EFFECTS OF ENVIRONMENTAL STRESSES ON THE SURVIVAL AND PRODUCTION OF DIARRHOEAL ENTEROTOXINS BY BACILLUS CEREUS FROM SOME FOOD SOURCES
EFFECTS OF ENVIRONMENTAL STRESSES ON THE SURVIVAL AND PRODUCTION OF DIARRHOEAL ENTEROTOXINS BY BACILLUS CEREUS FROM SOME FOOD SOURCES
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Date
2014-10
Authors
BELLO, SAKINA
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Abstract
This research work was conducted with the aim of studying the effects of environmental stresses on the survival and production of the Haemolysin BL (HBL) diarrhoeal enterotoxin by Bacillus cereus food isolates. A total of 387 food samples were purchased from street food vendors and some markets in Kaduna north and Zaria local government areas of Kaduna state. Samples which comprised of 43 samples each of unpasteurized cow milk, locally fermented milk (nono), raw meat, roast meat products (suya, soye and balangu), uncooked rice, Jollof rice and boiled rice were analyzed for presence of B. cereus. Appropriate 10 fold dilutions of each food sample was pour plated onto sterile Mannitol Egg-yolk Polymyxin agar (MYP)(OXOID) plates and incubated at 37oC for 24-48 h. Presumptive B.cereus isolates were biochemically characterized with MicrogenTm Bacillus identification kits (Microgen Bioproducts). To study the effects of varying pH level, sodium chloride (NaCl) concentration (W/W) and temperature on HBL production by isolates, confirmed B.cereus isolates; R3MI, F5MI, Bln3g2 and F2 from raw milk, nono, balangu and boiled rice were selected. Mcfarland standard 1( about 8.5 log units) of each isolate was inoculated into sterile freshly prepared corresponding food samples and stored at varying NaCl concentrations (0- 2%), pH levels (6.3,6.4,6.7,6.8 for milk and 3.7, 3.8, 4.2,4.3 for nono), and temperature (4-10oC, 26-28oC, 37oC). These isolates were assayed for HBL enterotoxin production using BCET–RPLA toxin detection kits (OXOID), and Polymerase Chain Reaction (PCR) studies conducted determine the presence of HBL and BceT (bceT) genes in the isolates. Enterotoxin producing isolates were subjected to antibacterial susceptibility tests using the Kirby-Bauer method against 10 antimicrobial agents (OXOID); Ampicillin (10μg), Amoxicillin – clavulanic acid
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(20/10 μg), Cefpodoxime (10μg), Ceftriaxone (30μg), Cephalothin (30μg), Amikacin (30μg), Tetracycline (30μg), Erythromycin (30μg), Ciprofloxacin (5μg) and Trimetoprim–Sulfamethoxazole (1.25/23.75μg). A total of 47 of 109 isolates from retail food samples were confirmed as B. cereus, and mean B.cereus counts ranged from 1.1±0.2 log10 cfu/ml for unpasteurized milk samples to 2.1±0.3log10 cfu/ml for soye. Counts fell within safe limits (102 to <103), and were not significantly different amongst producers (P>0.05) for all samples. Food storage studies revealed decreases in B.cereus cells from initial levels of 8.5 log units in boiled rice samples at NaCl levels of 1.5 and 2%, at refrigeration temperature (4 – 10oC) for 24 h. Counts were significantly different when analyzed across NaCl concentrations, and storage period (P<0.05). Decreases in B.cereus counts from initial levels of 8.5 log units were sustained in pasteurized milk samples (pH 6.3 and 6.8) for 48 h at refrigeration temperature (4 – 10oC), and counts were significantly different across pH levels (P<0.05). Isolates from pasteurized milk at pH 6.8 following 48 h of refrigerated storage tested negative (< 2ng/ml) for HBL production, whereas most rice isolates had toxin titres of ≥ 64ng/ml. PCR studies revealed that both enterotoxin and non-enterotoxin producing isolates encoded for at least one of the hbl genes. The bceT gene was detected in 70% of rice isolates, and in only about 38% of pasteurized milk isolates. Isolates from pasteurized milk at pH 6.8 following 72 h of refrigerated storage (4 – 10oC) were sensitive to Cephalothin, in contrast to other isolates. All but 96% of B.cereus balangu isolates were sensitive to Erythromycin, while none of B.cereus nono isolates appeared to be sensitive to Tetracycline. This study has established the presence of B.cereus in some ready –to-eat (RTE) and uncooked foods sold in some streets and markets of Kaduna metropolis. Food
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storage studies showed B. cereus is inhibited in boiled rice at NaCl levels above 1% at refrigeration temperature (4-10oC), for up to 48 h. Results also showed sustained inhibitory effects are exerted on B.cereus cells at pH levels of 6.3 and 6.8 in pasteurized cow milk for up to 48 h. Findings of this study also indicate storage conditions suppress the expression of the hbl genes in some B.cereus isolates from pasteurized milk. Decreased resistance to particular antibacterial agents is also induced in these isolates following 72 h of refrigerated storage (4 – 10oC). Results highlight the need for enactment and enforcement of regulations for food service operators and vendors by the appropriate agencies with a view to providing safer RTE foods in Nigeria. There is also the need to carry out more researches to understand the mechanisms of survival and pathogenicity of food pathogens in locally consumed foods. Results of such researches also need to be incorporated in mandatory enlightenment programmes for food service operators to reduce the risk of foodborne illnesses associated with RTE foods.
Description
A DISSERTATION SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES, AHMADU BELLO UNIVERSITY, ZARIA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY IN MICROBIOLOGY DEPARTMENT OF MICROBIOLOGY FACULTY OF SCIENCE AHMADU BELLO UNIVERSITY, ZARIA NIGERIA
Keywords
EFFECTS,, ENVIRONMENTAL,, STRESSES,, SURVIVAL,, PRODUCTION,, DIARRHOEAL,, ENTEROTOXINS,, BACILLUS CEREUS,, FOOD SOURCES.