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Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 13-18 Global Science Publications INCIDENCE AND ANTIBIOTIC SENSITIVITY OF BACILLUS CEREUS ISOLATED FROM READY TO EAT FOODS SOLD IN SOME MARKETS IN PORTHARCOURT, RIVERS STATE, NIGERIA O. K. AGWA, C.I. UZOIGWE AND E.C. WOKOMA Department of Microbiology, University of Port Harcourt, Rivers State, Nigeria Department of Crop and Soil Science, Faculty of Agriculture, University of Port Harcourt, Nigeria (Received 20 August 2011; Accepted 30 September 2011) Key words : Antibiotic sensitivity, Bacillus cereus isolates, Microbiological standards, Ready-to-eat indigenous Abstract - Sixty-four food samples of “cooked rice”, “masa”, “agidi” and “epiti” purchased from localmarkets in Obio-Akpor Local Government Area of Port Harcourt, Nigeria were examinedmicrobiologically. Plate count analysis on mannitol egg-yolk bromothymol blue polymyxin B (MYP) agarrevealed that “cooked rice” had the highest frequency of occurrence of Bacillus cereus isolates (29.51%),“masa” (26.23%), “agidi” (22.95%) and “epiti” had the least frequency of 21.31%. All Bacillus cereusisolates tested were found to be susceptible to rifampin (30µg), chloramphenicol (20µg), erythromycin(30µg), ciprofloxacin (10µg), streptomycin (30µg), gentamycin (10µg) and lincocin (30µg) and 100%resistance against norfloxapin (30ug), floxapen (30µg) and ampiclox (30µg). The study unveils thepresence of Bacillus cereus in food samples sold in Port Harcourt especially in ‘cooked rice” and “masa”which may pose serious threat to the health of consumers and should not be ignored.
Dierick et al., 2005). Bacilli cause symptoms leading toillness, relatively mild lasting up to 24h, but could Present economic conditions have resulted in be severe leading to hospitalisation and in some situations, where food vending has become cases death. The organism is found in a wide range increasingly important in most countries; of habitat including air, water, dust and soil, from contributing significant income inflow for where spores are introduced into cereal crops, households involved in selling these foods vegetables, animal hair, fresh water and sediments.
(Mosupye and vonholy, 1999; FAO/WHO, 2003; Raw plant foods especially rice, potatoes, beans, FAO/WHO, 2005 ). Hygiene and sanitation practices peas, and spices are the most common sources of obtained during preparation and marketing of Bacillus cereus (Eglezos et al., 2010). The occurrence of these foods provide ample opportunities for the the organism within the environment enables it to proliferation of these food with food-borne enter the food chain through raw materials and is pathogens (Desai and Varadaraj, 2009). Among the a major problem in convenience foods and mass food- borne pathogens, strains of Bacillus cereus are catering (Beathe and William, 2000; Jensen et al., of significance because of its ubiquitous nature and 2005; Guinebretiere et al., 2006). Because of the role ability to occur in a wide range of foods (Ehling- of microorganisms in spreading diseases, the need schulz, et al., 2004a; Oguntoyinbo and Oni, 2004; to assess the safety and quality of foods is very important, in order to ensure safety of supply, The unique properties of Bacillus cereus includes clean, wholesome and high quality delivery to the heat resistant endospore forming ability, toxin public. When the food hygiene system fails, a production in varieties of foods and the psychro- batch of food is contaminated with high level trophic nature makes the organism a prime cause microbes potential for food borne disease outbreak, of public health hazard (Griffith and Schraft, 2002; its distribution cannot be controlled, changes in *Corresponding author - Obioma Agwa, E-mail : [email protected] public health and deterioration and increases food sample, aseptically transferred into 90mL of susceptibility rate (Sahota et al., 2008).
tryptone broth to give an initial 1:10 dilution.
In several studies that have been conducted on Aliquots of 0.1mL from 10-5dilutions were surface the incidence of food-borne pathogens, emphasis plated on sterile dried Mannitol egg-yolk had been on Bacillus cereus (Yusuf et al., 1992; Umoh bromothymol blue polymyxin B (MYP) agar plates.
and Odoba, 1999; Umar et al., 2006; Desai and The plates were incubated at 37C for 24h examined, Varadaraj, 2009). The microbiological safety of foods then left for another 24h at room temperature and sold in the market is of major concern because of the re-examined. Sixteen presumptive colonies of environment in which they are prepared, often in Bacillus cereus were randomly selected and counted places that may have poor sanitation, coupled with based on characteristic colony feature. The colonies use of containers which expose the food to were purified on freshly prepared MYP agar plates.
numerous potential contaminants such as heavy After incubation, typical colonies of bluish green - metals and pesticides. Some of the food samples blue colonies with zones of egg yolk precipitate on have been tested for various microorga-nisms of the medium were picked, streaked out to obtain public health concern and Bacillus cereus was among pure cultures and finally maintained on nutrient agar slopes in the refrigerator (4C). The This study was carried out to evaluate the presence of Bacillus cereus in ready- to -foods morphological characteristics. The morphological purchased from markets in Port Harcourt and test includes appearance of cell, shape and characterize the organism isolated in terms of pigmentation. Further identification of the biochemical reaction and antibiotic susceptibility.
organisms using biochemical characteristics areGram’s reaction, position of spores, motility, citrate utilisation, oxidase, indoleproduction, urease,voges-proskauer reaction, hydrolysis of starch, nitrate reduction, gelatine hydrolysis, productionof gasandacid from glucose, sucrose, mannitol and A total of sixty four food samples being sold in lactose (Cappuccino and Sherman, 2004).
various sales points in urban markets at Mile I,Mile III, Oil mill, and Rumuokoro areas of Port Harcourt were enumerated for the viable counts of All Bacillus cereus isolates were tested for their Bacillus cereus. The food samples comprised of sensitivity to antibiotics by means of a disc Sixteen samples each of “cooked rice”, “masa”, diffusion method (Bauer et al., 1966). The organisms “agidi” and “epiti”(cereal preparation) were were investigated using antibiotics disc containing collected from the food vendors in sterile rifampin (10µg), chloramphenicol (20µg), erythromycin (30µg), ciprofloxacin (10µg), laboratory for microbiological analysis. Analysis of streptomycin (30µg), gentamycin (10µg) ), floxapen (30µg), ampiclox (30µg), norfloxapin (30ug),and lincocin (30µg). The antibiotics disc werespaciously placed on Mueller - Hinton agar plates Table 1. Ingredients of the food samples analysed previously seeded with 6h-broth cultures of Bacillus cereus isolates. The plates were incubatedat 37oC for18-24h. The different zones of inhibition were measured to the nearest millimetre and interpreted as sensitive, moderate sensitive and resistant based on the interpretation table recommended by the disc manufacturer (Oxoid, The results of Bacillus cereus in some cereal based A homogeneous sample was prepared with 10g of ready to eat foods sold in Port Harcourt is depicted Incidence and Antibiotic Sensitivity of Bacillus Cereus Isolated from Ready to Eat Foods Sold in in Fig. 1 and Table 2. Among the various markets sampled, Oil mill market had the highest microbial organism naturally with total percentage count of load of the organism, followed by rumuokoro, mile 26.23%. Masa was followed by agidi with 5.6 × 105 3 and finally mile 1 market. A total of sixty-four cfu/g and a total percentage count of 22.95% and food samples “cooked rice”, “masa”, “agidi” and lastly epiti (5.3×105cfu/g) with total percentage “epiti” were assessed. The average count from the count of 21.31%. The variations in the value of food samples showed that about 7.2 x 105cfu/mL bacterial count percentages may be attributed to were obtained from cooked rice, 6.4 x105cfu/mL from masa, 5.6 x 105cfu/mL from agidi and 5.3 x organism, as most of the food samples were 105cfu/mL from epiti. Sixteen presumptive colonies prepared in places with poor sanitation practices, of Bacillus cereus isolated revealed an irregular edge, poor storage and transport conditions. In most large, flat, rough bluish green to blue colonies with cases, food vendors do not have adequate bathing zones of egg yolk precipitate. Antibiotic sensitivity facilities, atimes starts their day without taking a test of the isolates determined by the disc diffusion bath, therefore foods and ingredients are exposed to method in accordance with the instructions of the repeated contamination from unwashed hands antibiotics disc manufacturer are shown in Table 3.
and materials used for wrapping such as leaves, The isolates were highly resistant to ampiclox, newspaper and reusable polyethylene bags.
norfloxacin & floxapen, sensitive to erythromycin, Purchasing ready-to-eat foods from markets chloramphenicol, streptomycin rifampin and exposes one to a considerable risk of public health ciprofloxacin and less sensitive to lincocin and due to poor hygiene practices (Mosupye and Vonholy, 1999; Ehirim et al., 2001; FAO/WHO, 2003;FAO/WHO, 2005). Agidi and epiti which had the Table 2. Cumulative plate count and total percentage lowest percentage of the isolates could be attributed to the method of processing the corn grain andplantain (drying on moderately clean floor and machine milling) (AOAC, 1995). The limited time & temperature exposure during agidi preparation is equally insufficient to destroy Bacillus cereus spores.
Bacillus cereus food poisoning is principally associated with temperature abuse during the storage of food. Temperature abuse can result in spore germination and multiplication of the vegetative cells leading to hazardous levels of vegetative cells or toxins in the food at the time of consumption (Granum, 2001). The extremeresistance of the spores of this pathogen to heat, desiccation, sanitizers and irradiation contributesto their distribution, survival and persistence The public health significance of Bacillus cereus is of within the entire food chain from raw agricultural high concern in view of the organism being commodities to finished retailed products (Eglezos outbreaks. The isolation of Bacillus cereus from all The results of the antibiotic susceptibility of the the food samples could be explained by the isolates are shown in Table 2. The antibiotic ubiquitous distribution of this organism and its susceptibility was determined by the disc agar ability to form endospores (Kotiranta et al., 2000; diffusion method in accordance with the instru- McKillip, 2000). A total of 64 samples (cooked rice, ctions of the antibiotic disc manufacture (Oxoid).
masa, agidi and epiti) were analysed. From the All the Bacillus cereus isolates were susceptible to results obtained in the study, there were high Streptomycin (100%) Chloramphenicol (100%), contamination levels of Bacillus cereus found in cooked rice (7.2×105cfu/g) this is not surprising Ciprofloxacin (100%) Gentamycin (100%) and less because the organism is a normal flora of rice followed by Masa (6.4×105cfu/g), a cereal The findings of the present study correspond Table 3. Antimicrobial sensitivities of Bacillus cereus isolates from food sample Zones of inhibition in diameter mm status Keys: CR = Cooked rice ; AG = Agidi ; S = Sensitive ; EP = Epiti ; R = Resistant;MS = Masa ; MS = Moderately Sensitive Fig. 1 Distribution of the food samples within the markets analyzed with those obtained by other researchers (Umar et isolates, drug resistance transfer and the overall al., 2006; Whong and Kwaga, 2007). Previous works wide spread use of the antibiotics in the have shown that antimicrobial susceptibility of environment. The development of drug resistance Bacillus cereus were highly susceptible to may be due to the use of these drugs in medical and streptomycin, chloramphenicol, erythromycin, veterinary practice to treat infections and misuse of ciprofloxacin, and less susceptible to ampicillin, the drugs in the society, such practices can lead to ampiclox, cotrimazole, cloxacillin (Umar, et al., drug resistance strains.The antimicrobial pattern 2006). Variations in the percentages may be due to of resistance of Bacillus cereus from foods is useful the differences in the concentrations of antimicro- in epidemiological studies but its effectiveness bial agents used, differences in the source of decreases due to the negligence in utilisation policy Incidence and Antibiotic Sensitivity of Bacillus Cereus Isolated from Ready to Eat Foods Sold in Beattie, S.H. and Williams, A.C. 2000. Detection of The following strategies should be undertaken to toxins. In :Encyclopedia of Food Microbiology (Vol. 1).
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