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.

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.

Aeromonas-associated diarrhoea in north India.

The incidence of Aeromonas-associated diarrhoea in patients attending the hospital attached to the Dayanand Medical College, Ludhiana, India was investigated during May 1984-May 1987. Microbiological examination of single faecal samples from 2,661 patients showed the presence of various enteropathogens of which details of only Aeromonas isolations are given and discussed in the present article. A small control sample of 105 age-matched healthy volunteers were also examined. Only 55 patients (2.1% of all patients) showed Aeromonas spp. in their stool samples on culture. Aeromonas as a single pathogen was isolated from 37 patients and, from 18 patients it was mixed with other enteropathogens. Stools of healthy controls did not contain any pathogen. Seven of 25 isolates of A. caviae, all 18 isolates of A. sobria and all 7 isolates of A. hydrophilla were shown to be enterotoxigenic by rabbit ileal loop test. There was no immunological cross-reaction between Aeromonas enterotoxin and Escherichia coli labile toxin antiserum. The Aeromonas isolates were susceptible to most of the commonly used antimicrobial agents except ampicillin and cephalothin.

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.

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.

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).

Effects of aflatoxin on the immune system of the chick.

A decreased antibody response to injected sheep red blood cells and weight losses to the extent of 25-38% were observed in the bursa Fabricii and thymus in chicks fed an aflatoxin diet (10% w/w) for 3 weeks. The immune organs did not show any significant histopathological changes. The antibody titre decreased from day 6 of treatment onward until the 15th day, when the antibody titre had disappeared completely in the test chicks. The body weight gain was also less in test chicks (9.7-37.3%) given test feed or test feed along with Salmonella pullorum in water (10(8) cfu ml 1) in comparison to chicks given toxin-free diet and water after 3 weeks of feeding trials. The disappearing component in the test chicks' liver homogenate was found to be antigenic by immunoelectrophoretic studies.

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.

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.

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.

Bacteriocin (klebocin) typing of clinical isolates of Klebsiella pneumoniae.

Three-hundred-forty-two clinical isolates of Klebsiella pneumoniae were subjected to bacteriocin (klebocin) typing using six standard klebocin-producer strains (153-158). The overall typability was 72.8 per cent. The predominant klebocin types found were 244 (14.3 per cent), 313 (13.7 per cent) and 113 (7.6 per cent). Klebocin types 314 and 111 each contributed 5.2 per cent to the total number of isolates. No significant correlation was observed between the source of isolation and the klebocin type.

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 certain chelating agents on the antibacterial action of silver nitrate.

EDTA and EGTA when used in conjunction with AgNO3 enhanced the antibacterial action of the latter significantly, so that strains of Klebsiella pneumoniae and Staphylococcus aureus resistant to 70 micrograms/ml of AgNO3 were observed to became sensitive to 10 micrograms/ml of this compound. The synergistic effect of EDTA appears to be due to a mechanism other than the removal of lipopolysaccharide from outer membrane, as its effect could be observed in even non-LPS containing gram positive S. aureus cells. Penicillamine, another potent chelator had an opposite effect so that it decreased the toxicity of silver ions.

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.

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.

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.