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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 37o
C 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
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Submitted by: Charles Bantz, Provost and Senior Vice President Academic Affairs JOSEPH F. YOUNG, SR. PSYCHIATRIC RESEARCH AND TRAINING PROGRAM PROPOSED BUDGET FISCAL YEAR 2003 It is recommended that the Board of Governors approve the proposed FY 2003 budget for theJoseph F. Young, Sr. Psychiatric Research and Training Program to be allocated to program Approved Proposed Progr
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