Proteins of the outer layer of the vitelline membrane of hen's eggs.

A study has been made of the proteins in the vitelline membrane of hen's eggs before and after mechanical separation into the inner and outer layers. The membranes were dissolved in detergent (sodium dodecyl sulphate) and chromatographic fractions were examined by gel electrophoresis. The separated inner and outer layers were compared by gel electrophoresis. The outer layer contained (i) enzymically active lysozyme (EC 3.2.1.17) (about 60% dry weight), (ii) an insoluble ovomucin complex and (iii) a new protein, VMOI (vitelline membrane outer I). These account for most of the protein. In addition, some minor constituents were detected by gel electrophoresis but were not isolated. Except for ovomucin, the constituents of the outer layer could be dissolved from the membrane at high ionic strength (greater than 0.5 M sodium chloride), resulting in a loss of its structure. On lowering the ionic strength the soluble proteins recombined with the membrane, partially regenerating the original structure. Ovomucin appears to form the skeleton of the outer layer, but the salt-soluble proteins, especially lysozyme, are responsible for its integrity. The function of the newly-recognized protein (VMOI) is not known. Its molecular weight is 17,500 according to gel electrophoresis in detergent and it contains no methionine. The inner layer consists largely of the proteins GPI, GPII and GPIII isolated by Kido et al. (Kido, S., Janado, M. and Nunoura, H. (1975) J. Biochem. 78, 261-268) from the whole membrane.

Drug resistance patterns and susceptibility to aflatoxin B1 of strains of Escherichia coli and Staphylococcus aureus.

The antibacterial properties of aflatoxin B1 have been evaluated against antibiotic-resistant clinical isolates of Escherichia coli and Staphylococcus aureus. The inhibition of growth ranged from 11.5 to 60.0% and 4.5 to 18.5% in the strains of S. aureus and E. coli, depending on the extent of drug resistance. Aflatoxin-B1 binding varied with toxin concentration, the presence of surfactants (Tween-80 or EDTA) as well as with the antibiotic-resistance pattern; binding was maximal in antibiotic-sensitive strains and least in the most resistant strains. Binding of aflatoxin B1, correlated with growth inhibition. Aflatoxin B1 also caused leakage of cell contents and decrease in inulin uptake, effects which were also concentration dependent.

Biochemical and immunological effects of aflatoxins in rabbits.

Aflatoxicosis caused impairment of specific and nonspecific immune responses to varying degrees in rabbits. However, the impairment was of lesser extent as compared to the earlier reports in other laboratory animals. The nonspecific response, as determined by phagocytic index was 60% of the control in test rabbits. Aflatoxicosis caused a decrease in protein content (22%), alkaline phosphatase activity (75%) and an increase (1.5-3 times) in the level of cholesterol, total lipids and acid phosphatase activity in test rabbits sera. The changes were statistically significant (P less than 0.05) in test rabbits. The test rabbits had a tendency to recover from the effects of aflatoxicosis after 3-4 weeks.

Phospholipase A production by isolates of Salmonella species.

The incidence of intra- and extra-cellular phospholipase-A production by Salmonella isolates from human, poultry and environmental material was investigated. Nearly 17 per cent (15/90) of the strains tested produced phospholipase A. Phospholipase production was encountered in S. typhi, S. paratyphi A, S. typhimurium, S. seftenberg, S. bareilly, S. weltevredeen, S. newport, S. adelaide, S. alachua and S. gallinarum. Maximum (10/15) phospholipase producers were isolated from the human material. Phospholipase positive human isolates exhibited a high incidence of simultaneous multiple drug resistance, enterotoxigenicity and cell surface hydrophobicity.

Low temperature direct methylation of lipids in biological materials.

The procedure for low temperature methylation of fatty acids in lipids by sulfuric acid-methanol has been adapted to direct methylation of fatty acids of lipids in biological materials without prior extraction of the lipids. Successful application requires a solution or a suspension of fine particles of the lipid bearing material in ether. Concentrated sulfuric acid is added to the solution or suspension at low temperatures followed by addition of absolute methanol. The acid is neutralized by methanolic KOH and the esters extracted. Application of the method to prepare methyl esters of lipids in cream, blood serum, swine liver and kidney tissue, and cells of yeast onStaphylococcus aureus show that fatty acid composition based on this method compares with that determined by methylation of extracted lipids.

Microbiological quality & incidence of organisms of public health importance in food & water in Ludhiana.

Bacteriological analysis of 713 samples of various types of foods and related articles and potable water samples from different places in Ludhiana, Punjab was carried out. The highest counts ranging from 2.5 x 10(6)-7.5 x 10(8) organisms/g were observed in raw vegetables and fruits, followed by 3 x 10(6)-9.8 x 10(7)/ml, 8.3 x 10(4)-8.9 x 10(7)/g and 1 x 10(3)-6.7 x 10(7)/g in fruit juice, milk and its products, and salty/non milk snacks respectively. Fresh chapati, dal, rice, cooked vegetables and karhi etc., showed no microbial contamination. However, samples of these articles from road side cafes gave counts up to 1 x 10(7) organism/g. The most probable number of coliforms and Escherichia coli/100 ml of water ranged from < 1 to > 1100. Although 1332 isolates of 16 types of organisms of public health significance were obtained those of proven enteropathogenicity were enterotoxigenic Esch. coli (55), Esch. coli O157 (3), enteropathogenic Esch. coli (1), enterotoxigenic Klebsiella (23), Streptococcus faecalis (152), Bacillus cereus (133), Staphylococcus aureus (125), Aeromonas spp (47), Salmonella spp (10), Shigella spp (4) and Yersinia enterocolitica (2). Poor quality of potable water and widespread occurrence of enteropathogens in food consumed by the common man in Ludhiana was evident.

Seasonal fluctuations in the occurrence of enteroinvasive Escherichia coli diarrhoea.

Enteroinvasive Esch. coli (EIEC) was implicated in 2.1 per cent (57 out of 2661) diarrhoeal patients investigated at Ludhiana (Punjab). The predominant serogroups isolated were 028, 0147, 0124 and 0112. High prevalence of EIEC infection was noticed among infants (50.6%) followed by children up to 10 yr (19.3%). The peak incidence (24.5%) occurred during the hot and dry month of May as compared to 1.7 per cent in February. The disease incidence was positively correlated with mean minimum temperature and mean maximum temperature. All strains gave Sereny's test positive and were mostly susceptible to cephalothin, gentamicin, nalidixic acid, furazolidine and neomycin.

Bioecological factors & rotavirus diarrhoea.

Rotavirus was detected (using Rotalex) in 11.72 per cent (120 of 1024) children below 3 yr age with diarrhoea. None of the 25 healthy control children excreted rotavirus in their faeces. Group specific ELISA for 89 Rotalex positive samples revealed 30 subgroup I and 43 subgroup II whereas 10 were untypable. Rotavirus infection ranged from 2.8 to 22.20 per cent in different months. There was no correlation with mean minimum and maximum temperatures. However, the incidence showed a negative correlation (r = -0.645) with relative humidity. Children between the age of 10 to 12 months had the maximum incidence of rotavirus infection. Male patients were found to be more susceptible to infection than females (3.3:1).

ELISA for detection of heat stable enterotoxin producing Escherichia coli strains.

Among 557 strains of Esch. coli isolated from patients with acute diarrhoea, 392 (70.4%) isolates demonstrated ST production by ELISA. Predominant ST producing serogroups were 020 (45), 078 (40), 0128 (21), 061 (19), 0149 (9), 04, 055, 0106 and 0114 (8 each). The inhibition ELISA range was between 10.5 and 40.5 per cent. Visual difference between a negative and a positive ELISA test was distinct. A comparison of ELISA with classical suckling mouse assay for 100 strains showed 88 and 80 positive strains respectively for ST. ELISA proved a more specific, rapid and sensitive assay which may be useful for screening large number of isolates in epidemiological studies.

Survival of Salmonella typhimurium and Staphylococcus aureus in eggs cooked by different methods.

Shell eggs inoculated with Salmonella typhimurium and Staphylococcus aureus were cooked by recommended procedures for boiling, poaching, and frying. Except for poaching, the recommended procedures were inadequate in destroying the inoculum placed in the yolk. Boiling for 7 min was necessary for complete destruction of S. typhimurium and it took 12 min of boiling to destroy Staph. aureus. Cooking time-temperature relationship for complete kill depended on the cooking method with fried eggs. Four minutes and 70 C were needed for covered eggs, 3 min on each side at 64 C for turned over eggs, while cooking for 7.5 min at 64 C for sunnyside eggs was not sufficient for destruction of both of the test organisms. None of the test organisms could be recovered from omelets baked by the recommended procedure (86 C for 25 min). Scrambling for 1 min at 74 C was required for the complete destruction of S. typhimurium and 2 min at 78 C for Staph. aureus.

Effect of various inducing agents on bacteriocin (klebocin) production by Klebsiella pneumoniae.

The influence of various inducing agents on growth, synthesis and release of klebocin by Klebsiella pneumoniae was studied. A significant level of klebocin was detected only after induction. The highest level of klebocin was achieved with mitomycin C followed by rifampicin and polymyxin B. Chloramphenicol and UV irradiation did not show any effect on klebocin production. Maximum klebocin release occurred after 8 h of induction with all the agents. Concentration of mitomycin C did not show any significant effect on klebocin production.

Effect of medium on the bacteriocin production by Klebsiella pneumoniae.

The influence of growth media and media constituents on bacteriocin production by Klebsiella pneumoniae was studied. Among the standard laboratory media used trypticase soy broth (TSB) showed the maximum production and poor yields resulted from growth in peptone water and nutrient broth. A number of peptones differed in their bacteriocin production. Best yields were observed in tryptone and proteose peptone water. Addition of 1% yeast extract to TSB further stimulated bacteriocin production. However, activity was low when glucose, glycine, sodium mercaptoacetate or bile salt mixture were added to the medium. Suppression of synthesis by certain agents as well as inhibition of formed bacteriocin by the others appears to affect the bacteriocin yield. No proteinase activity was detected during the entire incubation period.

Evidence for a chromosomally determined bacteriocin production by Klebsiella pneumoniae.

Location of the genes responsible for pneumocin production in Klebsiella pneumoniae was examined by classical procedures. Conjugal intrageneric transfers, elimination experiments with various curing agents, high temperature and plasmid isolation procedures showed that this strain did not harbour any plasmid. Hence chromosomal location of the genetic determinants is suggested.

Susceptibility of multidrug-resistant isolates of Klebsiella pneumoniae to silver nitrate.

Multidrug-resistant (MDR) clinical isolates of Klebsiella pneumoniae were checked for their sensitivity toward silver nitrate by the tube-dilution method. Nearly 75% of MDR strains could be successfully inhibited by 5 mg/L of silver nitrate. A significant correlation was observed between incidence of silver and trimoxazole resistance and silver and kanamycin resistance in these isolates. The genetic linkage of these two properties could not be proved since simultaneous curing and co-transfer studies gave negative results.

Factors affecting intra- and extracellular phospholipase A1 production by Salmonella newport.

The effect of various physico-chemical factors on production of intra- and extracellular phospholipase A1 by Salmonella newport was investigated. Maximum intracellular enzyme levels were observed when cells were grown in brain heart infusion broth, after 12 h of incubation at 37 degrees C. Highest level of extracellular phospholipase A1, however, was seen in synthetic medium (pH 7.0) after 24 h of incubation at 37 degrees C. Agitation during incubation had no effect on the intracellular enzyme synthesis but enhanced extracellular enzyme levels. Addition of surfactants to the growth media significantly decreased both intra- and extracellular phospholipase A1 production.

Effect of nutritional factors on aflatoxin B1 inhibition of growth in Escherichia coli and Salmonella typhi.

The extent of growth inhibition by aflatoxin B1 in S. typhi and E. coli was greater in the presence of sodium citrate or sodium phosphate, palmitic and stearic acid than aflatoxin B1 alone. The addition of amino acids (glycine or glutamic acid) stimulated growth in E. coli and inhibited in S. typhi in the presence of aflatoxin B1. Other nutrients, such as yeast extract, lactose, or salt addition did not alter aflatoxin B1 antibacterial activity but decreased growth was observed in the presence of peptone.

Effect of fatty acids on aflatoxin production by Aspergillus parasiticus.

The effect of saturated and unsaturated fatty acids on aflatoxin production was studied in a synthetic medium. The aflatoxin production decreased (10-75%) in the presence of lauric acid and palmitic acid but the addition of behenic and sebacic acid stimulated aflatoxin production by 125-541%. Linolenic and linoleic acids effected aflatoxin production and mycelium growth. An 34-fold increase in aflatoxin production was observed with 50 mM linoleic acid. An inverse relationship was observed between aflatoxin production and mycelium mass, irrespective of the nature of the fatty acid.

Effect of metal ions on aflatoxin production by Aspergillus parasiticus.

The effect of iron, copper, cobalt, cadmium, zinc, molybdenum, magnesium and manganese salts was studied on aflatoxin production in relation to mycelial mass. Iron, copper and cadmium salts decreased the aflatoxin production to different levels but a mixed trend was observed depending on salt concentration, with molybdenum, magnesium and manganese. Cobalt and zinc salts stimulated aflatoxin production at all concentrations studied. The maximum increase in aflatoxin production, 655% and 519% was observed in the presence of zinc sulfate and sodium molybdate, respectively. A negative correlation was observed between aflatoxin production and vegetative growth of fungus.