Introduction

Bread is eaten all over the world by almost peoples of every culture. If we traveled to the other side of the planet we would probably find a culture very different from our own, yet with its own version of bread [1]. Therefore; bread is a food product that is universally accepted as a very convenient form of food that has desirability to all population rich and poor, rural and urban. Thus, none of any food types compute with bread in line with consumption in the world. In Nigeria, bread has become the second most widely consumed non-indigenous food product after rice [2]. Also in Ethiopia, next to Injera the bread is highly consumed and loaves among most people, particularly in urban area daily and on holiday. Although bread is a prominent food for the world population, it is affected the health of the people in case of contaminated with pathogenic microorganisms. Basically, the surface of a fresh baked bread free of viable microorganisms; however, it is subject to contamination by mould spores and bacteria from the air, improperly sanitized utensils, and handlers, transporting equipments and wrapping materials. Ehavald and Estonia [3] explained that more than 90% of bread contamination occurs during cooling, transporting, slicing and wrapping operation. Moreover, it has been reported that mould spores in proofers’ cloths in bakeries can build up enough heat resistance to survive baking [4]. Ogundare and Adetuyi [5] reported from Nigeria, freshly baked bread, after ten minutes had been contained bacterial species, include Bacillus cereus and Staphylococcus spp. and after 48 and 96 h Staphylococcus cohnii and Bacillus firmus were isolated, respectively. Similarly, after 10 min mould such as Aspergillus flavus, Aspergillus niger and Penicillium citrinum were reported. Daniyan and Nwokwu [6] reported total aerobic bacterial count ranged from 2.85 × 104 CFU/g to 6.21 × 106 CFU/g, Coli form from 1.19 × 104 CFU/g to 2.05 × 106 CFU/g, Staphylococcus from 2.00 × 104 CFU/g to 5.52 × 105 CFU/g and fungi count ranged from 4.0 × 103 to 1.40 × 106 CFU/g and the highest frequency occurrence of Staphylococcus, Escherichia coli and Bacillus spp., respectively were recorded from the beaked bread which are currently emerging to resist various types antibiotics. Even though, the bread is highly consumed as daily meal in home, cafeteria, and tea shops in Ethiopia [7], the microbial safety of this delicious food is still not documented. On the other hand, the bread is eaten by nearly all of the world population but some groups of the consumers are criticized about the microbial safety of the bread because of unhygienic of transporting material, handlers and storages place of bread [8], which is presently appeared in Jimma town. Hence, having this scenario insight, the present study was designed to assess the microbial load of shop and cafeterias sold bread, and evaluate their antibiotic susceptibility.

Materials and Methods

Description of the study area

The study was conducted in Jimma town, Oromia regional state, located at 353 km southwest of Addis Ababa. The microbial analysis was carried out at Jimma University, Biology department, research and Post graduate Laboratory. The geographical location of the town is 7o 41’N latitude, 36o 50’E longitude, and an average altitude of 1,780 m above sea level. The average of minimum and maximum temperature of the town is of 14 and 30°C, respectively with the annual rainfall ranges from 1138 to 1690 mm [9].

Sample collection

A total of 90 samples were collected from baked bread selling shops of Jimma town for past two years. The food samples were purchased from bread sellers at time between 8:00 and 12 am. The purchased food samples were added into sterile polyethene bag by bread sellers and transported to Research and Postgraduate Lab. The microbial analysis was conducted within one to three hours of collection. The food samples were kept in the refrigerator at 4o C until microbial analysis was conducted.

Sample preparation and microbial enumeration

Sample preparation: A 25 g of bread samples were suspended in 225 mL; of buffered peptone water (BPW), and homogenized in Erlenmeyer flasks for 5 min using shaker at 160 rpm. A 1 mL of homogenized sample was transferred into 9 mL of BPW, and mixed thoroughly by using vortex mixer. The homogenized food sample was serially diluted from 10-1 to 10-6 and 0.1 mL aliquot of appropriate dilution was plated on pre-solidified plates and incubated at appropriate temperature and period. The colonies was counted from plate containing microbial colonies between 30 and 300 and expressed in colony forming units per gram (CFU/ g).

Microbial enumeration: From appropriate serial dilutions, 0.1 mL of the aliquot was plated on Plate Count Agar (PCA), Violet Red Bile Agar (VRBA) plates [10], MacConkey agar [11], Mannitol Salt Agar (MSA) [12] and incubated at 32°C for 48 h for count Aerobic mesophilic bacteria, Coliform, Enteriobacteriaceae and Staphylococci, respectively, and also for count of Aerobic bacterial spore formers, appropriate serially diluted sample was heat treated in a water bath adjusted at 80o C for at least 10 min. Thereafter, 0.1 mL aliquot was plated on pre-solidified surface of PCA and incubated at 35o C for 48 h [12]. Moreover, after, 0.1 mL aliquot was plated on pre-solidified surfaces of Potato Dextrose Agar supplemented with 0.1 g chloramphenicol and incubated at 25o C for 5-7 days [11]. Smooth (non-hairy) colonies without extension at periphery were counted as yeasts whereas hairy colonies with extension at periphery were counted as mould. Microbial analysis: From appropriate aerobic mesophilic countable plate, 10 to 15 colonies with distinct morphological differences were randomly picked from plates and aseptically transferred in to a test tube containing 5 ml of nutrient broth. Then, incubated at 32o C for 24 h. The repeatedly sub-culturing isolates were characterized up to genus level based on John [13] bacterial classification manual. The cell morphology of the bacterial pure isolates was examined microscopically after gram staining, and also the motility of isolates were checked using motility medium. Moreover, after repeatedly purified the agar block of mold culture on PDA, the morphology of hyphal culture was stained using lacto phenol cotton blue and observed under the microscope for identification of fungal genera. The biochemical test including KOH (potassium hydroxide), Catalase, Oxidation fermentation (O/F), Cytochrome oxidase tests were conducted, accordingly.