Jayant R. Bandekar

Lipopolysaccharide nonresponder cells: The C3H/HeJ defect

Since its initial discovery as endotoxin resistant, the C3H/HeJ mouse has been extensively studied and used as a comparative model to help reveal the mechanism under genetic control which governs host responses to endotoxin. Most of the research has focused on the B lymphocyte and macrophage of this strain which fail to be activated by LPS. Recently, specific LPS binding proteins have been isolated on lymphocytes and other cells; however a receptor which transduces an activation signal has not been isolated as yet from responder cells which is missing or altered on C3H/HeJ nonresponder cells. Investigations into the signal transduction pathways used by C3H/HeJ B cells when they are activated by a protein mitogen have been found to be similar to those used by LPS responder cells when activated by LPS. Protein kinase C and tyrosine kinase, which phosphorylate signal proteins in cells have been found to be operative in C3H/HeJ and C3H/OuJ B cells. In both cases, DNA synthesis is shut off by either PKC or PTK blockade; however, PTK inhibition will also block activation of PKC stimulated DNA synthesis, indicating tyrosine kinase initiated phosphorylation may regulate the PKC signal pathway. Further analysis of the proteins that are phosphorylated in LPS responder and LPS nonresponder B cells is needed before conclusions can be drawn as to whether the defect in C3H/HeJ cells resides in the signal pathway leading to gene activation and proliferation. Nevertheless, the notion of a missing or defective signal receptor still remains as a working hypothesis to explain C3H/HeJ cell hyporesponsiveness to LPS. Isolation of the Lpsn gene and its product will provide the evidence needed for a clearer understanding of how LPS reacts with cells.

Multiple-Stress Tolerance of Ionizing Radiation-Resistant Bacterial Isolates Obtained from Various Habitats: Correlation Between Stresses

Isolation of five ionizing radiation (IR)-resistant bacteria by screening of isolates from various habitats classified as common and stressed is reported. IR-resistant isolates exhibited varying degrees of resistance to γ-radiation and were classified as highly and moderately radiation resistant. Resistance to ultraviolet (UV) radiation correlated well with γ-radiation resistance, whereas a comparable desiccation resistance for all the highly and moderately radiation-resistant isolates was observed. However, salt tolerance failed to correlate with IR resistance, indicating a divergent evolution of the salt tolerance and radiation resistance. Characterization of isolates by the amplified rDNA restriction analysis profiling attested to the clustering of these isolates with their stress phenotype. 16S rRNA gene-based analysis of the isolates showed that the bacteria with similar-resistance physiologies clustered together and belonged to related genera. Hydrogen peroxide resistance and mitomycin survival patterns of the isolates indicated the roles of oxidative-stress tolerance in desiccation survival and recombination repair in higher radiation resistance, respectively.

Effectiveness of Radiation Processing in Elimination of Salmonella Typhimurium and Listeria monocytogenes from Sprouts

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.

Radiation Processing To Ensure Safety of Minimally Processed Carrot (Daucus carota) and Cucumber (Cucumis sativus): Optimization of Dose for the Elimination of Salmonella Typhimurium and Listeria monocytogenes

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.

Microbiological evaluation of sprouts marketed in mumbai, india, and its suburbs

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.

Rapid, sensitive, and validated method for detection of Salmonella in food by an enrichment broth culture – Nested PCR combination assay

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.

Prevalence, Characterization, and Antimicrobial Resistance of Aeromonas Strains from Various Retail Food Products in Mumbai, India

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.

Radiation processing for elimination of Salmonella typhimurium from inoculated seeds used for sprout making in India and effect of irradiation on germination of seeds.

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.

Deinococcus piscis sp. nov., a radiation-resistant bacterium isolated from a marine fish

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)).

Radiation resistance of Deinococcus radiodurans R1 with respect to growth phase

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.