Nigerian Food Journal Nigerian Institute of Food Science and Technology ISSN: 0189-7241 Vol. 25, Num. 1, 2007, pp. 178-183

¹Department of Microbiology, Ahmadu Bello University, Zaria
²Department of Veterinary Public Health and Preventive Medicine,
Ahmadu Bello University, Zaria*Address for correspondence:Final: clementwhong@yahoo.com

Code Number: nf07018

A total of 300 isolates of B cereus from nine of some Nigeria food were tested for their sensitivity to ten commonly used antibiotics. An average of 25 isolates were obtained per food sample. All B. cereus isolates tested were found to be susceptible to ciprofloxacin (5 mg), chloramphenicol (30 mg), ofloxacin (10 mg) and less than 10% were resistance to gentamicin (10 mg) and nalidixic acid (10 mg). Overall resistance to penicillin G (82%), cefotaxime (56.7%), ceftriaxone (53.3%) and ampicillin (44%) were most frequent, whereas isolates were least resistance to tetracycline (6.7%) nalidixic acid (3%) and gentamicin (1%). The predominant antimicrobial resistance patterns irrespective of food source were cefotaxime, penicillin, ampicillin and ceftriaxone (23%), penicillin (16.3) and cefotaxime, penicillin and ceftriaxone (10.3%).

Keywords: Bacillus cereus isolates, antimicrobial agents, Nigerian foods.

Bacillus cereus is a gram-positive, sporeforming, facultatively anaerobic bacterium. Differentiation of B. cereus from its closely related organisms depends upon the absence of toxin crystal (from B. thuringiensis), hemolysis activity (from B. anthracis), and nonrhizoid growth and motility from B. mycoides (Yamada et al., 1991). B.cereus causes two principal types of food poisoning, emetic and diarrhoeal syndromes. The emetic type is effected by a small cyclic heat-stable peptide, which cause vomiting a few hours after ingestion and is most frequently associated with farinaceous foods such as fried and boiled rice dishes, macaroni and pasta based foods (Lund, 1990; Drobniewski, 1993).

Diarrhoeal types are attributed to enterotoxins, a group of heat-labile proteins causing abdominal pain and diarrhoea after incubation for 8 to 16 hours and vegetative growth of the bacteria in the intestine (Granum, 2001). Diarrhoeal syndrome is most commonly associated with proteinaceous foods such as meat-based dishes, vegetables and salad (Lund, 1990; Brooks et al; 1995). B. cereus is a soil bacterium and has been isolated from cooked and uncooked rice, meat, vegetables, spices “kunun zaki”, milk and dairy products and infant formulae (Kim et al., 2000; Whong et al., 2006). This paper reports the antibiotic resistance patterns of B. cereus isolated from various Nigerian foods to ten antimicrobial agents.

MATERIALS AND METHODS

Sources of Bacillus Isolates

A total of three hundred (300) previously identified B. cereus isolates from fermented milk (Nono), sweetened cereal beverage (kunun zaki), cooked and uncooked rice, roasted beef (suya), spinach (Amaranthus hybridus), ginger (Zingiber officinale), garlic (Allium sativum), and pepper (Capsicum annuum) were purchased from retail outlet in Shika, Samaru and Sabon gari markets, Zaria (Whong et al., 2006).

Isolation and Enumeration of Bacillus cereus

A 0.1ml aliquots of dilution 1:10; 1:100 and 1:1000 were surface plated in duplicates on Bacillus cereus selective Agar.(oxoid, cm617) The plates were incubated at 30⁰C for 24 h. Following incubation, the plates were examined, left at room temperature for another day and re-examined. Presumptive Bacillus cereus colonies were crenated, about 5 mm in diameter and had a distinctive rough turquoise to peacock blue colour surrounded by egg yolk precipitate of the same colour were counted. The total number of colony forming unit (cfu) per ml or g of specimen of presumptive B. cereus was determined. Representative colonies of B. cereus with lecithinase activity were gram-stained. The catalase and grampositive rods purified on nutrient agar plates were stored on nutrient agar slants until they were required for biochemical tests.

Characterisation of Bacillus cereus

Characterisation of B. cereus isolates were based on motility test, hemolysis on sheep blood cells, methyl red reaction, Voges-Proskauer reaction, gelatin hydrolysis, starch hydrolysis, citrate utilization, hemolysin Blproduction and acid production from mannitol, arabinose, glucose, mannose, xylose and salicin. These tests were carried out as described by Cowan and Steel (1974); Harmon (1978); Beecher and Wong (1994).

Antibiotic susceptibility testing

The antibiotic discs (oxoid) and concentrations tested were as follows: ofloxacin (10 mg), cefotaxime (30 mg), penicillin G (10 units), ampicillin (10 mg), gentamicin (10 mg), tetracycline (30 mg), nalidixic acid (30 mg), ceftriaxone (30 mg), ciprofloxacin (5 mg) and chloramphenicol (30 mg).

Susceptibility testing was conducted by the disc diffusion method of Bauer et al. (1966). Bacillus cereus ATCC 14579 and Staphylococcus aureus ATCC 17079 were provided byDr. M.M. Lecadet, Louis Pasteur’s Institute, France and Professor P.F. Olurinola, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, served as positive and negative controls, respectively, for each test run. The antibiotic disc were spaciously placed on inoculated Mueller-Hinton agar plates. The plates were inverted and incubated aerobically at 37⁰C for 16 – 18 h. Zones of inhibition were measured to the nearest millimeter and interpreted as sensitive, intermediate and resistant based on the interpretation table recommended by the disc manufacture (oxoid, 1998).

All the Bacillus cereus isolates were susceptible to ciprofloxacin, chloramphenicol, ofloxacin and less than 10% were resistant to gentamicin and nalidixic acid. No isolates was simultaneously resistant to all ten antimicrobial agents (Table 1). Majority of B. cereus isolates were resistant to penicillin (80.0%), cefotaxime (56.7%), cefriaxone (53.3%) and ampicilin 44.0%. There were twenty-two patterns of resistance (Table 2). Our studies indicate that B. cereus isolates were highly susceptible to of loxacin (100%), ciprofloxacin (100%), chloraphenicol (100%), gentamicin (99.0%), nalidixic acid (97.0%) and tetracycline (93.3%). The B. cereus isolates were less sensitive to cefotaxine (56.7%), penicillin G (82.0%) and ceftriaxone (53.3%). The findings of the present study regarding susceptibilities to these agents are similar to those obtained in other countries (Schiemann, 1978; Chung and Sun, 1986; Wong et al., 1988; Drobniewski, 1993). Previous work has shown that the antimicrobial susceptibility of B. cereus isolates were highly susceptible to chloramphenicol, erythromycin, streptomycin and tetracycline and less sensitive to cotrimoxazole and ampicilin.(Umoh et al; 1995) The variations in the values of percentage susceptibilities in this study may be attributed to the differences in the concentration of antimicrobial agents used, differences in the sources of isolates, drug resistance transfer and the wide spread use of the antibiotics in the environment. The development of drug resistance is multifactorial. The high percentage of isolates resistant to cefotaxime, ceftriaxone, penicillin G and B-lactam antimicrobial agents may be due to the frequent use of these agents in sublethal doses in medical and veterinary practices to prevent or treat infections. This could be a reflection of use and misuse of antibiotics in the society. Such practices could lead to development of drug resistant strains. The antimicrobial resistance patterns of B, cereus from foods is useful in epidemiological studies (Kwaga and Adesiyun,1984). There was a higher proportion of single and multiple resistance patterns among the B. cereus isolates obtained from foods and spices. The multiple resistance patterns that were observed may provide a picture that these antimicrobial agents are highly abused or used at sublethal dose in this environment and also due to the differences in the sources of the isolates. The effectiveness of any antibiotics decreases in the absence of a rational utilization policy, which unfortunately is virtually nonexistent in Nigeria (Bulger and Sherris, 1968).

Based on the results of the in vitro testing, the most effective antimicrobial agents against B. cereus isolates were notably ciprofloxacin, ofloxacin (tarivid), chloramphenicol, gentamicin and nalidixic acid.

REFERENCES

• Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Turck, M. (1966).Antibioticsusceptibilitytesting by a standardized single disk method. American Journal of Clinical Pathology 36: 493-496.
• Beecher. D.J. and Wong, A.C.L. (1994). Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in Blood Agar. Applied and Environmental Microbiology 60:1646-1651.
• Brooks, G. E., Butel, J. S and Morse, S. A. (1995). Spore forming, gram-positive Bacilli: Bacillus and Clostridium species. Jewtz, melnick and Adelberg’s Medical Microbiology International edition (21 edition). Prentice Hall International Inc, USA. p 182-189:
• Bulger, R.I. and Sherris, J.L. (1968). Decreased incidence of antibiotic resistance among Staphylococcus aureus. A study in a university hospital over a 9-year period. Ann. Intern Med. 69:1099-1108.
• Chung, K-T and Sun, H-L (1986). Distribution and characteristics of Bacillus cereus isolated from rice in Taiwan. Journal of Food Science 51: 1208-1212.
• Cowan, S.T. (1974). Cowan and Steel’s manual for the identification of medical bacteria. Second edition, Cambridge University Press, Cambridge. Drobniewski, F.A. (1993). Bacillus cereus and related spcies. Clinical Microbiology Reviews 6: 324-338.
• Granum, P.E. (2001). Bacillus cereus: In food microbiology. Fundamental and Frontiers. (Eds. M.P. Doyle, L.R. Beuchat and T.J. Montville). p. 373-381. ASM Press, Washington, D.C. Harmon, S.M. (1978). Bacillus cereus. Pp. 161
• 166. In: Bacteriological analytical manual, 5th Ed. Food and Drug Administration, Washington, D.C.
• Kwaga, J.K.P. and Adesiyun,A. A.(1984).Antibiograms of Staphylococcus aureus isolates from some ready – to –eat. Products.JournalofFoodProtection47:865-867.
• Kim, Y. Czajka, J. and Batt, A.I. (2000). Development of a fluorogenic probe-based PCR Assaydetectionof Bacilluscereus in Nonfat Dry Milk. Applied and Environmental Microbiology 66: 1453-1459.
• Lund, B.M.(1990). Foodborne illness: Foodborne disease due to Bacillus and Clostridium species. The Lancet 336; 982-986.
• Schiemann, D.A. (1978). Occurrence of Bacillus cereus and the Bacteriological Quality of Chinese” Take-out”Foods. Journal of Food Protection41:450-454.
• Umoh, V.J.,Yusuf, Z.I. andAhmad,A.A. (1995). Antibiograms of Bacillus cereus isolates from flour commonly used in stiff-porridge preparation. Nigerian Food Journal, P31-39.
• Whong, C.M.Z., Kwaga, J.K.P., Umoh, V.J. and Ameh, J.B. (2005). Densities of Bacillus cereus in some Nigerian foods and food ingredients.: Nigerian Journal of Microbiology, Volume 18:
• Wong, H.-C, Chang, M and Fan, J. (1988). Incidence and characterisation of Bacilluscereus isolates contaminating dairy products. Applied and Environmental Microbiology 65: 14831490.
• Yamada, S., Ohashi, E., Agata, N. and Venkateswaran, K. (1999). Cloning and nucleotide sequence analysis of ggrB of bacillus cereus, B. thuringiensis, Baciullus mycoides and B. anthracis and their application to the detection of bacillus cereus in rice. Applied and Environmental Microbiology 65: 1483-1490.

© Copyright 2007 - Annals of African Medicine.

The following images related to this document are available:

Photo images