INCIDENCE AND ANTIBIOTIC SENSITIVITY OF …

1 Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 13-18 Global Science PublicationsINCIDENCE AND ANTIBIOTIC SENSITIVITY OF BACILLUSCEREUS ISOLATED FROM READY TO EAT FOODS SOLD INSOME MARKETS IN PORTHARCOURT, RIVERS STATE, NIGERIA O. K. AGWA, UZOIGWE AND WOKOMAD epartment of Microbiology, University of Port Harcourt, Rivers State, NigeriaDepartment 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 indigenousAbstract - 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 ( ), masa ( ), agidi ( ) and epiti had the least frequency of All Bacillus cereusisolates tested were found to be susceptible to rifampin (30 g), chloramphenicol (20 g), erythromycin(30 g), ciprofloxacin (10 g), streptomycin (30 g), gentamycin (10 g) and lincocin (30 g) and 100%resistance against norfloxapin (30ug), floxapen (30 g) and ampiclox (30 g).

2 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.*Corresponding author - Obioma Agwa, E-mail : economic conditions have resulted insituations, where food vending has becomeincreasingly important in most countries;contributing significant income inflow forhouseholds involved in selling these foods(Mosupye and vonholy, 1999; FAO/WHO, 2003;FAO/WHO, 2005 ). Hygiene and sanitation practicesobtained during preparation and marketing ofthese foods provide ample opportunities for theproliferation of these food with food-bornepathogens (Desai and Varadaraj, 2009). Among thefood- borne pathogens, strains of Bacillus cereus areof significance because of its ubiquitous nature andability to occur in a wide range of foods (Ehling-schulz, et al.)

3 , 2004a; Oguntoyinbo and Oni, 2004;Reyes et al., 2007).The unique properties of Bacillus cereus includesheat resistant endospore forming ability, toxinproduction in varieties of foods and the psychro-trophic nature makes the organism a prime causeof public health hazard (Griffith and Schraft, 2002;Dierick et al., 2005). Bacilli cause symptoms leading toillness, relatively mild lasting up to 24h, but couldbe severe leading to hospitalisation and in somecases death. The organism is found in a wide rangeof habitat including air, water, dust and soil, fromwhere spores are introduced into cereal crops,vegetables, animal hair, fresh water and plant foods especially rice, potatoes, beans,peas, and spices are the most common sources ofBacillus cereus (Eglezos et al., 2010). The occurrence ofthe organism within the environment enables it toenter the food chain through raw materials and isa major problem in convenience foods and masscatering (Beathe and William, 2000; Jensen et al.

4 ,2005; Guinebretiere et al., 2006). Because of the roleof microorganisms in spreading diseases, the needto assess the safety and quality of foods is veryimportant, in order to ensure safety of supply,clean, wholesome and high quality delivery to thepublic. When the food hygiene system fails, abatch of food is contaminated with high levelmicrobes potential for food borne disease outbreak,its distribution cannot be controlled, changes in14 AGWA ET health and deterioration and increasessusceptibility rate (Sahota et al., 2008).In several studies that have been conducted onthe INCIDENCE of food-borne pathogens, emphasishad been on Bacillus cereus (Yusuf et al., 1992; Umohand Odoba, 1999; Umar et al., 2006; Desai andVaradaraj, 2009). The microbiological safety of foodssold in the market is of major concern because of theenvironment in which they are prepared, often inplaces that may have poor sanitation, coupled withuse of containers which expose the food tonumerous potential contaminants such as heavymetals and pesticides.

5 Some of the food sampleshave been tested for various microorga-nisms ofpublic health concern and Bacillus cereus was amongthem (Tomlins et al., 2004). This study was carried out to evaluate thepresence of Bacillus cereus in ready- to -foodspurchased from markets in Port Harcourt andcharacterize the organism isolated in terms ofbiochemical reaction and ANTIBIOTIC AND METHODSS ampling site and collectionA total of sixty four food samples being sold invarious sales points in urban markets at Mile I,Mile III, Oil mill, and Rumuokoro areas of PortHarcourt were enumerated for the viable counts ofBacillus cereus. The food samples comprised ofSixteen samples each of cooked rice , masa , agidi and epiti (cereal preparation) werecollected from the food vendors in steriledisposable polyethylene bags brought tolaboratory for microbiological analysis. Analysis ofsamples were performed within 60mins ofcollectionTable 1.

6 Ingredients of the food samples analysedSamplesIngredientsCooked riceRice, meat, groundnut oil,Seasonings(salt, onions, pepper,tomato paste)MasaRice and raw milkAgidiPowdered corn grain & seasoningsEpitiPowdered corn grain, plantain flour,palmoil, of Bacillus cereusA homogeneous sample was prepared with 10g offood sample, aseptically transferred into 90mL oftryptone broth to give an initial 1:10 of from 10-5dilutions were surfaceplated on sterile dried Mannitol egg-yolkbromothymol blue polymyxin B (MYP) agar plates were incubated at 37C for 24h examined,then left for another 24h at room temperature andre-examined. Sixteen presumptive colonies ofBacillus cereus were randomly selected and countedbased on characteristic colony feature. The colonieswere purified on freshly prepared MYP agar incubation, typical colonies of bluish green -blue colonies with zones of egg yolk precipitate onthe medium were picked, streaked out to obtainpure cultures and finally maintained on nutrientagar slopes in the refrigerator (4C).

7 Thepresumptive isolates were identified bymorphological characteristics. The morphologicaltest includes appearance of cell, shape andpigmentation. Further identification of theorganisms using biochemical characteristics areGram s reaction, position of spores, motility, citrateutilisation, oxidase, indoleproduction, urease,voges-proskauer reaction, hydrolysis of starch,nitrate reduction, gelatine hydrolysis, productionof gasandacid from glucose, sucrose, mannitol andlactose (Cappuccino and Sherman, 2004). ANTIBIOTIC SENSITIVITY testAll Bacillus cereus isolates were tested for theirsensitivity to antibiotics by means of a discdiffusion method (Bauer et al., 1966). The organismswere investigated using antibiotics disc containingrifampin (10 g), chloramphenicol (20 g),erythromycin (30 g), ciprofloxacin (10 g),streptomycin (30 g), gentamycin (10 g) ), floxapen(30 g), ampiclox (30 g), norfloxapin (30ug),andlincocin (30 g).

8 The antibiotics disc werespaciously placed on Mueller - Hinton agar platespreviously seeded with 6h-broth cultures ofBacillus cereus isolates. The plates were incubatedat 37oC for18-24h. The different zones of inhibitionwere measured to the nearest millimetre andinterpreted as sensitive, moderate sensitive andresistant based on the interpretation tablerecommended by the disc manufacturer (Oxoid,1998).RESULTSThe results of Bacillus cereus in some cereal basedready to eat foods sold in Port Harcourt is depicted15 INCIDENCE and ANTIBIOTIC SENSITIVITY of Bacillus Cereus Isolated from Ready to Eat Foods Sold inin Fig. 1 and Table 2. Among the various marketssampled, Oil mill market had the highest microbialload of the organism, followed by rumuokoro, mile3 and finally mile 1 market. A total of sixty-fourfood samples cooked rice , masa , agidi and epiti were assessed. The average count from thefood samples showed that about x 105cfu/mLwere obtained from cooked rice, x105cfu/mLfrom masa, x 105cfu/mL from agidi and x105cfu/mL from epiti.

9 Sixteen presumptive coloniesof Bacillus cereus isolated revealed an irregular edge,large, flat, rough bluish green to blue colonies withzones of egg yolk precipitate. ANTIBIOTIC sensitivitytest of the isolates determined by the disc diffusionmethod in accordance with the instructions of theantibiotics disc manufacturer are shown in Table isolates were highly resistant to ampiclox,norfloxacin & floxapen, sensitive to erythromycin,chloramphenicol, streptomycin rifampin andciprofloxacin and less sensitive to lincocin 2. Cumulative plate count and total percentagefrom various food samplesS/NoSampleNo. ofTotal% totaltestedsamplescounts(x106cfu/g) : CR = cooked rice, MS = masa,AG = agidi; EP = epiti DISCUSSIONThe public health significance of Bacillus cereus is ofhigh concern in view of the organism beingimplicated in a number of food poisoningoutbreaks. The isolation of Bacillus cereus from allthe food samples could be explained by theubiquitous distribution of this organism and itsability to form endospores (Kotiranta et al.)

10 , 2000;McKillip, 2000). A total of 64 samples (cooked rice,masa, agidi and epiti) were analysed. From theresults obtained in the study, there were highcontamination levels of Bacillus cereus found incooked rice ( 105cfu/g) this is not surprisingbecause the organism is a normal flora of ricefollowed by Masa ( 105cfu/g), a cerealpreparation from rice which harbour theorganism naturally with total percentage count Masa was followed by agidi with 105cfu/g and a total percentage count of andlastly epiti ( 105cfu/g) with total percentagecount of The variations in the value ofbacterial count percentages may be attributed tonumerous contaminations potential of theorganism, as most of the food samples wereprepared in places with poor sanitation practices,poor storage and transport conditions. In mostcases, food vendors do not have adequate bathingfacilities, atimes starts their day without taking abath, therefore foods and ingredients are exposed torepeated contamination from unwashed handsand materials used for wrapping such as leaves,newspaper and reusable polyethylene ready-to-eat foods from marketsexposes one to a considerable risk of public healthdue to poor hygiene practices (Mosupye andVonholy, 1999; Ehirim et al.