Serotyping, PCR, phage-typing and antibiotic sensitivity testing of Salmonella serovars isolated from urban drinking water supply systems of Nepal.

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.

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 gamma-radiation and were classified as highly and moderately radiation resistant. Resistance to ultraviolet (UV) radiation correlated well with gamma-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.

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.

FT-IR spectroscopic studies of polyurethanes: IV. Studies of the effect of the presence of processing aids on the hemocompatibility of polyurethanes.

Many types of proprietary processing aids are used by the manufacturers of polymer medical devices, which are difficult to completely eliminate from the end product. In such cases, it is important to investigate how such processing aids affect the properties of the materials. One such material is Pellethane and a most commonly found processing aid is wax. We investigated the effect of the presence of such wax on hecompatibility properties, particularly on the adhesion and activation of human platelets, on a group of Pellethane samples with varying amounts of wax. The effectiveness of cleaning agents, like Freon, was examined for wax removal. The type and quantity of wax present within the near-surface regions of the Pellethane tubings was estimated by using the FT-IR-ATR technique. The presence of bis amide processing wax was found to affect the hemocompatibility properties of the Pellethane samples. Correlation between hemocompatibility and the amount of wax was made; platelet activation, as well as amount and density of fibrin formation, showed a linear correlation.

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.

Suppression of C3H/HeJ cell activation by lipopolysaccharide endotoxin.

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.

Immunomodulation of C3H/HeJ cells by endotoxin associated protein and lipopolysaccharide endotoxin.

Protein kinase C plays a vital role in the activation of C3H/HeJ B lymphocytes by endotoxin associated protein; however, it is unlikely that G proteins are involved in the early signals stimulated by EP. On the other hand, LPS suppresses C3H/HeJ B cell DNA synthesis induced by EP which may be the result of PKC down regulation. LPS inhibits C3H/HeJ B cells from progressing through the G1 phase of the cell cycle blocking RNA synthesis within the first 12 hr after the cells are stimulated. Finally, this inhibition extends to activation of the arachidonic acid metabolism in C3H/HeJ macrophages and T cell proliferation to a limited extent.

Roles of protein kinase C and G proteins in activation of murine resting B lymphocytes by endotoxin-associated protein.

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.

Vibrational analysis of crystalline tri-L-alanine.

We have found that tri-L-alanine (Ala3) can crystallize in a parallel-chain beta structure in addition to the previously known antiparallel-chain beta structure. Although the chain conformations in each structure are essentially similar, the ir and Raman spectra are distinctively different. We have calculated the normal modes of each structure, and can account in significant detail for these differences. This demonstrates the essential validity of our empirically refined force fields, as well as showing that deeper insights into polypeptide and protein structure can be achieved through the rigorous analyses of normal mode calculations.

Biological activities of lipid A from Vibrio parahaemolyticus: stimulation of murine peritoneal macrophages.

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.

Stimulation of macrophages by lipopolysaccharide of Vibrio parahaemolyticus.

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.

Antitumor activity of lipopolysaccharide and radio-detoxified lipopolysaccharide of Vibrio parahaemolyticus.

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.

Stimulation of macrophages and antitumor activity of radiodetoxified endotoxin.

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)).

Vibrational analysis of peptides, polypeptides, and proteins. XXX. Normal mode analyses of gamma-turns.

The normal modes have been calculated for three kinds of low energy gamma-turn structures resulting from recent conformational energy calculations by Némethy. Frequencies have been computed for a gamma-turn, a mirror-related gamma-turn, and an inverse gamma-turn of CH3-CO-(L-Ala)n-NH-CH3, with n = 3 and n = 5, and for certain 14C and 15N derivatives of the n = 3 molecule. Correlations are evident between amide frequencies and gamma-turn structures, and it is found that only amide I modes of peptide groups in the turn are relatively insensitive to the lengths of attached chains.

Conformations of cyclo(L-alanyl-L-alanyl-epsilon-aminocaproyl) and of cyclo(L-alanyl-D-alanyl-epsilon-aminocaproyl); cyclized dipeptide models for specific types of beta-bends.

Conformational energy calculations indicate that the peptide backbones of the low-energy conformations of the cyclized dipeptide derivatives cyclo (L-alanyl-L-alanyl-epsilon-aminocaproyl) and cyclo (L-alanyl-D-alanyl-epsilon-aminocaproyl) are constrained to form beta-bends of types I + III and II, respectively. Thus, the two compounds can serve as models for the spectroscopic properties of beta-bends of these types. The coupling constants obtained from 1H n.m.r. spectra in DMSO-d6 are consistent with the dihedral angeles of the computed lowest-energy conformations. Differences in 13C chemical shifts between the two compounds can be correlated with differences in shielding by C=O groups in bends of various types. 1H and 13C chemical shifts suggest association of cyclo (L-Ala-L-Ala-Aca) but not of cyclo (L-Ala-D-Ala-Aca) in dimethylsulfoxide. The different tendencies to associate can be explained in terms of the difference in conformation. The circular dichroism spectra of the two compounds are quite different. In methanol, trifluoroethanol and water, the L-Ala-L-Ala derivative has a positive extremum near 190 nm and two negative extrema near 206 and 220 nm, whereas the L-Ala-D-Ala derivative has a positive extremum at about 203 nm and negative extrema at about 187 and 229 nm. The spectra can be used to estimate the contribution of various bend types in a related series of compounds. A normal mode analysis of the vibrations of the computed low-energy conformations was compared with solid state infrared and Raman spectra, in order to determine the predominant conformations. The bend types determined by this comparison fully agree with the predictions of the theoretical computations for both derivatives.

Induction of resistance to ascites tumor in mice with MFS-180 cells treated with glutaraldehyde, lipopolysaccharide, and concanavalin A.

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.