PaPer 336 Ital. J. Food Sci., vol. 27 - 2015 - Keywords: foodborne pathogens, Bacillus cereus, Escherichia coli, proteolytic enzymes, 16SrRNA - Prevalence and antibiotic resistance of food borne bacterial contamination in some egyPtian food samy selim1,2*, mona Warrad3, el fatih el doWma1 and mohamed abdel aziz2 1Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Aljouf University, Sakaka, P.O 2014, Saudi Arabia 2Botany Department, Faculty of Science, Suez Canal University, Ismailia, P.O. 41522, Egypt 3Department of Medical Laboratory Sciences, College of Applied Medical Science in Al qurait, Aljouf University, Al qurait, Saudi Arabia *Corresponding author: sadomm2003@yahoo.com AbstrAct this study was undertaken to investigate the prevalence and antibiotic resistance of food borne bacterial contamination in some Egyptian food. total viable bacteria and total coliform bacteria were isolated from different sources of food; carbohydrates (bread, flour and basbousa), vegetables (outer and inner tissues of potato and outer and inner tissues of cucumber) and proteins (minced meat, cheese and milk). the study resulted in maximum value of total viable bacteria found in outer tissue of potato 68X104±1.0, while the minimum value found in inner tissues of potato and cucumber. the study resulted in total coliform was maximum value in minced meat 6.4X103±0.3. basbousa and inner tissue of potato and cucumber were free from coliforms. the ability of isolates to producing proteolytic enzymes was tested, we found that 326 isolate (63.92%) from all isolates had this ability, thus we selected most 2 potent proteolytic isolates. the two isolates were identi- fied as Bacillus cereus and Escherichia coli. the identification confirmed by microlog 34.20 system and 16srrNA for two isolates and the same result was founded. sensitivity tested for the most po- tent proteolytic species to 12 of the most commonly used antibiotics in the Egyptian pharmacy. the results showed that all species were sensitive to most of antibiotics, except B. cereus which was strongly susceptible to azteronam and ceftazidim. the data showed that raw meat, cooked food products, and raw milk were most commonly contaminated with foodborne pathogens and many pathogens were resistant to different antibiotics. the study provided useful information for assessment of the possible risk posed to consumers, which has significant public health impact. mailto:sadomm2003%40yahoo.com?subject= Ital. J. Food Sci., vol. 27 - 2015 337 INtroductIoN It has been estimated that as many as 30% of people in industrialized countries suffer from a food borne disease each year and in 2000 at least two million people died from diarrheal disease worldwide (WHo, 2002a). Foods are not only of nutritional value to those who con- sume them but often are ideal culture media for microbial growth, chemical reactions that cause offensive and sensory changes in foods are mediated by bacteria that use food as car- bon and energy source. some of the major bac- terial genera which cause food born infection and intoxication (PuNdIr and JAIN, 2011). con- tamination of food can affect a large number of populations. About 2.5 million people die every year from water born diseases. More than 40% of total population of Indonesia & 60% in thai- land suffered from gastroenteritis per year. A total of 32.7% outbreak was involved with res- taurant catering bakery products (NAzIr and IslAM, 2007). Fruits and vegetables carry microbial flo- ra while passing from the farm to the table. the produce is exposed to potential microbi- al contamination at every step including culti- vation, harvesting, transporting packing, stor- age and selling to the final consumer (FdA, 2000). sources of environmental microbial contamination include raw materials, process- ing equipment, manufacturing activities, san- itation and maintenance practices, workers, waste, animal and insect pests, and microbi- al growth niches embedded in equipment and in structural components of the building. the survival and growth of microorganisms in a food-processing environment may lead to con- tamination of the finished product that may, in turn, result in a reduction of microbiologi- cal safety and quality. Most food plants have locations that can promote the growth of path- ogens and spoilage microorganisms that may be transferred directly on to product or car- ried into additional niches. the origins of these growth habitats are mainly unhygienic design, construction, and maintenance and repair ac- tivities that prevent easy cleaning and disin- fection. the presence of water and nutrients (food product) is required to form a microbial growth niche and the chemical composition of the food and conditions of water activity, pH, temperature, etc., will select the “normal” or- ganisms that can grow there. Microbial growth niches may be established when water is used to clean dry processing environments not de- signed for wet cleaning and not all points in the equipment are promptly and completely dried (JAy, 1996, tEsFAyE et al., 2011). VAN- dErzANt and sPlIttstoEssEr, (1992) dem- onstrated that the microbial growth on equip- ment for processing perishable foods is gov- erned mainly by the ecology of the food, the process, packing room temperature, presence of food residue on the equipment, and efficacy of cleaning and disinfection. recontamination of a biocidaly treated food may increase the risk of foodborne illness if the food is not heat- ed to destroy pathogens before consumption. Perishable foods that do not receive a biocid- al treatment in the final container may be de- contaminated by spoilage microorganisms be- fore packing (VANdErzANt and sPlIttstoEss- Er, 1992, HAIlEsElAssIE et al., 2013). the research project will deal with investigate the prevalence and antibiotic resistance of Ba- cillus cereus, Escherichia coli contamination in some Egyptian food. 2. MAtErIAls ANd MEtHods 2.1. Food samples All food samples were collected from Mansoura city, Egypt. they collected from different shops, super market, groceries and butchers. ten sam- ples were taken from each of bread, flour, bas- bousa, potatoes, cucumber, minced meat, cheese and milk. 2.2. Preparation of samples twenty five grams of each of the following sam- ples bread, flour, basbousa, outer tissue and in- ner tissue of potatoes and cucumber, minced meat, cheese and ten ml of raw milk (unboiled/ unpasteurized) was homogenized in 225 ml ster- ile physiological saline solution (0.85% Nacl) in 500 ml conical flask using a plender for 1-2 minutes, then decimal dilutions were prepared. 2.3. Isolation of bacteria one ml of appropriate dilution was inoculat- ed on both of Nutrient agar medium and Mac- conkey agar medium; the plates were incubat- ed aerobically for 24h at37oc. total viable bac- teria (t.V.b) were enumerated on Nutrient agar medium using pour plate technique. total col- iform (t.c) bacteria were counted on Macco- nkey agar medium by using pour plate tech- nique also. the plates were incubated aerobi- cally for 24h at 37oc. 2.4. Purification After the incubation period (24h), the grow- ing colonies were enumerated for counting. Af- ter counting a sterile wire loop was used to pick the isolate from the plate and was streaked on freshly prepared nutrient agar medium then in- oculated for 24h at 37oc in order to get pure cul- ture. the growing colonies were purified and ex- amined by using cultured morphological appear- ance and Gram reaction. 338 Ital. J. Food Sci., vol. 27 - 2015 2.5. Proteolytic assay Proteolytic activity was carried out according to casein - Pholine method (rAMAlAksHMI et al., 2012). culture media was centrifugated at 7200 rpm for 10 min and supernatant was used as enzyme source. However, 1% casein (in 0.1 M phosphate buffer and pH 7.0) was used as sub- strate. 1 ml each of enzyme and substrate was incubated at 50°c for 60 min. the reaction was terminated by adding 3 ml of trichloroacetic acid (tcA). one unit of protease activity was de- fined as the increase of 0.1 unit optic density at 1 h incubation period. then it was centrifuged at 5000 rpm for 15 min. From this, 0.5 ml of su- pernatant was taken, to this 2.5 ml of 0.5M so- dium carbonate was added, mixed well and in- cubated for 20 min. then it was added with 0.5 ml of folin phenol reagent and the absorbance was read at 660 nm using spectrophotometer. the amount of protease produced was estimated and expressed in microgram of tyrosine released under standard assay conditions. based on the tyrosine released the protease activity was ex- pressed in microgram of tyrosine released by 1 ml of enzyme in 30 minutes at 300c on tyros- ine equivalent. 2.6. Identification of bacterial isolates 2.6.1. conventional methods the appearance of cultures, cell morphologi- cal characteristics and physiological character- istics of the purified selected identified isolates were studied. Media and reagent were prepared according to standard and procedures as de- scribed by (MAcFAddIN, 1980). the identifica- tion was carried out by traditional characters and biochemical tests for isolates according to (krieg et al., 1994) and confirmed out by biolog micolog 34.20 system for most potent proteo- lytic bacterial species. characterization of the most potent isolates were completed and con- firmed by biolog Microlog 34.20 system at the unit of Identification of Microorganisms and bi- ological control unit of Agriculture reasearch center, Giza, Egypt. 2.6.2. Molecular method the polymerase chain reaction (Pcr) meth- ods based on 16s rrNA gene for identification of isolates were used. Genomic dNA was ex- tracted and purified by using Qiagen kit (Qia- gen company). the purity was assessed from the A260/A280 ratios: cultures of bacteria were streaked on tryptic soy agar medium and incubated at 37°c for 24 h. A single colony of each pathogen was grown in (lb) broth medi- um in Erlenmeyer flask and incubated at 37°c for 24 h. culture was harvested by centrifuga- tion at 4°c for 10 min, dNA was extracted from pellets according Qiagen kit instructions. Full length 16s rrNA (1500 bp) were amplified from isolates by Pcr using universal forward prim- er 518F    (5’–ccAGcAGccGcGGtAAtAcG-3’) and 800r (5’-tAccAGGGtAtctAAtcc-3’). op- timum conditions (denaturation 94-1 min, an- nealing 63-45 s and extension 72-2 min, 35 cycles). Amplified 16s rrNA was purified from 0.8% melting point agarose gel. bands ob- tained from Pcr product were eluted and pu- rify by (Qiagen elution kit) Pcr instructions, dNA band desired was excised from ethidi- um bromide stained agarose gel with a razor blade, transferred to Ependorf tube. dNA was sequenced directly using specific primer with concentration 20 pmol Macrogen sequencing company, korea. 2.7. Antibiotic susceptibility test 2.7.1. Antibiotic disks Antimicrobial susceptibility profile of identi- fied bacterial species, Bacillus cereus and E.coli against different antibiotics ampicillin, aztreon- am, cefadroxil, ceftazidime, chloramphenicol, ciprofloxacin, erythromycin, imipenem, neomy- cin, norfloxacin, streptomycin and vancomy- cin were studied. the antibiotic discs used in this research were purchased from oxoid ltd., England. 2.7.2. disc diffusion agar method Antibiotic susceptibility test for the bacterial isolates was carried out by disc diffusion tech- nique according to bAurE et al., (1966). the technique was done by inoculation of pure col- onies of the tested organism into 5 ml of sterile nutrient broth and incubation at 37°c for 24h. then 0.1ml of bacterial suspension (0.5 McFar- land turbidity) was spreading by sterile swabs on nutrient agar plates. duplicate plates were prepared for the strain. Antibiotic discs were ap- plied to the surface of plates at constant distanc- es. the plates were incubated at 37°c for 24h. At the end of incubation period zones of inhibi- tion were measured as (mm). the entire diame- ter of the zone was measured including the di- ameter of the disc. the end point of the reading was taken as complete inhibition of the growth to the naked eye. our (+++) or (++++) indicate high inhibitory effect (large diameter of clearing zone) and (-) indicate no inhibitory effect (good growth). 2.8. Statistical analysis the variations between experiments were es- timated by standard deviations, and statistical significance of changes was estimated by stu- dent’s t-test. only the probability P ≤ 5% was regarded as indicative of statistical significance. Ital. J. Food Sci., vol. 27 - 2015 339 3. rEsults 3.1. Isolation of total viable bacteria from different types of food All growing isolates were enumerated, collect- ed, purified and tabulated. All growing isolates were collected from investigated types of food. ta- ble 1 includes the isolates numbers and sourc- es of collected isolates. total viable bacterial counts (tVb) and total coliform bacterial count (tcb) of three main groups of food were tabulat- ed in table 4. In carbohydrates, the highest count 40.5x103cfu/gm was recorded in bread. the to- tal viable bacterial count in flour was 35.3x103c- fu/gm. the lowest count 1.1x103 cfu/gm was re- corded in basbousa. both Fig. 1 and table 4 show these results. the count of total coliform bacteria (tcb) was 5.4x103 and 5.3x103 cfu/gm in flour and bread respectively. this group of bacteria (tcb) was not recorded in basbousa (table 1). the counts of t.V.b were 68x104 and 52.7x104 cfu /ml in the epidermis of both of potato and cucumber respectively. While the count was re- duced to a lowest count 1.0x102 cfu /ml of the inner tissues of both potato and cucumber. It table 1 - counts of total viable bacteria (t.V.b) and total coliform (t.c.b) & log10 cfu/ ml for samples of food collected from Al-Mansoura city, Egypt. T.C.B T.V.B Types of Food Counts Log 10 Counts Log 10 (cfu/ml) (cfu/ml) I-Carbohydrates Bread 40.5x103± 0.5 4.61 5.3x103± 0.4 3.72 Flour 35.3x103± 0.9 4.55 5.4x103± 0.4 3.73 Basbousa 1.1x103± 0.1 3.03 0.0 0.0 II-Vegetables Potato Outer tissue 68x104± 1.0 5.83 3.2x103± 0.3 3.51 Inner tissues 0.1x103± 0.1 1.98 0.0 0.0 Cucumber Outer tissue 52.7x104± 2.5 5.72 1.8x103± 0.2 3.26 Inner tissues 0.1x103± 0.0 2.0 0.0 0.0 III-Proteins Minced meat 28x103± 3.5 4.44 6.4x103± 0.3 3.80 Cheese 42.7x104± 2.3 5.63 2.1x103± 0.2 3.32 Milk 39.7x104± 0.6 5.60 2.33x103± 2.5 3.37 All values are the means of triplicate plate. Fig. 1 - the percentage of proteolytic and non-proteolytic isolates from different kinds of food is 340 Ital. J. Food Sci., vol. 27 - 2015 is important to notice that tcb was not record- ed in inner tissues of potato and cucumber. the counts of tcb were 3.2x103 and 1.8x103 cfu/ ml from the outer tissue of potato and cucum- ber respectively. the total viable bacterial count in milk was 39.7x104cfu/ml while the tcb was 2.33x103 cfu/ml. From table 1 we notice that the highest count of total viable bacteria was 42.7x104 in cheese. And also the lowest count of tcb was 2.1x103cfu/ml in cheese. From table 4, the count of t.V.b was 28x103 cfu/ml, while the count of tcb was 6.4x103 cfu/ml. 3.2. Screening test for detection of most potent proteolytic bacterial species Fig. 1 showed the potency of proteolytic activ- ities of all purified isolates. the proteolytic iso- lates were 326 isolates (63.92%), while the non- proteolytic isolates were 184 isolates (36.08%). It also shows that the largest clearing zone was 21 mm in case of isolates 62 and 412 (Fig. 2). this indicates that these isolates were the most potent of proteolytic activity. 3.3. Presumptive and confirmation identification the cultural study, morphological appear - ance, Gram reaction and physiological charac- teristics of four most potent proteolytic isolates table 2 - Morphological and biochemical features of strains 62 and 412. Character and biochemical tests Code of Isolate 62 412 Growth Aerobic or Facultative anaerobic Facultative anaerobic Morphology of colony Colony smooth, convex, circular and creamy in color Colony smooth, convex, circular and creamy in color Gram stain + - Cell shape Straight rods, arranged in paires Straight rods, arranged singly or in pairs Motility Motile Motile Flagella arrangement Peritrichous Peritrichous Oxidase -- Gelatine hydrolysis + - D-Glucose, acid production + + D-Glucose, gas production - + Nitrate reduction + + Catalase production + + Oxidation- fermentation F F Voges-Proskaure NT - Arginine dihydrolase NT - Acid production: L-Arabinose - + Lactose NT + Maltose NT + Trehalose NT + D-Mannitol - + D-Xylose - + Indole production NT + Methyle red NT + Citrate (Simmons) NT - H 2 S production NT - NT= not tested for this isolate because of results of another biochemical tests, F= fermentative. Fig. 2 - Example of proteolytic activity of isolated species. were studied according to krIEG et al. 1994. these results were tabulated in table 2. the pre- sumptive identification of the two most potent proteolytic activities. Isolate numbers 62 and 410 were identified as Bacillus cereus and Es- cherichia coli respectively. the most potent bac- terial isolates were confirmed by using biolog Mi- crolog 34.20 system for identification. 16s rrNA gene bands which were detect by specific prim- er at 1500 bp. the 16s rrNA sequences for two isolates were blasted with genebank sequence Ital. J. Food Sci., vol. 27 - 2015 341 database (table 3) and found closet to the same isolates identified by conventional methods. 3.4. Antibiotic susceptibility test of different antibiotics the antibiotic susceptibility test was obtained on the bacterial isolates by using 12 different an- tibiotics by disc diffusion method (table 4). Ba- cillus cereus was resistant (non susceptible) to Aztreonam and ceftazidime. Escherichia coli (gram negative bacteria) were resistant (non suscepti- ble) to vancomycin. While gram positive organ- isms Bacillus cereus was non susceptible (resist- ant) to vancomycine. Bacillus cereus was resist- ant to ampicillin, while Escherichia coli was sus- ceptible (sensitive) to ampicillin. Bacillus cere- us was resistant to ampicillin while Escherichia coli was non susceptible (sensitive) to ampicillin. the studied bacterial species both gram positive and gram negative were sensetive to 8 antibiot- ics (chloramphenicol, cephadroxil, erythromycin, norfloxacin, imipenem, neomycin, ciproloxacin and streptomycin). Escherichia coli was sensitive to three more (ampicillin, aztreonam and ceftazi- dime) i.e. it was sensitive to 11 antibiotics, thus Escherichia coli was the most sensitive bacterial species. Bacillus cereus was sensitive to one more vancomycin and ampicillin respectively i.e. each of them was sensitive to 9 antibiotics. the small- est inhibition zone was 8mm which was record- ed in Escherichia coli due to the effect of erythro- mycin and ceftazidime respectively. table 4 - Antimicrobial susceptibility profile of studied mi- croorganisms against different antibiotics expressed as di- ameter of clearing zones. Antibiotics (Conc.) Bacillus Escherichia cereus coli Ampicillin AM10 0 10 Aztreonam ATM30 0 17 Cefadroxil CFR30 22 16 Ceftazidime CAZ30 0 8 Chloramphenicol C30 23 24 Ciprofloxacin CIP5 20 20 Erythromycin E15 23 13 Imipenem IPM10 37 25 Neomycin N30 20 17 Norfloxacin NOR10 21 20 Streptomycin S10 20 15 Vancomycin VA30 17 0 table 3 - blAst analysis of 16s rrNA sequences of the representative isolates. Closest Validly Described Species Identities Isolate Description Accession number Match Total % Similarity 62 Bacillus cereus KM007-1 KF055368 481 485 99 412 Escherichia coli Xuzhou21 CP001925 1465 1469 99 4. dIscussIoN the bacterial count is considered an index of quality that gives an idea about the hygien- ic measures during processing and helps in the determination of keeping quality of the product (AbErlE et al., 2001). this work will emphasize on the counts and characteristics of bacteri- al genera that is considered to be important in healthy foods giving an attention to their classifi- cation and identification. the main scope of this work is to count the different bacterial species found in the different food sources and study the antibiotic susceptibility patterns of these bac- terial species which are isolated from Al-Man- soura city, Egypt. bacterial counts of foods in- cludes t.V.b and t.c.b are similar with the re- sults of PrAdNyA and soNAlI (2008) who found the counts of t.V.b and t.c.b were in range 9-10 log cfu/ml for local open market in India. All isolated bacterial species are common compo- nents of the bacterial flora of mammals, birds, insects’ reptiles and are commonly found in soil, on plants, water and foods as normal flora (GIl- MorE et al., 2013). these groups of bacteria were isolated by PrAdNyA and soNolI, 2008; NAzIr and Is- lAM, 2007; EAsA, 2010; oluFEMI and AkINy- ErA, 2011; kudJAWu et al., 2011. Presence of Escherichia coli and t.c.b in food usually indi- cates lack of hygiena in handling and post pro- cess contamination, therefore Escherichia coli & t.c.b enumeration are used as food quality parameter (GoNzAlz et al., 2003). the present study was initiated by collection of food samples. All isolates were selected and purified and ini- tial morphologically identified (cocci 46.7% and rods 53.3%) and (Gram’s stain as gram posi- tive rods & cocci 60.3% and Gram negative rods 39.7%). For some species, the range is wide and the growth occurs in a variety of substrates (as a true for coliform bacteria) but for others (e.g. many of pathogens) can grow in limited kinds of substrates. thus, the bacteria found in food differ according to their ability of utilization of energy. the foods that are most often involved in staphylococcal food poisoning differ widely from one country to another (bENNEtt and lAN- cEttE, 1995). the present study was concerned with isolation of the bacterial content of differ- ent food samples, which collected from open markets in Al-Mansoura city, Egypt. All isolates were selected, purified and initial morphological- 342 Ital. J. Food Sci., vol. 27 - 2015 ly identified by shape and gram stain as 46.7% cocci and 53.3% rods and gram positive cocci and rods 60.3% and gram negative rods 39.7%. 16s rrNA gene sequencing will continue to be the gold standard for the identification of bacte- ria, and the automation of the technique could enable it to be used routinely in clinical micro- biology laboratories, as a replacement of the tra- ditional phenotypic tests. Modern technologies have made it possible to construct a high den- sity of oligonucleotide arrays on a chip with ol- igonucleotides representing the 16s rrNA gene sequence of various bacteria. such a design will facilitate automation of the annealing and de- tection of the Pcr products of 16s rrNA gene amplification, and hence routine identification of most clinical isolates will be possible. the use of 16s rrNA gene sequencing has several advan- tages. First, the turnaround time is short. be- cause amplification of the 16srrNA gene takes only four to six hours, and the annealing and detection of Pcr products takes only another few hours, theoretically the identification can be completed within one day. second, it can be used for slow growing bacteria, unlike most commer- cially available kits that are based on phenotyp- ic tests that require the detection of growth of the organism in the presence of certain specific substrates, and hence the slow growing bacte- ria are usually ‘‘unidentified’’ when the growth control shows a negative result. third, the prob- lem of ‘‘unidentifiable strains’’ will be overcome and there would be minimal misidentification – the identification of a clinical strain is clearly de- fined by the number of base differences between it and the existing species. Fourth, oligonucleo- tides representing all bacterial species, includ- ing those rarely encountered clinically, can be included in the array, making it easy to identify the rare species. lastly, such a technique will be applicable not only to pyogenic bacteria, but also to other organisms such as mycobacteria (El-HAdEdy and Abu El-Nour, 2012). Antibiotic resistance (Gram positive and Gram negative bacterial species) from food sources are important and serious problem in clinical field (El-AIdy, 2007). the antibiotic sensitivity against bacteria is assayed by disc-diffusion method and in our study as well (sElIM, 2011; sElIM et al., 2012; 2013)). In this study, the antibiotics sus- ceptibility patterns of potent proteolytic bacteri- al species (Bacillus cereus and Escherichia coli) against 12 different antibiotics were investigat- ed. Antibiotics include ampicillin (AM), aztreon- am (AtM), cefadroxil (cFr), ceftazidime (cAz), chloramphenicol (c), ciprofloxacin (cIP), eryth- romycin (E), imipenem (IPM), neomycin (N), nor- floxacin (Nor), streptomycin (s) and vancomy- cin (VA). In this study Bacillus cereus was re- sistant to 3 antibiotics (ampicillin, azactam and cerazdime). beta-lactam antibiotics also bind to inhibit the action of other cytoplasmic pro- teins that had a role in peptidoglycan synthesis and turn over (AbIGAl and dIXE, 1994). trans- peptidation reactions that cross links the pep- tide side chain of polysaccharide peptidoglycan back bone. transpeptidase and other proteins were called “penicillin binding protein”. the net result of beta- lactam binding to this protein was to stimulate endogenous enzyme that degrade peptidoglycan (AbIGAl and dIXE, 1994). the in- hibitory effect of vancomycin was similar to the effect of beta-lactam antibiotic. our results showed that Bacillus cereus and Escherichia coli were sensitive to cefadroxil, chlo- ramphenicol, ciprofloxacine, erythromycin, imi- penem, neomycin, norfloxacin and streptomy- cin. Neomycin, aminoglycoside antibiotic; eryth- romycin, as macrolid antibiotic have the ability to bind to the 50s or 30s ribosomal subunit (in- hibit protein synthesis). Also, inhibitory effect of ciprofloxacin and norfloxacin, as quinolones an- tibiotic, may be due to having the ability to in- hibit bacteria by interfering with their ability to make dNA with diverse targets dNA gyrase, this inhibition effect leads to preventation of multiply of bacteria. the present results showed that all tested Escherichia coli strains were resistant to vancomycin antibiotic, while srinivasona et al., (2007) who found that all tested Escherichia coli strains were resistant to two or more antimicro- bial used in veterinary medicine. Bacillus cere- us was resistant to aztreonam and ceftazidime. Antibiotic resistance can be categorized in three types: natural or intrinsic resistance; mu- tational resistance and extrachromosomal or aquired resistance. the resistance of isolates to beta-lactam antibiotic may be due to drug inac- tivation: i.e. Ampc cephalosporinase (beta lac- tamase enzyme that open the beta-lactam (ring) as an intrinsic resistance. target site modifica- tion (i.e. change in PbPs- penicillin binding pro- teins-) as mutational resistance represented in drug inactivation (AbIGAIl and dIXIE, 1994). Moreover the resistance of isolates to aminogly- coside antibiotic and erythromycin macrolides antibiotics may be due to inaccessibility of the target as an intrinsic resistance, reduced perme- ability or uptake as mutational resistance and aquired resistance represented in drug activa- tion (dIAb et al., 2002; 2004). the resistance of isolates to furadantine antibiotic may be due to chromosomal or plasmide mediated and inhi- bition of nitrofuran reductase. Also the resist- ance of isolates to fluoroquinolones antibiotics may be due to reduced permeability or uptake as mutational resistance (FANGE et al., 2009). the mode of action of ciprofloxacin and norflox- acine as a quinolones antibiotic as accumulat- ed with the explanation of FANGE et al., (2009). In our present study, four studied bacterial species which isolated from food sources were investigated to all selected antibiotics. Most of the selected antibiotics represent the following classes ; beta-lactam, aminoglycoside, macrolide and quinlones. total bacterial counts is con- Ital. J. Food Sci., vol. 27 - 2015 343 sidered an index of quality which gives an idea about the higienic measures during processing and help in the determination of keeping qual- ity of the product (AbErlE et al., 2001). In this study, the identified bacterial species were Ba- cillus cereus (13.72%) and Escherichia coli (18.3). these results had a strong support of many re- searches. these bacterial species are common components of the microbial flora community. Generally, the methods of production, transpor- tation, handling and sale of food entirely unhy- gienic and entirely depend on the traditional system, such system could pose favorable en- vironment for bacterial contamination. the ex- istences of these bacterial species which isolat- ed in different food sources. these results are agreement with FdA, 2000. Moreover the stud- ied food sources are considered as reservoir for some pathogenic bacteria (MANGEs et al., 2006). the Bacillus species are of the soil origin and may contaminate bread through the raw mate- rial and bakery requirements used. Bacillus ce- reus is widely distributed in the environment and is found almost everywhere including, dust, water and decaying matter. the microbiotas in dried traditional vegetables sold in open market in parts of Ghana are dominant by aerobic mes- ophilic bacteria including mainly bacillus, lactic acid bacteria, coliform and moulds. Many stud- ies provided evidence that Escherichia coli is a frequently occurring organism in milk (sooM- ro et al., 2002), this evidence agrees with the obtained results. Presence of Escherichia coli and t.c in food usually indicates lack of hy- giene in handling, storing food and production inadequate storage and post process contamina- tion. therefore Escherichia coli and t.c enumer- ation are used as food quality parameter (GAN- zAlEz et al., 2003). Escherichia coli was isolat- ed from the samples and also has been detect- ed in many studies rEFErENcEs Aberle E.d., Forrest J., Gerrard d.E. and Mills E.W. 2001. Principles of meat science (4th ed). Hunt Puplishing co., kendall, usA. Abigail A.s. and dixie d.W. 1994. Antibiotics: Mechanisms of action and mechanism of bacterial resistance. In: bac- terial pathogenesis a molecular approach: Abigail, A. s. and dixie, d. W. (Eds); (AsM Press), 8th Ed. 97-110. bauer A.W., kirby W.M.M., sheriss J.c. and turck M. 1966. Antibiotic susceptibility testing by standarised single method. Am. J. clin. Pathol., 45:493-6. bennett r.W. and lancette G.A. 1995. Staphylococcus au- reus. In : bacteriological Analytical Manual. 8 Ed. Gaith- ersburg. P. 12.01-12.05. diab A.M., Abdel Aziz M.A. and selim, s.A. 2002. Plasmid encoded transferable antibiotic resistance in gram-nega- tive bacteria isolated from drinking water in Ismailia city. Pak J biol sci. 5(7):774-779. diab A.M., Abdel Aziz M.H., selim s.A., El-Alfay s. and Mou- sa M.A. 2004. distribution, Involvement and Plasmid characterization of Aeromonas spp. Isolated from Food staffs and Human Infections. Egyptian Journal of biol- ogy 6:12-20. Easa s.M.H. 2010. Microorganisms found in fast and tradition- al fast food. Journal of American science 6(10):515-531. El-Aidy E.F. (2007). Antibiotic resistance of some microor- ganisms isolated from cancer patients. M.sc thesis, Fac- ulty of science, zagzig uni. Egypt. El-Hadedy d. and Abu El-Nour s. (2012). Identification of Staphylococcus aureus and Escherichia coli isolated from Egyptian food by conventional and molecular meth- ods. Journal of Genetic Engineering and biotechnolo- gy 10:129-135. Fange d., Nilsson k., tenson t. and Ehrenberg M. 2009. drug efflux pump deficiency and drug targets resistance masking in growing bacteria Proc. Natl. Acad. sci. usA 106:8215-8220. FdA (Food and drug Administration) 2000. Guide to min- imize microbial food safety hazards for fresh fruits and vegetable. Gilmore M.s., lebreton F. and van schaik W. 2013. Genom- ic transition of enterococci from gut commensals to lead- ing causes of multidrug-resistant hospital infection in the antibiotic era. curr opin Microbiol. 16(1):10-16. Gonzalez r.d., tamagnini l.M., olmos P.d. and de sousa G.b. 2003. Evaluation of a chromogenic medium for to- tal coliforms and Escherichia coli determination in ready- to-eat foods. Food Microbiology. 20:601-604. Jay J.M. 1996. Modem food microbiology, 5th. ed. Interna- tional thomson Publishing New york, 661. Haileselassie M., taddele H., Adhana k. and kalayou s. 2013. Food safety knowledge and practices of abattoir and butchery shops and the microbial profile of meat in Mekelle city, Ethiopia. Asian Pacific Journal of tropical biomedicine. 3(5):407-412. krieg r.N., Holt G.J., sneath P.H.A. and Williams s.t. 1994. bergey’s Manual of determinative bacteriology. Williams & Wilkins baltimore u.s.A. Ninth Eddition. kudjawu b., sakyi-dawson E. and Amoa-Awua W.k. 2011. the microbiota of dried traditional vegetables produced in the sudan savannah and Guinea savannah agro-eco- logical zone of Ghana. International Food research Jour- nal 18:101-108. MacFaddin, J.F. 1980. biochemical tests for identification of medical bacteria. the Williams & Wilkins company, baltimore. u.s.A. Manges A.r., Natarajan P., solberg o.d., dietrich P.s. and riley l.W. 2006. the changing prevalence of drug-resist- ant Escherichia coli clonal groups in a community: evi- dence for community outbreaks of urinary tract infec- tions. Epidemiol Infect. 134:425-31. Nazir k. H. and Islam t. 2007. Association of bacteria in stored bakery foods of retailers’ shops in Mymensin- gh, bangladesh. J bangladesh soc Agric sci technol. 4(1&2):21-24. olufemi A. and Akinyera b. 2011. Microbial quality of prawns offered for sales at some locations and sales outlets in Ibadan south Western Nigeria. J. Microbiol biotech res. 1 (1):46-51. Pradnya A.J. and sonali P.P. 2008. Microbiological analy- sis of fresh vegetables & fruits and effect of anti-micro- bial agents on microbial load. department of microbiol- ogy, birla college of Arts, science and commerce, kaly- an-421 304, India. Pundir r.k. and Jain P. 2011. Evaluation of five chemical food preservatives for their antibacterial activity against bacterial isolates from bakery products and mango pick- les. J. chem. Pharm. res. 3(1): 24-31. ramalakshmi N., Narendra d., ramalakshmi M., roja s., Archana b.k.N., Maanasa G. 2012. Isolation and charac- terization of protease producing bacterial from soil and estimation of protease by spectrophotometer. the Exper- iment 1 (1):1-7. selim s.A. 2011. chemical composition, antioxidant and an- timicrobial activity of the essential oil and methanol ex- tract of the Egyptian lemongrass Cymbopogon proximus stAPF. International Journal of Fats and oils (Grasas y Aceites) 62 (1):55-61. selim s.A., El Alfy s., Al-ruwaili M., Abdo A., Al Jaouni s. 2012. susceptibility of imipenem-resistant Pseudomonas 344 Ital. J. Food Sci., vol. 27 - 2015 aeruginosa to flavonoid glycosides of date palm (Phoe- nix dactylifera l.) tamar Growing in Al Madinah, saudi Arabia. African Journal of biotechnology 11(2):416-422. selim s.A., Abdel Aziz M.H, Mashait M.s., Warrad M.F. 2013. Antibacterial activities, chemical constitutes and acute toxicity of Egyptian Origanum majorana l., Peganum har- mala l. and Salvia officinalis l. essential oils. Journal of pharmacy and pharmacology 7(13):725-735. soomro A.H., Arian, M.A., khaskheli M. and bhutto b. 2002. Isolation of Escherichia coli from raw milk and milk products in relation to Public Health sold under mar- ket conditions at tandojam. Pakistan Journal of Nutri- tion 1(3):151-152. srinivasana V., Gillespiea b.E., lewisa M.J., Nguyena l.t., Headricka s.I., schukkenb y.H. and olivera s.P. 2007. Phenotypic and genotypic antimicrobial resistance pat- Paper Received January 28, 2014 Accepted October 21, 2014 terns of Escherichia coli isolated from dairy cows with mastitis. Food scien. and technol. 50(7):767-773. tesfaye A., Mehari t. and Ashenafi M. 2011. Inhibition of some foodborne pathogens by pure and mixed lAb cul- tures during fermentation and storage of Ergo, A tradi- tional Ethiopian fermented milk. ArPN J Agric biolog sci 6 (4):13-19. todar k. 2008. todar’s online textbook of bacteriology. uni- versity of Wisconsin Madison department of bacteriology. Vanderzant c. and splittstoesser F.d. 1992. compendium of Methods for the Microbiological Examination of Foods. 3 rd. ed. American Public Health association, Washing- ton, dc., 1219. WHo (World Health organization) 2002a. Food safety and foodborne illness. World Health organization Fact sheet, Geneva, 237.