seeds inoculated with 4 log CFU/g of Salmonella Typhimurium were treated with radiation doses of 1 and 2 kGy. A reduction in counts of Salmonella Typhimurium in inoculated seeds after radiation treatment was observed. A radiation dose of 2 kGy resulted in the complete elimination of 4 log CFU/g of Salmonella Typhimurium from the inoculated seeds. However, on sprouting for 48 h, the count of Salmonella Typhimurium reached 8 log CFU/g for the control seeds and the seeds treated with a 1-kGy radiation dose. The aerobic plate counts for seeds were 2.0 to 2.6 log CFU/g, which were reduced to 0.9 to 1.2 log CFU/g on treatment with a 2-kGy radiation dose. On sprouting for 48 h, the aerobic plate count reached 8 log CFU/g for both the control and radiation-treated seeds. The study demonstrates that irradiation can control bacterial levels on seeds but not contamination introduced during posttreatment handling. Therefore, radiation processing of the final product (sprouts) is recommended, rather than of the seeds.

Aims: To study the occurrence and diversity of Salmonella serovars in urban water supply systems of Nepal., Methods and Results: Occurrence of Salmonella was detected in 42 out of 300 water samples by enrichment culture technique in selenite F broth followed by plating on Salmonella Shigella agar. A total of 54 isolates identified to genus level by standard tests were subsequently confirmed by serotyping, phage typing and PCR detection of virulence genes (inv A and spv C). The predominant serotype was Salmonella Typhimurium, followed by Salm. Typhi, Salm. Paratyphi A and Salmonella Enteritidis. Most of the Salm. Typhi isolates were E1 phage type followed by UVS4, A and UVS1. All isolates of Salm. Paratyphi A and Salm. Enteritidis were an untypable (UT) phage type. The majority of isolates were multi-drug resistant as revealed by Kirby-Bauer disc diffusion technique. Ceftriaxone resistant isolates of Salm. Enteritidis indicated the presence of one of the ESBL genes, blaSHV, whereas the genes blaTEM and blaCTX were absent., Conclusions: The microbiological quality of the urban water supply is poor and indicates possibility of fatal outbreaks of enteric fever and related infections in Nepal., Significance and Impact of the Study: The present study will be useful in water borne disease control and prevention strategy formulation in Nepal and in the global context.

The microbiological quality of market samples of minimally processed (MP) pineapple was examined. The effectiveness of radiation treatment in eliminating Salmonella Typhimurium from laboratory inoculated ready-to-eat pineapple slices was also studied. Microbiological quality of minimally processed pineapple samples from Mumbai market was poor; 8.8% of the samples were positive for Salmonella. D(10) (the radiation dose required to reduce bacterial population by 90%) value for S. Typhimurium inoculated in pineapple was 0.242 kGy. Inoculated pack studies in minimally processed pineapple showed that the treatment with a 2-kGy dose of gamma radiation could eliminate 5 log CFU/g of S. Typhimurium. The pathogen was not detected from radiation-processed samples up to 12 d during storage at 4 and 10 degrees C. The processing of market samples with 1 and 2 kGy was effective in improving the microbiological quality of these products.

A study was undertaken to assess the microbiological quality of sprouts marketed in Mumbai and its suburbs. A total of 124 sprout samples of four different legumes--mung (Phaseolus aureus), matki (Phaseolus aconitifolius), chana (Cicer arietinum), and vatana (Pisum sativum)--were analyzed over a period of 12 months for aerobic plate counts, coliforms, yeast and mold counts, staphylococci, Salmonella, Listeria monocytogenes, Escherichia coli, E. coli O157:H7, and coagulase-positive Staphylococcus aureus. Aerobic plate counts ranged from 7.6 to 8.9 log CFU/g, coliform counts ranged from 5.4 to 7.9 log CFU/g, yeast and mold counts ranged from 3.6 to 7.3 log CFU/g, and staphylococci counts ranged from 3.3 to 6.6 log CFU/ g. Nonpathogenic E. coli was detected in 13% of the mung, 26% of the matki, 40% of the chana, and 19% of the vatana samples. Salmonella Typhimurium was detected in 21% of the mung, 40% of the matki, and 4% of the chana samples. Salmonella Dublin was detected in 2% of the mung samples, and Salmonella Washington was detected in 4% of the matki samples. L. monocytogenes and E. coli O157:H7 were not detected in any of the samples examined. Coagulase-positive S. aureus was detected in 4% of the mung, 11% of the matki, and 4% of the chana samples. The results indicated that the marketed sprouts were of poor microbiological quality; therefore, further processing, such as radiation processing, is needed to ensure their safety.

The effectiveness of radiation treatment in eliminating Salmonella Typhimurium and Listeria monocytogenes on laboratory inoculated ready-to-eat sprouts was studied. Decimal reduction doses (D10-values) for Salmonella Typhimurium and L. monocytogenes in dry seeds of mung (green gram), matki (dew gram), chana (chick pea), and vatana (garden pea) ranged from 0.189 to 0.303 kGy and 0.294 to 0.344 kGy, respectively. In sprouts made from these seeds, the D10-values ranged from 0.192 to 0.208 kGy for Salmonella Typhimurium and from 0.526 to 0.588 kGy for L. monocytogenes. Radiation treatment with a 2-kGy dose resulted in complete elimination of 10(4) CFU/g of Salmonella Typhimurium and 10(3) CFU/g of L. monocytogenes from all the four varieties of sprouts. No recovery of Salmonella Typhimurium and L. monocytogenes was observed in the radiation treated samples stored at 4 and 8 degrees C up to 12 days. Radiation treatment with 1 kGy and 2 kGy resulted in a reduction of aerobic plate counts and coliform counts by 2 and 4 log CFU/g, respectively; the yeast and mold counts and staphylococci counts decreased by 1 and 2 log CFU/g, respectively. However, during postirradiation storage at 4 and 8 degrees C, aerobic plate counts, coliform counts, yeast and mold counts, and staphylococci counts remained constant throughout the incubation period. This study demonstrates that a 2-kGy dose of irradiation could be an effective method of processing to ensure microbial safety of sprouts.

Minimally processed vegetables are in demand, because they offer convenience to consumers. However, these products are often unsafe because of possible contamination with pathogens, such as Salmonella, Escherichia coli O157:H7, and Shigella species. Therefore, this study was carried out to optimize the radiation dose necessary to ensure the safety of precut carrot and cucumber. Decimal reduction doses (D-values) of Salmonella Typhimurium MTCC 98 were ca. 0.164 kGy in carrot samples and 0.178 kGy in cucumber samples. D-values of Listeria monocytogenes were determined to be 0.312 and 0.345 kGy in carrot and cucumber samples, respectively. Studies of inoculated, packaged, minimally processed carrot and cucumber samples showed that treatment with a 1-kGy dose of gamma radiation eliminated up to 4 log CFU/g of Salmonella Typhimurium and 3 log CFU/g of L. monocytogenes. However, treatment with a 2-kGy dose was necessary to eliminate these pathogens by 5 log CFU/g. Storage studies showed that both Salmonella Typhimurium and L. monocytogenes were able to grow at 10 degrees C in inoculated control samples. Neither of these pathogens could be recovered from radiation-processed samples after storage for up to 8 days.

A radiation-resistant, Gram-negative and pleomorphic bacterium (CON-1) was isolated from a contaminated tryptone glucose yeast extract agar plate in the laboratory. It was red pigmented, nonmotile, nonsporulating, and aerobic, and contained MK-8 as respiratory quinone. The cell wall of this bacterium contained ornithine. The major fatty acids were C16:0, C16:1, C17:0, C18:1 and iso C18:0. The DNA of CON-1 had a G+C content of 70 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that CON-1 exhibited a maximum similarity (94.72%) with Deinococcus grandis. Based on the genotypic, phenotypic and chemotaxonomic characteristics, the bacterium CON-1 was identified as a new species of the genus Deinococcus, for which the name Deinococcus mumbaiensis sp. nov. is proposed. The type strain of D. mumbaiensis is CON-1 (MTCC 7297(T)=DSM 17424(T)).

Earlier studies in our laboratory showed that the lipopolysaccharide (LPS) of Salmonella typhi, which fails to activate B lymphocytes of C3H/HeJ mice, can suppress proliferation and polyclonal antibody synthesis by these cells when they are stimulated by polyclonal activators. In order to determine what stage of the cell cycle was blocked, resting B cells from C3H/HeJ spleens were activated by using different mitogens in the presence of inhibitory concentrations of LPS and analyzed by flow cytometry, using acridine orange to stain DNA and RNA. LPS was found to inhibit the progression of cells into the G1 stage of the cell cycle. Furthermore, [3H]uridine uptake studies showed that RNA synthesis is inhibited during the early phase of activation. These results indicate that inhibition by LPS of the signalling process occurs during a critical period of the cell cycle when the cells become susceptible to the inhibitory effects of LPS. To examine whether LPS acts only on B cells or whether it can suppress other immunocompetent cells from C3H/HeJ mice, studies were carried out on activated thymocytes and macrophages. LPS was found to inhibit thymocyte proliferation stimulated by concanavalin A or the combination of phorbol myristate acetate and ionomycin. Prostaglandin E2 synthesis by macrophages was also blocked by LPS. Thus, LPS is a potent inhibitor of the functioning of the major immunocompetent cells of C3H/HeJ mice.

Endotoxin-associated protein (EP) from the outer membrane of gram-negative bacteria is a potent immunomodulator. To examine the mechanism of EP stimulation, the protein kinase C inhibitors H7 and staurosporine were used. Both DNA and RNA synthesis of EP-stimulated murine resting B cells were completely inhibited when inhibitors were added at 0 h, whereas 55 to 76% inhibition of DNA synthesis was observed when H7 was added after 12 h of stimulation. In contrast, HA 1004, which blocks protein kinase A and protein kinase G activity, was relatively ineffective even at high concentrations, suggesting that the activity of protein kinase C is a primary mechanism of EP-induced murine B-cell proliferation. To examine the role of G proteins in EP-induced DNA synthesis in B cells, the effects of pertussis toxin (PT), which inactivates certain G proteins, and the B oligomer of PT (PTB), which does not, were also examined. PT was found to inhibit EP-induced DNA synthesis in a dose-dependent manner. However, PTB also caused equivalent inhibition, suggesting that PTB may be responsible for most of the inhibitory effect seen with the holotoxin. These results serve to question whether G proteins are involved in the signal transduction that occurs during EP-induced DNA synthesis in murine B cells.

The antitumor activity of lipopolysaccharide (LPS) and radio-detoxified LPS of Vibrio parahaemolyticus was tested against S180 cells in Swiss mice. The toxicity of the LPS was 200 times less than that of Salmonella typhimurium LPS. The V. parahaemolyticus LPS could be detoxified by exposure to gamma radiation. Both LPS and the irradiated LPS exhibited antitumor activity, though the irradiated LPS was less effective than the native LPS. These observations indicated that exposure to gamma radiation caused significant detoxification of V. parahaemolyticus LPS and the detoxified LPS still possessed considerable antitumor activity.

Immunization with MFS-180 vaccine prepared in the presence of glutaraldehyde (0.05%), lipopolysaccharide (LPS) (100 micrograms/ml), and concanavalin A (Con A) (200 micrograms/ml) could protect Swiss mice against a subsequent challenge by 1 X 10(6) MFS-180 cells. The sequence of attachment of LPS and Con A to glutaraldehyde-treated cells as found to determine the efficacy of the vaccine. LPS coupled with glutaraldehyde-treated cells before Con A could effect 100% survival, while LPS attached after Con A treatment to glutaraldehyde-treated cells showed only 60% survival. The protection was specific for syngeneic cells.

The effect of lipopolysaccharide (LPS) from Vibrio parahaemolyticus on the biochemical and phagocytic activities of murine peritoneal macrophages was determined. Intraperitoneal treatment with different doses (0.5-25 micrograms) of V. parahaemolyticus LPS markedly increased the cellular RNA content as well as lysosomal enzyme activities of peritoneal macrophages. The treatment also stimulated the phagocytic activities of macrophages. These observations suggest that V. parahaemolyticus LPS causes stimulation of murine peritoneal macrophages.

Lipopolysaccharide of Salmonella typhimurium was irradiated with gamma radiation at 10, 15, and 30 kGy doses. A dose of 30 kGy significantly detoxified the LPS (180 times). Mice were injected intraperitoneally with the radiodetoxified LPS, and it was found that it stimulated peritoneal macrophages as was evident from the enhancement of their acid hydrolases and cellular RNA content. Both LPS and radiodetoxified LPS exhibited antitumor activity against S180 cells in Swiss mice. Treatment with 20 micrograms/mouse of either LPS or 30 kGy LPS gave maximum survival of the mice (90%). These mice were found to resist the challenge of S180 cells (1 X 10(6)).

The toxicity and macrophage stimulating property of Vibrio parahaemolyticus lipid A was studied. The LD50 dose of lipid A in galactosamine-sensitized mice was found to be 0.6 micrograms when injected intraperitoneally. Administration of lipid A resulted in stimulation of peritoneal macrophages as evident by increase in their cellular RNA contents and lysosomal enzyme activities. The treatment also caused enhancement in the phagocytic activity of macrophages.