Bacillus Cereus isolation and load from raw cow milk sold in Markets of Haramaya District, eastern Ethiopia
© The Author(s). 2017
Received: 31 August 2017
Accepted: 23 October 2017
Published: 26 October 2017
Bacillus cereus is a spore-forming bacterium that is widely distributed in the environment and can be detected in a variety of raw milk and milk products especially in vegetative form, which exposed directly in contact with the soil and produces toxins which caused food borne illness and considered as a significant public health hazard. A cross-sectional study was carried out to determine the prevalence and load of Bacillus cereus vegetative cells in raw bulk milk sold in market places at Bate, Haramaya and Aweday Towns, Eastern Ethiopia as well as to assess the in-vitro antimicrobial susceptibility of isolates. A total of 103 raw milk samples were collected and examined for the presence of Bacillus cereus by using Bacillus cereus selective agar media. The isolated presumptive colonies were further analyzed using standard microbiological methods to confirm the presence of Bacillus cereus. Spreading plate method was employed to count the bacteria from milk samples based on 10 fold serial dilutions. For antimicrobial susceptibility test, the disc diffusion test was employed using commercially available 10 antimicrobial discs.
The overall prevalence’s of Bacillus cereus in raw milk samples was 38.8%. Milk collected from Aweday market showed higher occurrence (46.9%) than from Bate (40%) and Haramaya town (31.7%). Analysis of bacterial count showed that, 60% of Bacillus cereus positive milk samples were with bacterial load above recommended limit (>105CFU/ml) for human consumption. However, there was no statistically significant difference (p > 0.05) on the occurrence of Bacillus cereus and bacterial load among different areas. Based on disc diffusion test, B. cereus isolates showed high resistance to Penicillin (100%), Ampicillin (100%), Amoxicillin (80%) and Cefoxitin (80%).
The study indicated that milk samples from market areas were highly contaminated with Bacillus cereus, with potential risk for human consumption. This suggests the need for effective hygienic measures to be introduced during milk production and distribution to avoid public health hazards.
Bacillus cereus is a facultative anaerobic, motile, gram-positive, catalase positive, does not ferment mannitol, rod-shape, beta-hemolytic and spore-forming bacterium; that is widely distributed in the environment due to its ability to resist hostile conditions (Ceuppens et al. 2013) and growth temperature ranging from 10 to 48 °C with optimal growth between 28 °C and 35 °C, pH values of 4.9 to 9.3 and water activities of 0.92 to 1.0 (OSPBH 2005). Another important trait of Bacillus cereus is their ability to grow at the storage temperature of milk (4–7 °C), which mainly determines the shelf life of pasteurized milk and derived milk products (TeGiffel et al. 1995; Granum 2005).
Bacillus cereus is commonly present in food production environments by virtue of its highly adhesive endospores, spreading to all kinds of foods. It produces a range of virulence factors that may cause unpleasant disease in humans when present in food or the gastrointestinal tract and it is one of the major food-borne pathogenic bacteria, although in most cases disease is mild and of short duration. Interestingly, the spectrum of potential B. cereus toxicity ranges from strains used as probiotics for humans to highly toxic strains reported to be responsible for food-related fatalities (Hong et al. 2005). The unique nature of Bacillus cereus like heat resistance, endospore forming abilities, toxin production and psychrotrophic feature (ability to growing below 7 °C) gives ample scope for this organism to be a prime cause of public health hazard (Griffiths and Schraft 2002).
The distribution of Bacillus cereus is worldwide (Logan and Rodrigez-Diaz 2006). Diseases caused by B. cereus are commonly found in places where there is improper food handling. The prevalence of B. cereus induced food-borne illnesses is difficult to determine, because the symptoms associated with B. cereus infections or intoxication are mild, so it is conceivable that many B. cereus infections are not reported and that the prevalence of these infections is largely under estimated (Granum 2007). B. cereus illness recognized that there may be significant under reporting due to the generally mild, short duration and self-limiting symptoms, in addition to its being infrequently tested for in routine laboratory analyses of stool samples (Hall et al. 2005) and due to lack of effective surveillance, B. cereus associated food poisoning may be largely under reported, and probably confused with Staphylococcus aureus and Clostridium perfringens food poisoning due to similar symptoms (Stenfors et al. 2008).
This organism can be detected in a variety of raw milk and milk products especially in vegetative form, which exposed directly in contact with the soil (Bennett and Belay 2001) and produces toxins which caused food borne illness and considered as a significant public health hazard. However, the isolation of high levels of B. cereus was suggested to be involved in food poisoning and causes gastroenteritis motility (Madigan et al. 2009).
The consumption of raw milk and its derivatives is common in Ethiopia, which is not safe for consumer health as it may lead to the transmission of various diseases (Wubete 2004; Shunda et al. 2013). Moreover, from experience of the researchers in Haramaya districts, the consumption of raw milk and milk products was a common practice. However, detailed information on the occurrence and load of Bacillus cereus is limited in the district. Thus, to get information on the pathogen trends, there should be isolation of the organism and profiling of its antibiotic sensitivity patterns. Therefore, the objectives of the study were; to determine the occurrence of Bacillus cereus in raw bulk milk samples collected from market areas at Haramaya district; to evaluate Bacillus cereus load in raw milk samples and assess the susceptibility patterns of Bacillus cereus isolates to commercially available antimicrobials.
Study area and materials
The study was conducted from November 2015 to March 2016 at Haramaya district (Aweday, Haramaya town, Bate towns) in Eastern Hararghe zone, Ethiopia. The area is located at 2000 m altitude above sea level and receives 492 mm average annual rain fall ranging from 118 to 866 mm. The maximum and minimum temperatures are 24 0c and 90c respectively. Ecologically, the area has 65% midland and 35% lowland zones. The two predominant soil types are 60% rigo soils and 40% heavy black clay soil. The relative humidity of the area is 65% (HADB 2014). The study materials were bulk milk samples collected from market areas of the mentioned towns. In the market areas, raw milk was mostly handled by plastic containers with capacity of 3 litters and sellers sat on sides of main access roads to market places which were exposed to dusts from the passengers and vehicles.
Study design and sampling techniques
A cross-sectional study was carried out to study the occurrence and load of B. cereus and their antimicrobial susceptibility profile in milk samples collected from market places of the study areas. Due to the availability of materials and reagents, the sample size was estimated to consist at least 30 from each subject according to the suggestion given by Singh (2006).
Milk sample collection for microbiology analysis
Milk samples were collected into sterile bottles aseptically from bulk milk (market). Before taking samples from bulk, the containers were gently homogenized to allow uniform distribution of the constituents in the milk and then taken 10 ml of milk samples using universal bottle. The universal bottles were clearly labeled by water proof ink using coding system for each sample. Finally, the collected samples were kept in ice box and transported to Haramaya University, College of Veterinary Medicine, Microbiology Laboratory and processed up on arrival.
Laboratory analysis of milk samples
Bacillus cereus isolation and identification
Bacillus cereus selective agar base (CM0167, Oxoid) was the media used to isolate vegetative cells of Bacillus cereus from milk samples. The medium was prepared based on egg yolk, mannitol supplementation. The medium was made selective by addition of Polymyxin B Supplement (SR99) which gives a final concentration of 100 IU of polymyxin B per ml of medium. After18–24 h of incubation at 37 °C in aerobic conditions, the growth of Bacillus cereus was determined based on the colonial morphology, precipitation of hydrolyzed lecithin around colonies and the failure of Bacillus cereus to utilize mannitol sugar according to Mossel et al. (1967) and Fricker et al. (2008). Generally, colonies appearing as crenate, about 5 mm in diameter and have a distinctive turquoise to peacock blue color surrounded by egg yolk precipitation of the same color were presumptively considered as Bacillus cereus. The isolates were confirmed by microscopic examination of Gram’s reagent stained smear and biochemical tests. On gram staining Bacillus cereus was expected to appear as Gram-positive (purple colored), rod shaped cells with short to long chains. The growth of other organisms (contaminants) was ruled out by their colonial appearance and bacterial characteristics on gram staining.
Bacillus cereus isolates were tested for different biochemical tests such as, catalase test, oxidase test and oxidation-fermentative test. Due to the selective nature of the media no further tests were conducted as it enables to differentiate the organism based on these tests.
Enumeration of Bacillus cereus
Where: N = total viable colony counts; ∑C = sum of colonies counted from all plates; n1 = number of plates counted at first dilution; n2 = number of plates at second dilution; C = number of colonies counted; d = dilution factor from which the first counts obtained (least counted dilution).
Antimicrobial susceptibility test of isolates
Guideline for antimicrobial discs used for susceptibility test of B. cereus with their respective concentrations
Disc potency (μg)
Zone diameter, nearest whole mm
Data management and analysis
Data collected from laboratory result was stored on Microsoft excel spread sheet program, and analysis was done by using SPSS Version 20 software program. The total prevalence was calculated by dividing the number of B. cereus positive milk samples by the total number of milk sample tested. Chi-square test was performed to assess the association of different variables with the occurrence and load of Bacillus cereus. A p-value of less than 0.05 (p < 0.05) was considered as statistically significant association.
Prevalence of Bacillus cereus
The prevalence of Bacillus cereus in raw milk from different areas
Number of samples examined
Number of positive samples
Bacillus cereus load of raw milk samples
Bacillus cereus load of raw milk samples collected from different sources
Number of positive samples
Number of samples with above recommended level (%)
In-vitro antimicrobial susceptibility of isolates
Summary of antimicrobial susceptibility profile of Bacillus cereus isolates (n = 10)
Number of isolates
Moderately susceptible (%)
The present study was conducted on raw milk sample collected from market areas with the objectives; to determine B. cereus prevalence and evaluate B. cereus load in raw milk. The overall prevalence of B. cereus in raw bulk milk in Haramaya district was 38.8%. In their study Organji et al. (2015) reported far higher prevalence (66.6%) of B. cereus isolated from of raw milk samples in Egypt. This variation could be due to the small sample size examined by the researchers. However, it is evident that milk could serve as good media for human exposure.
This high prevalence of B. cereus in milk samples from market areas could be due to improper collection, transportation and handling of the milk as it was observed that almost all personnel’s use dirt containers and there is high risk of exposure environmental contamination due to frequent opening and closing of containers. The prevalence of B. cereus from bulk milk was higher in Aweday as compared to other districts. But there was no statistically significant difference (P > 0.05). This could be due to similar condition under which milk is handled for sale in the districts.
In the present study, the Bacillus cereus load was estimated based on the legal limit which is safe for human consumption. Thus, 60% of Bacillus cereus positive milk samples were having load above the recommended level. Generally, the prevalence and load of Bacillus cereus was not significantly varied between different factors. This could be due to similar exposure rate and similar patterns of occurrence of the organism in the environment in the districts. Moreover, it is evident that, Bacillus cereus is ubiquitous organism (Bottone 2010).
The present study showed that, B. cereus isolates showed high resistance to penicillin (100%), ampicillin (100%), amoxicillin (80%) and cefoxitin (80%) which is supported by the findings of Luna et al. (2007), who showed that majority of B. cereus were resistant to ampicillin (95%) and penicillin (95%). This high resistance pattern could be due to irrational and widespread use of few antimicrobials. Agreeably, Organji et al. (2015) from Egypt reported that 100% of tested isolates showed resistance to Penicillin G. Moreover, concurring the present finding, Organji et al. (2015) reported that, 100% of tested isolates were susceptible to Erythromycin and Vancomycin.
The present study presents basic data on the occurrence and load of Bacillus cereus in raw milk ready for market. From the present study report it is evident that, the prevalence of Bacillus cereus was high (38.8%) and majority (60%) of positive samples were having bacterial load above the recommended level for human consumption. The organism showed resistance and moderate susceptibility to majority (80%) of tested antimicrobials. Thus, there could be challenges in the treatment of infections associated with organism. The high prevalence of Bacillus cereus recorded in the markets is quite alarming as these organisms are causative agents of food poisoning and spoilage. Therefore, adequate equipment and facilities for milk storage should be used necessary to minimize microbial contaminations and milk for public consumption should be properly boiled at appropriate temperature.
The authors would like to acknowledge the College of Veterinary Medicine, Haramaya University, for allowing access to Microbiology Laboratory and provision of materials and reagents for the isolation of the organism.
There is no fund for the activity. The research was done by the initiation of the Staffs of College of Veterinary Medicine and externship student (for fulfillment of the DVM degree) with the help of Haramaya University Materials and reagents (bacterial media, biochemical test kits, antibiotic discs).
Availability of data and materials
All necessary data supporting our findings can be found in the repository.
AA carried out the conception of the research concept and designed the methodology, data analysis and interpretation and preparation of the manuscript for publication. TB and SA carried out the sample collection and laboratory work and revision of the manuscript. YM give critical comment on the proposal methodology and reviewed the manuscript for publication. All authors read and approved the final manuscript.
AA: Doctor of Veterinary Medicine (DVM), MSc in Veterinary Microbiology, Assistant Professor at College of Veterinary Medicine, Haramaya University. TB: Doctor of Veterinary Medicine (DVM). SA: Bachelor of Veterinary Science, Lecturer at College of Veterinary Medicine, Haramaya University. YM: Veterinary laboratory technologist (VLT), MSc in Veterinary Microbiology, Lecturer at College of Veterinary Medicine, Haramaya University.
Ethical approval and consent to participate
There was no involvement of animals and humans for sample taking as the study was conducted on milk samples taken from containers which were ready for sale on non-standardized market systems.
Consent for publication
In our study, we don’t have individual participants image, video, etc.
The authors declare that there is no financial or non-financial competing interest from anybody or institute. We also want to assure that we did not receive any technical assistant in developing the research concept or preparation of the manuscript.
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