Isolation and antibiotic resistance of bacillus cereus from spices collected in markets in Bogotá Colombia

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(1)Isolation and antibiotic resistance of Bacillus cereus from spices collected in markets in Bogotá Colombia. Juan Camilo Farfán Esquivel, María Consuelo Vanegas López, Angélica Garzón Boada.. Abstract Bacillus cereus is a foodborne pathogen that could cause two types of intoxications. There are associated to the consumption of spices, rice, pasta, salads, and related. This bacterium is reported to develop resistance to Beta lactam antibiotics. In Colombia some outbreaks were reported in different departments but there are not current investigations on its antibiotic resistance. In this study 79 samples of spices were analyzed and 16 of them (53.33%) showed presumptive colonies for B. cereus. 30 strains of B. cereus and related species were isolated and identified by biochemical reactions and PCR. Those strains were obtained mainly of oregano (17%), color (23%) and cumin (17%). Antibiotic susceptibility tests were tested in all strains finding multi-resistant strains: cefoxitin (63.33%), cefepime (100%), cefalotine (96.66%), trimethoprim\sulfamethoxazole (70%), penicillin (100%), tetracycline (20%) and ampicillin (100%). It was founded that all the strains isolated were resistant to some Beta lactam antibiotics in concordance with previous studies. Although B. cereus intoxications are not treated with antibiotics, antibiotic resistance could be important in means of resistance gene carrier. Key words: Bacillus cereus, antibiotic resistance, spices, foodborne pathogens. Colombia 1. Introduction Bacillus cereus is a foodborne pathogen that is worldwide distributed, like many other pathogenic bacteria (Carlin et al., 2010). It causes intoxications when the spores or cells, present in some raw, processed or cooked food, are consumed (UERIA, 2011). Intoxication is done by two types of toxins, diarrheic and emetic. The first type of toxins cause illness characterized by abdominal pain and diarrhea 6 to 18 hours after consume (Chaves, Pires, & Vivoni, 2011). Emetic toxin cause constant vomiting after 1 or 5 hours after the ingestion (Svensson, Monthán, Guinebretière, Nguyen-Thé, & Christiansson, 2007). Emetic toxin is produced by some strains that only grow at 15-40°C; this toxin can resist high temperatures. Meanwhile, diarrheic toxins are produced by strains that can grow at 4-10°C. Those toxins does not resist high temperatures and are produced in food (UERIA, 2011). Foodborne diseases have a great prevalence in least developed countries where population is truly affected. Children, neonates and the elderly are the risk group including immune-compromised patients in hospitals. (INFOSAN, 2008)..

(2) The capacity of survive in adverse conditions makes it very important for food industry and safety (Jensen, Hansen, Eilenberg, & Mahillon, 2003). Is usually associated to rice and flour related foods, due to its ability for hydrolyze starch. Nevertheless, these bacteria can be found in various additives, like sauces and spices (Samapundo, Heyndrickx, Xhaferi, & Devlieghere, 2011). B. cereus could be present in spices and herbs due to environmental contamination with spores or vegetative cells. Some outbreaks are reported worldwide, principally in rice containing foods (Stenfors et al., 2008). In the 20th century cases of diarrhoeal syndrome were present in Norway, Finland, Hungary, Taiwan and the Netherlands. In contrast, the United Kingdom and Japan preset more cases of emetic syndrome. All the cases were reported from different foods, not necessarily rice (Logan, 2011) However, B. cereus has a relative low incidence on developed countries belonging to a minority of reported foodborne outbreaks (EFSA, 2005). For not developed countries, some outbreaks are reported too that are related to rice and other foods. Some countries were contamination come from other sources rather than rice was: Argentina, Brazil, Chile, Costa Rica, Ghana, Niger and India. In India, an outbreak was related to rice but the source of contamination was the spices added to some preparations (UERIA, 2011). In Colombia, some outbreaks are reported but studies on real prevalence of B. cereus on spices are not available. According to SIVIGILA (Sistema Nacional de Vigilancia en Salud Pública), this bacteria is the third cause of foodborne diseases in the country and is involved in several outbreaks in different departments of Colombia. The most prevalent departments that reports outbreaks are: Tolima with 110 cases, Guajira with 42 cases, Caldas with 39 cases and Nariño with 31 cases. All outbreaks reported involved rice or preparations of rice, there are no information about cases that involves spices (UERIA, 2011). Under law of the Colombian Public Health Ministry spices may have a proper level of B. cereus. Legal acceptance on quality of spices admits 100 to 1000 CFU per gram (Ministerio de Salud Pública, 1991). Additionally, in the case of Bogotá, spices are classified as low risk foods. This status is for foods that does not represent significant risks in transmission of foodborne diseases. Those foods must be monitored to avoid contamination that represents serious problems in good manufacturing practices (Secretaria Distrital de Salud de Bogotá, 2010). Given that, studies on B. cereus in Colombia are important to elucidate epidemiology and prevalence of this foodborne pathogen. Some characteristics like potential production of toxins and relation with public health issues are needed to investigate. Isolation and identification of B. cereus are done by selective media followed by biochemical tests (Reekmans et al., 2009). For specific confirmation of foodborne pathogens PCR techniques are developed giving more precise results (Reekmans et al., 2009). Because B. cereus is part of the bacterial group named Bacillus.

(3) cereus or Bacillus cereus sensu lato, it is important to differentiate among other species that has similar biochemical properties (Stenfors Arnesen, Fagerlund, & Granum, 2008). Antibiotics are widely used, not only for treatment of human infections caused by bacteria. There are used in agriculture for avoid phytopathogens and in poultry industry in high doses. In those scenarios there is a probability of gene transfer with related bacteria (Phillips et al., 2004). Antibiotic resistance is done by some methods like efflux pumps, stress responses and production of hydrolytic enzymes that degrades antibiotics codified by certain genes. Resistance could be spread by plasmids, transposons or integrons to close or not related bacteria (Rather et al., 2012). In B. cereus gene transfer can be done between bacteria among its group and other Gram positive bacteria. The close relation with B. anthracis is of important concern given the difficult to differentiate between those species, ubiquity of B. cereus and exchange of virulence factors (Salter, 2011). Abuse of antibiotics for treating some infections select mutants that can resist antibiotics. There is a great concern about the wide dispersion of resistance and the limits in treatment. Those are of public health importance because it increasing frequency (Rather, Aulakh et al., 2012). Resistance in foodborne pathogens is important too, because some authors accept that it is causing an ecological imbalance. Done their ubiquity could spread resistance to bacteria that are not related to clinical strains (Roy et al., 2007). B. cereus is reported to be typically resistant to B-lactams and other antibiotics like ampicillin, cloxacillin, erythromycin, tetracycline, streptomycin (Rather, Aulakh et al., 2012) and rifampin (Vogler et al., 2002). The aim of this study is to isolate and identify B. cereus from different spices available on the Bogotá’s market. And evaluate if this bacteria could be a mechanism for antibiotic resistance by antibiotic susceptibility testing on agar. 2. Materials and methods 2.1 Isolation of Bacillus cereus from spices 79 samples of 16 different spices were obtained from markets in Bogotá. Those spices belonged to 11 different brands in 4 markets in Bogotá. This was a pilot study were sampling was done selecting samples randomly. Huge sampling could not be possible by time reasons. The isolation protocol was modified from the recommended by Food and Drug Administration (FDA). 10g of the sample were taken and poured in 90mL of peptone water 0.1%, the mix was homogenized with a vortex. 0.1mL of this was plated in a petri dish with solid Mossel agar. Petri dishes were incubated at 35°C, 24 hours for selection of presumptive colonies. This selection was done by mannitol negative criteria. Finally, presumptive strains.

(4) were incubated in Standard Plate Count Agar and stored in 4°C for further analysis (Tallent et al., 2001), (Vanegas et al., 2004). 2.2 Biochemical identification Biochemical test were selected from the FDA’s protocol for the B. cereus identification. For every isolated strain were tested: Gram stain, Methyl red-Vogues Proskauer test, starch hydrolysis and Beta hemolysis in Blood agar. For some strains that do not fulfill the biochemical criteria a Crystal for Gram positive bacteria (Becton, Dickinson & Co.) was tested (Valero, Hernandez-Herrero, & Giner, 2007). 2.3 DNA extraction for PCR Isolated strains were cultured in Brain Hearth Infusion broth (BHI) for 24 hours at 35°C. An aliquot of 1mL of the growth broth was pipetted into an eppendorf tube and centrifuged at 13000rpm (Sorvall RT) for 5 minutes. Two washes with 1mL of distilled sterilized water were performed, each with a centrifuge step at 13000x g for 5 minutes. Then the pellet was washed with 100µL of distilled sterilized water and centrifuged at 13000x g for 3 minutes. The eppendorf tubes were taken at 90°C in boiling water for 15 minutes. A final centrifugation step was made at 13000x g for 1 minute and then stored at -20°C (Ngamwongsatit et al., 2008). 2.4 16S PCR Genomic DNA samples extracted by boiling were used for PCR reactions. Amplifications of 16S rRNA gene were performed following the procedure proposed by Dojka et al., 2000. Products were reveled in an agarose 2% (w/v) gel with 0.5µL of GelRed (Promega). Results were visualized with the ChemiDoc XRS UV transilliminator system (Bio-Rad, Laboratories, Inc. USA) (Wehrle et al., 2009). 2.5 Sequence identification PCR products were sequenced in Macrogen Inc, Korea. Sequences were analyze and using Geneious software (Biomatters, Ltda.). Bioinformatic analysis and identification of strains were performed by nBLAST tool available in the NCBI (National Center for Biotechnology Information) website (http://blast.ncbi.nlm.nih.gov/). 2.6 Antibiotic resistance tests Antibiotic resistance tests were performed following recommendations and the protocol proposed by the British Society for Antimicrobial Therapy (BSAC) with the following antibiotics (Oxoid): Penicillin G 10µg, Ampicillin 10 µg, Imipenem 10 µg, Tetracycline 30 µg, Cefoxitin 5 µg, Cefalotine 5 µg, Chloramphenicol 10 µg,.

(5) Cefepime 5 µg, Kanamycin 30 µg, Ciprofloxacin 5 µg and Trimethoprimsulfamethoxazole 1.25 µg (Andrews, 2009). 3. Results 3.1 Isolation and biochemical identification of B. cereus Of 79 samples processed in this study, 75 (94.94%) presented growing of bacteria in Mossel agar. Of samples with bacterial growth, 16 (20.25%) contain presumptive colonies. Finally 30 strains suspicious for B. cereus were isolated from these positive samples. Samples do not have equal distribution of strains isolated. All strains were pre-selected by mannitol negative criteria and presumptiveness of colony morphology Table 1. There were more strains isolated from color (23.33%), oregano (16.67%) and cumin (16.67%) Table 2. Sample. 3 4. Spice. Biochemical Strain identification (1) (%). Oregano BC 3-1 Color. BC 4-1 BC 4-2 BC 4-3 BC 43-2* BC 4-4. 6. 7 10. 11. 16 17. Cumin. BC 6-1. BC 6-2 BC 6-3 Color BC 7-1 BC 7-2 BC 10Cumin 1 BC 102 Other BC 11spices 2 BC 113 BC 114 BC 16Pepper 1 BC 17Oregano 2. 100 100 100 100. Crystal Percentage Result (%) B. cereus 100. 100 66,67. B. cereus. 99,97. 100 100 100. 66,67 100 100. B. cereus. 0.0. 99. B. cereus B. cereus B. cereus. 0.0 0.0 0.0. 99 100 99. B. cereus. 0.0. 99. B. cereus. 0.0. 99. B. cereus B. cereus B. cereus B. cereus. 5,00E106 0.0 0.0 0.0 0.0. 99 100 99 100. B. thuringiensis. 0.0. 99. B. cereus. 0.0. 99. B. cereus. 0.0. 99. B. cereus. 0.0. 97. B. cereus. 0.0. 99. B. cereus. 0.0. 99. B. cereus. 0.0. 99. B. cereus. 100 100 100 100 100. 66,67. Sequence Analysis EIdentity Result Value (%). B. cereus B. cereus. 100 99,99. 100.

(6) 18. Garlic. BC 185. B. weihenstephanensis. 100. 0.0. 99. BC 23G. 6,00E80 1 100 stearothermophilus 135 BC 2525 Garlic B. cereus 0.0 99 2 100 BC 3333 Basil L. fusiformis 0.0 99 2 100 BC 3737 Curry B. thuringiensis 0.0 100 2 100 BC 4343 Oregano B. cereus 0.0 99 1 100 BC 435,00EB. cereus 98 2 100 168 BC 439,00EB. cereus 80 3 100 10 BC 47B. 47 Pepper B. cereus 0.0 99 1 66,67 cereus 99,99 BC 47B. B. cereus 0.0 99 2 66,67 cereus 100 BC 47B. cereus 0.0 100 3 100 Table 1, Number of strains by sample, its denomination and identification. (1). the first number corresponds to the number of sample and the second is related to the number of strain isolated of this sample. *The third number is done by founding two types of colonies on BC 4-3 strain, this was the second. 23. Other spices. Spice Percentage. Oregano 16.67. Color 23.33. Cumin 16.67. Pepper 13.33. Garlic 10.00. Basil 3.33. Curry 3.33. Other Spices 13.33. Table 2, Percentage of strains by spices. Of 30 strains isolated, 5 was from oregano, 7 from color, 5 from cumin, 4 from pepper, 3 from garlic, 1 from basil, 1 from curry and 4 from various spices.. 3.1.1 Confirmation by Crystal tests 5 Strains were negative for mannitol and have a suspicious colony but some biochemical tests were not congruent. 4 strains show negative or slow results for Vogues-Proskauer reaction. Another strain show negative result for starch hydrolysis. Those results were not correspondent with B. cereus ATCC 11778 positive control. Crystal panels for Gram positive bacteria (Becton, Dickinson & Co.) were used for confirmation including B. cereus as positive control. Results for this test show a great probability of B. cereus identification for all strains tested. Only 3 of them show other percentages different to 100%, but close to that Table 1..

(7) 3.2 PCR 16S identification 3.2.1 PCR 29 strains were congruent with the control strain (B. cereus ATCC 11778) showing an approximate weight of 1400 to 1500pb. 1 strain showed a different weight over 1500pb. 3.2.2 Bioinformatic sequence analysis After sequence analysis in Geneious software a nucleotide mega BLAST algorithm was performed. Of 30 strains, 28 (93.33%) strains belonged to Bacillus spp. and 2 strains (6.66%) were identified as another genus. Strain BC 37-2 and strain BC 232 were identified as Lysinibacillus fusiformis and Geobacillus stearothermophilus respectively. Of 28 Bacillus spp strains 25 (83.33%) were identified as B. cereus and 3 (10%) strains were identified as other Bacillus species: BC 23-1 strain as B. weihenstephanensis and BC 43-1 strain, BC 10-1 strain as B. thuringiensis Table 1. BC 47-2 strain does not show any results with nucleotide mega BLAST algorithm. For this strain a less restrictive algorithm was used. This algorithm show results for somewhat similar sequences. 3.3 Antibiotic resistance In accordance with Mietke et al., 2010 resistance was considered if the inhibition zone was greater or equal than 15mm. For the antibiotics tested it was founded that every strain has resistance for more than 4 antibiotics. The majority of strains resist 6 antibiotics followed by 5 and 4. As we expected, all strains were resistant for penicillin, and some strains were resistant for other B-lactam antibiotics. Susceptibility for kanamycin, aminoglycoside, carbapenem and quinolone was showed by the majority of the strains isolated. In contrast, some antibiotics showed different results among the strains like cefoxitin, trimethoprim-sulfamethoxazole and tetracycline Table 3. Those results show that some strains of B. cereus isolated are developing resistance to other antibiotics that are not common in these bacteria. Strain. IMP. FOX. FEP. CF. SXT. K. P. TE. AMP. CIP. C. BC 03-1. S. S. R. R. R. S. R. S. R. S. S. BC 04-1. S. R. R. R. R. S. R. S. R. S. S. BC 04-2. S. S. R. R. R. S. R. S. R. S. S. BC 04-3. S. R. R. R. R. S. R. S. R. S. S. BC 04-3-2. S. R. R. R. R. S. R. S. R. S. S. BC 04-4. S. R. R. R. R. S. R. S. R. S. S.

(8) BC 06-1. S. R. R. R. R. S. R. S. R. S. S. BC 06-2. S. S. R. R. S. S. R. R. R. S. S. BC 06-3. S. S. R. R. R. S. R. R. R. S. S. BC 07-1. S. R. R. R. S. S. R. S. R. S. S. BC 07-2. S. R. R. R. R. S. R. S. R. S. S. BC 10-1. S. R. R. R. R. S. R. R. R. S. S. BC 10-2. S. R. R. R. S. S. R. R. R. S. S. BC 11-2. S. R. R. R. R. S. R. S. R. S. S. BC 11-3. S. S. R. R. S. S. R. R. R. S. S. BC 11-4. S. R. R. R. S. S. R. R. R. S. S. BC 16-1. S. S. R. R. R. S. R. S. R. S. S. BC 17-2. S. R. R. R. R. S. R. S. R. S. S. BC 18-5. S. R. R. R. R. S. R. S. R. S. S. BC 23-1. S. R. R. R. S. S. R. S. R. S. S. BC 25-2. S. S. R. R. R. S. R. S. R. S. S. BC 33-2. S. S. R. S. R. S. R. S. R. S. S. BC 37-2. S. R. R. R. R. S. R. S. R. S. S. BC 43-1. S. R. R. R. R. S. R. S. R. S. S. BC 43-3. S. S. R. R. R. S. R. S. R. S. S. BC 47-1. S. S. R. R. S. S. R. S. R. S. S. BC 47-2. S. R. R. R. R. S. R. S. R. S. S. BC 47-3. S. S. R. R. S. S. R. S. R. S. S. BC 72-1. S. R. R. R. S. S. R. S. R. S. S. BC 72-3. S. R. R. R. R. S. R. S. R. S. S. CONTROL. S. R. R. R. R. S. R. S. R. S. S. Table 3, Resistance in strains isolated. Imipenem (IMP), Cefoxitin (FOX), Cefepime (FEP), Cefalotine (CF), Trimethoprim\sulfamethoxazole (SXT), Kanamycin (K), Penicillin (P), Tetracycline (TE), Ampicillin (AMP), Ciprofloxacin (CIP), Chloramphenicol (C). Susceptible (S), Resistant (R).. 4. Discussion In this study the majority of samples (94.94%) presented any type of bacterial growth. Early identification of strains was made on Mossel agar. This agar is not only designed for B. cereus growth since other Bacillus spp can grow and other gram positive bacteria (Tallent et al., 2001). Due that, biochemical identification was important in order to select presumptive colonies from the media. From 75 samples, 16 (53.33%) samples were positive for B. cereus using biochemical criteria. From all the samples 30 strains were isolated. In accordance with literature here cited the majority of strains isolated in this study were related to positive.

(9) control in terms of biochemical results. There was an accurate selection of strains since all strains belong to Bacillus spp, and by confirmation some were B. cereus. Only 3 strains (10%) showed slow results for Vogues-Proskauer test and 2 (6.66%) negative results. 1 (3.33%) strain show negative result for starch hydrolysis. Even with a good biochemical selection this could be a problem because interpretation for some samples could be missed, increasing wrong reports. Specific and reliable techniques are needed for isolation and formal identification of this pathogen. In recent years some molecular methodologies are developed (Wehrle et al. 2009). The principal characteristic of these methods is precision in results making more accurate for identification of microorganisms. Nevertheless, PCR and related are not too important for food industry and in clinics like biochemical traditional tests (Reekmans, Stevens, Vervust, & De Vos, 2009). Under PCR reaction we confirmed 28 (93.33%) strains belonging to Bacillus spp and from them 25 (83.33%) were confirmed for B. cereus. Other results among Bacillus spp was expected since B. cereus is part of a group named as Bacillus cereus sensu lato. In this group there are other bacteria that were identified here (Samapundo et al., 2011). B. thuringiensis is a bacterium that could be isolated from soil and from the gut of some arthropods. This bacterium is used for biological control for arthropods (Jensen et al., 2003).Those strains were isolated from oregano and cumin. Presence in plant spices could be possible from a soil contamination with this bacterium. The bacteria could be present in the leaves of the plant and pass through all the manufacturing process. One strain was identified as B. weihenstephanensis that is also a bacterium belonged to B. cereus group. This bacterium can grow at low temperatures and, like B. cereus, is recognized its ability to produce diarrheic and emetic toxins (Thorsen et al., 2009). B. cereus group shares also ecological properties. This could explain the presence of B. weihenstephanensis in spices, possibly for association with soil or plants (Stenfors et al., 2008). Similarity between those bacteria with B. cereus makes identification more imprecise. PCR targeting the 16S rRNA gene has the problem that all bacteria share the same gene. Similarity between Bacillus spp strains is done also at this level. The final identification of B. cereus could be done with more specific molecular methodologies like Random Fragment Length Polymorphism (RFLP) and Random Amplification of Polymorphic DNA (RAPD) (Chaves et al., 2011). Diseases that are caused by food borne pathogens are of great concern in not developed countries. Intoxications by B. cereus are not reported since is not part of the most searched foodborne pathogenic bacteria (Anonymous, 2007). Especially when foods are not cooked or are ready to eat, the bacteria could be ingested (Rather et al., 2012).In addition, B. cereus can survive many adverse conditions which make it prevalent in foods and food industries (Jensen et al., 2003). Here, we are concluding that spices are important sources of B. cereus. Strains were.

(10) isolated from different spices. The most prevalent isolations were from oregano, color and cumin. From oregano only B. cereus strains were isolated. This could be realized since this spice does not show another type of colony growing. Moreover, high contamination were not founded in this spice like others were uncountable colonies were reported. In uncountable samples, serial dilutions were needed until isolation of strains was possible. Those results indicate an important source of Bacillus spp. present in spices. As we cited before, no reports or investigations on the prevalence of B. cereus was done in Colombia (UERIA, 2011). Carefully analysis of spices in outbreaks will give information about importance of them in public health. In those food additives sometimes flour is added. This addition decreases humidity levels and increases starch content. These factors are important for B. cereus growth or viability in foods (Lebert et al., 2005). Spices are widely used for improve flavor in different foods. Then, the consumer could ingest the bacterium with a great probability to develop a disease. Other samples containing starch must be proved in order to search B. cereus and make a more accurate report of incidence of this foodborne pathogen in foods. Beta lactam resistance for strains isolated was coincident with other reports. All strains showed resistance to penicillin, ampicillin and cefepime. Resistance to other B-lactam antibiotics like cefalotine (96.99%) and cefoxitin (63.66%) were done by a great percentage of strains isolated. Finally other groups of antibiotics like sulfonamide and tetracycline were resisted: trimethoprim\sulfamethoxazole (70%) and tetracycline (20%). Not all strains were equal in resistance, it is important to determine to which antibiotics B. cereus strains are resistant. And not all the cephalosporins were resisted for all strains. Cases of intoxications are not treated with antibiotics and hospitalization are not required (Mandell, 2010). These factors could be part of the explanation why B. cereus clinical characteristics are not recognized or studied in Colombia (UERIA, 2011). B-Lactam resistance in B. cereus is showed in a wide manner. It is reported resistance to penicillin and derived antibiotics, resistance to cephalosporins and others are not studied (Rather et al., 2012). Resistance to B-lactam antibiotics and susceptibility to kanamycin, aminoglycoside, carbapenem and quinolone was expected. Resistance to penicillin, ampicillin and some other cephalosporins was showed by all strains. Furthermore, resistance of some strains for tetracycline was not expected and could be an important issue for searching other types of resistance genes. The Blactam group is composed by some different antibiotics like penicillins, modified penicillins, cephalosporins of first to fourth generation, monobactams and carbapenems. It is important to decide if all of these antibiotics are resisted by B. cereus strains isolated from food. Resistance could be a resent event for some antibiotics in this genus and antibiotic resistance surveillance must be done. In addition, Bacillus spp. may share an antibiotic resistance profile especially for B-.

(11) lactam antibiotics. The response for searching resistance in B. cereus could be done because there is a probability for gene transfer between this bacterium and other gram positive pathogens (Salter, 2011). Due the genetic similarity into the B. cereus group, gene transfer is very common. Virulence factors of the different bacteria that compose the group may be shared. Genetic transfer could be done in food matrices or more generally in the soil or the environment (Modrie et al., 2010). Some genes that regulate expression of hydrolytic enzymes leading to resistance could be present in B. cereus. And from this bacteria could be dispersed to other bacteria that do not have them. In the case of B. anthracis treatment it could be problematic. This pathogen, causing agent of anthrax, sometimes are treated with penicillin and penicillin derivate antibiotics (Mandell, 2010). But this feature is not well understood in this bacteria and its implication in public health is yet ignored (Rather et al., 2012). Studies on B. cereus antibiotic resistance must be done in order to evaluate it as a resistance genes carrier. Since plasmid or gene transfer strategies are similar in Gram positive bacteria, resistance genes could be wide spread. Not only among B. cereus group, other bacteria like Listeria monocytogenes or Staphylococcus aureus could be part of genetic material transfer for or from B. cereus (Modrie et al., 2010), (Rather, Aulakh et al., 2012). In conclusion, B. cereus is a foodborne pathogen that is present in spices where it can grow or survive. Prevalence on these food additives is important to evaluate prevalence of B. cereus in foods. For the identification are important select presumptive bacteria first by biochemical criteria and then confirmation with PCR techniques. Molecular methodologies like RFLP’s and RAPD’s are necessary if a strict differentiation among B. cereus group is needed. Antibiotic resistance must be evaluated for identify possible resistance gene transfer among Gram positive bacteria. B. cereus could be an important carrier of those resistance genes and surveillance must be improved. 6. Bibliography: Andrews, J.M.(2009). BSAC standardized disc susceptibility testing method (version 8). Journal of Antimicrobial Chemotherapy, 64, 454-489. Anonymous (2007). Food safety and foodborne illness. Media center, Fact Sheets. World Health Organization. Disponible en línea en: http://www.who.int/mediacentre/factsheets/fs237/en/ Carlin, F., Brillard, J., Broussolle, V., Clavel, T., Duport, C., Jobin, M., . . . Nguyen-Thé, C. (2010). Adaptation of Bacillus cereus, an ubiquitous worldwide-distributed foodborne pathogen, to a changing environment. Food Research International, 43(7), 1885-1894. doi: 10.1016/j.foodres.2009.10.024 Chaves, J. Q., Pires, E. S., & Vivoni, A. M. (2011). Genetic diversity, antimicrobial resistance and toxigenic profiles of Bacillus cereus isolated from food in Brazil over three decades. [Research Support, Non-U.S. Gov't]. Int J Food Microbiol, 147(1), 12-16. doi: 10.1016/j.ijfoodmicro.2011.02.029.

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