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Bacillus Cereus in Eggshell: Enterotoxigenic Profiles and Biofilm Production

ABSTRACT

A study was carried out with the objective of determining the presence of Bacillus cereus in eggshells commercialized in Mexico, the enterotoxigenic profile of the isolated strains, and the production of biofilms in different materials as well as in the eggshell. 1000 chicken eggs from four commercial brands were collected from markets and supermarkets located in the city of Chilpancingo, Mexico. Bacillus cereus was isolated from the eggshell. The molecular identification was by amplification of the gyrB gene and the enterotoxigenic profiles by the amplification of the cytK, ces, nheABC, and hblABD genes, in addition to the amplification of the tasA and sipW genes associated with the production of biofilms. In different materials and in eggshells, the production of biofilms was evaluated. The microbiological and molecular analysis of B. cereus yielded a frequency of 5.5% (55/1000), this was higher in brand III (11.6%, p=0.0001) and white eggshell (7.6%, 38/500, p≤0.001) and by marketing source, it was similar between market (5.2% / 26/500) and supermarket (5.8%, 29/500). The most common was the toxigenic profile A (23/55). Biofilm production is high in PVC in relation to other materials (p<0.0001), and the frequency of the related genes tasA and sipW was 72.7% and 40% respectively; the highest production was related to the tasA gene; in eggshell, most of the strains (54/55) were able to produce biofilm. Strains of B. cereus with toxigenic potential circulate and persist in this product, which shows the need for sanitary regulation in the country.

Keywords:
Bacillus cereus; eggshell; biofilm

INTRODUCTION

Eggs are used as an economical food source either as table eggs or eggshells, liquid, frozen or dehydrated products (Salfinger & Tortorello, 2015Salfinger Y, Tortorello ML. Compendium of methods for the microbiological examination of foods. Washington: American Public Health Association; 2015. Available from: https://doi.org/10.2105/MBEF.0222). Eggs and their products provide an important source of nutrition (Howard et al., 2012Howard ZR, O'Bryan CA, Crandall PG, Ricke SC. Salmonella Enteritidis in shell eggs: current issues and prospects for control. Food Research International 2012;45(2):755-764.) as well as being a functional part of other foods such as ice-cream, desserts, meats, breakfasts, and seafood (Kone et al., 2013Kone AZ, Jan S, Le Marechal C, Grosset N, Gautier M, Puterflam J, Baron F. Identifying risk factors for eggshell contamination by Bacillus cereus group bacteria in French laying farms. British Poultry Science 2013;54(3):298-305.). Mexico is the main consumer of fresh eggs worldwide; by the end of 2019, it was estimated that the per capita consumption of Mexican is 22.8 Kg of eggs per day (Sader, 2019). Regarding production during 2019, the laying flock of Mexican poultry farms obtained 2,949,782,315 tons of eggs for fresh consumption (SIAP, 2019). Considering the egg as an important part of the diet in Mexico.

Due to the wide use of eggs in the food chain, the sanitary quality of the product is imperative. In this sense, it has been described that contamination by eggshell bacteria can occur in a variety of routes, such as in egg formation in the hen’s reproductive system (Howard et al., 2012Howard ZR, O'Bryan CA, Crandall PG, Ricke SC. Salmonella Enteritidis in shell eggs: current issues and prospects for control. Food Research International 2012;45(2):755-764.) or due to poor hygiene and handling conditions; eggs can also become contaminated after breeding when exposed to contaminated conditions due to the inevitable accumulation of important microorganism in animal waste (Gentry & Quarles, 1972Gentry RF, Quarles CL. The measurement of bacterial contamination on egg shells. Poultry Science 1972;51(3):930-933.; Quarles et al., 1970); this contamination could be a risk for food safety, increasing the risk of disease in humans (Bencardino et al., 2017Bencardino D, Vitali LA, Petrelli D. High prevalence of clonally diverse spa type t026 Staphylococcus aureus contaminating rural eggshells. Journal of Medical Microbiology 2017;66(8):1196-1201.). The shell microflora is remarkably heterogeneous, with Gram-positive microorganisms being the most prevalent, probably due to their ability to tolerate stressful conditions (Siriporn, 2015). Regarding the last point, Bacillus cereus is a Gram-positive microorganism that can be found in multiple stressful environments due to the formation of endospores that resist heat, dehydration, and other physical agents (Drobniewski, 1993Drobniewski FA. Bacillus cereus and related species. Clinical Microbiology Reviews 1993;6(4):324-338.). Therefore, its presence in the eggshell has been previously reported (Koneet al., 2013Kone AZ, Jan S, Le Marechal C, Grosset N, Gautier M, Puterflam J, Baron F. Identifying risk factors for eggshell contamination by Bacillus cereus group bacteria in French laying farms. British Poultry Science 2013;54(3):298-305.; Siriporn, 2015). B. cereus being considered as an important microorganism transmitted by food; in this sense, various studies refer to the presence of B. cereus spores in raw milk and milk powders (Benahmed et al., 2020Benahmed M, Leguerinel I, Moussa-Boudjemaa B. Biodiversity, spoilage capacity and heat resistance of mesophilic aerobic spores isolated from milk powders marketed in Algeria. International Journal of Dairy Technology 2020;73: 771-780.; Pretorius & Buys, 2021Pretorius C, Buys EM. Extended shelf life milk processing: Effect of simulated cleaning in place on the germination and attachment of Bacillus cereus spores. International Journal of Dairy Technology 2021;74: 75-83.). Furthermore, the production of biofilms on surfaces of dairy production has been described (Alonso & Kabuki, 2019Alonso VPP, Kabuki DY. Formation and dispersal of biofilms in dairy substrates. International Journal of Dairy Technology 2019;72:472-478.). Its presence in dairy products as well as in other foods is important because it is related to its ability to produce a wide range of virulence factors that can cause disease in humans of short and moderate duration (Stenfors et al., 2008); these poisonings could not only be caused by the consumption of the egg, but by its usefulness in the production of other foods; therefore, the presence of microorganisms in chickens and poultry products, including eggs, potentially increases the entry of these microorganisms into the food chain, generating food poisoning (EFSA, 2011).

It is important not only to point out the presence of B. cereus in food, but also to characterize the main virulence factors associated with the two types of food poisoning that it causes. B. cereus produces the emetic toxin (cesoperon), which causes emetic syndrome and has been characterized as a small ring-shaped peptide (Ehling-Schulz et al., 2004Ehling-Schulz M, Fricker M, Scherer S. Bacillus cereus, the causative agent of an emetic type of food-borne illness. Molecular Nutrition & Food Research 2004;48(7):479-487.). In addition, it produces three enterotoxins that have been implicated in diarrheal syndrome: pore-forming hemolysin BL (Hbl), non-hemolytic enterotoxin (Nhe), and cytotoxin K (CytK) (Beecher & Macmillan, 1991Beecher DJ, Macmillan JD. Characterization of the components of hemolysin BL from Bacillus cereus. Infection and Immunity 1991;59(5):1778-1784.; Lund et al., 2000Lund T, De Buyser ML, Granum PE. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molecular Microbiology 2000;38(2):254-261.; Lund & Granum, 1996). Finally, B. cereus has been described as capable of producing biofilms in a variety of environments associated with food production (Evans et al., 2004Evans JA, Russell SL, James C, Corry JEL. Microbial contamination of food refrigeration equipment. Journal of Food Engineering 2004;62(3):225-232.; Gunduz & Tuncel, 2006Gunduz GT, Tuncel G. Biofilm formation in an ice cream plant. Antonie van Leeuwenhoek 2006;89(3-4):329-336.; Storgårds et al., 2006Storgårds E, Tapani K, Hartwall P, Saleva R, Suihko ML. microbial attachment and biofilm formation in brewery bottling plants. Journal of the American Society of Brewing Chemists 2006;64(1):8-15.; Marchand et al., 2012Marchand S, Block JD, Jonghe VD, Coorevits A, Heyndrickx M, Herman L. Biofilm formation in milk production and processing environments; influence on milk quality and safety. Comprehensive Reviews in Food Science and Food Safety 2012;11(2):133-147.; Alonso & Kabuki, 2019Alonso VPP, Kabuki DY. Formation and dispersal of biofilms in dairy substrates. International Journal of Dairy Technology 2019;72:472-478.) and that biofilm production could be a systematic contamination mechanism (Rajkovic et al., 2008Rajkovic A, Uyttendaele M, Dierick K, Samapundo S, Botteldoorn N, Mahillon J, et al. Risk profile of the Bacillus cereus group implicated in food poisoning. Brussels: Superior Health Council Belgium; 2008. p.80. Available from: https://www.health.belgium.be/sites/default/files/uploads/fields/fpshealth_theme_file/19060475/Risico-profiel%20voor%20Bacillus%20cereus%20Groep%20in%20voedsel%20toxi-infecties%3A%20situatie%20in%20België%20en%20aanbevelingen%20%5BBijlage%5D%20%28januari%202010%29%20%28HGR%208316%29.pdf.); However, it has been reported that it can produce biofilms directly in food and that it could also be a mechanism of persistence in food (Elhariry, 2011Elhariry HM. Attachment strength and biofilm forming ability of Bacillus cereus on green-leafy vegetables: Cabbage and lettuce. Food Microbiology 2011;28(7):1266-1274.). Currently, Mexico has no regulation which indicates the presence of this microorganism in these types of foods, but it has a history of the presence of the B. cereus in its products and its importance in the production chain. Therefore, the aim of this study was to expose the presence of B. cereus, the enterotoxigenic profile of the isolated strains, and the production of biofilms in different materials including the eggshell in eggs commercialized in Mexico.

MATERIAL AND METHODS

Collection of samples

A total of 1000 chicken eggs representing four commercial brands (250 eggs per brand, 125 brown eggs, and 125 white eggs) were collected from January to September 2019 from markets and supermarkets located in Chilpancingo City, Mexico. Those four brands represent some of the most popular brands in Mexico, two brands are commonly sold in supermarkets and two in markets. In supermarkets and markets, the eggs were stored at room temperature in unit tray packs (30 eggs per pack). A tray pack of each brand was collected each week from each store and transported to the laboratory (Laboratorio de Investigación en Patometabolismo Microbiano, Chilpancingo, México) at room temperature in a container. The experiments were carried out on the samples arrived at the laboratory; 10 eggs from each tray pack were randomly selected until the number of samples of each brand was completed.

Isolation and identification of Bacillus cereus in eggshell

The eggs were placed into a sterile plastic bag and 1 mL of brain heart infusion broth (BHI) was added, they were immediately rubbed manually for half an hour in order to recover all the microorganisms present in the eggshell. Once the time had elapsed, we spread 100 µL of the BHI broth on Mannitol Egg Yolk (MYP) Agar (Bioxon, México), and incubated under aerobic conditions at 30ºC for 24 h. We considered as suspicious colonies of B. cereus, the pink colonies with an opaque halo and confirmed by beta hemolysis in trypticase soy agar supplemented with sheep blood.

Molecular identification and enterotoxigenic profiles

From bacterial cultures, a thermal shock was performed to obtain the chromosomal DNA. In brief, cells from one colony were suspended in sterile water, heated to 95 ° C for 3 minutes, and placed on ice for 15 minutes. After centrifugation, the supernatant was used as a template for the molecular identification, the enterotoxigenic profile and genes that possibly participated in the production of the biofilm.

The differentiation of B. cereus group was targeted on gyrB gene (Wei et al., 2018Wei S, Chelliah R, Park BJ, Park JH, Forghani F, Park YS, et al. Molecular discrimination of Bacillus cereus group species in foods (lettuce, spinach, and kimbap) using quantitative real-time PCR targeting groEL and gyrB. Microbial Pathogenesis 2018;115:312-320.) and the toxin gene profiles from cytK, ces, nheABC y hblABD gene; tasA and sipW genes were also included due to previous reports of their participation in biofilm production (Caro-Astorga et al., 2015Caro-Astorga J, Pérez-García A, de Vicente A, Romero D. A genomic region involved in the formation of adhesin fibers in Bacillus cereus biofilms. Frontiers in Microbiology 2015;5.). The reaction mixes (50 µL) contained the following: 25 µL of REDTaqReadyMix DNA polymerase (Sigma-Aldrich), 11µL of sterile Milli-Q water, 0.5 µL of the genomic DNA template (concentration about 10-20 ng/µL), and 0.02 µM of each primer. The PCR cycling conditions and primers are shown in Table 1. A strain of B. subtilis was used as a negative control, this strain was previously characterized in the laboratory. B. cereus ATCC 14579 (diarrheagenic) and VK4 strain (emetic) were used as control strains.

Table 1
Polymerase chain reaction cycling conditions and primer sequences.

Electrophoresis was performed on 2% agarose gels at 80V for 120 minutes. The gels were stained with Midori Green (Nippon Genetics, Düren, Germany) and visualized with LED light. A 100 bp molecular weight marker was used in all electrophoresis (CSL-MDNA, Cleaver Scientific Ltd, Warwickshire, England, UK).

Production of biofilm in different materials

Prior to the determination of static biofilm in polyvinyl chloride (PVC), the PVC coupons were placed inside glass tubes. The coupons were pretreated to remove dust and other organic components. The biofilms were generated in brain heart infusion broth (BHI). Each glass tube containingthe PVC coupon was filled with 1 mL of BHI. The broths were inoculated with 5% volume of a 24 h culture (1 mL). The tubes were incubated at 30ºC in aerobiosis, for 48 h under static conditions. For the determination in glass and polyethylene, the procedure was similar without the PVC coupons and using tubes of each respective material. In the case of polystyrene, 96-well microplates were used, which were filled with 200 µL of the BHI. The incubation time was the same in all cases.

Next, the biofilm formation was measured by performing safranin assay. After incubation, the growth was analyzed by removing 200 µL of culture and reading it at an absorbance of 600 nm. Then, in the case of PVC, the coupons were carefully washed three times by dipping them in phosphate buffer saline (PBS) (Life Technologies, Carlsbad, CA, USA) using sterile tweezers. Because the PVC coupons were in glass tubes, they were considered as another material (PVC glass). In the case of the glass and polyethylene tubes, as well as the polystyrene plates, the medium was removed and they were washed three times with PBS. The adhered biofilm was stained with a 0.1% safranin solution (BD Difco, Franklin Lakes, NJ, USA) for 30 minutes. The coupons, as well as the glass and polyethylene tubes and the polystyrene plates, were washed again three times with PBS and were incubated with 70% ethanol for 30 minutes to release the biofilm- bound to safranin. The solubilized safranin was quantified by absorbance at a wavelength of 492 nm. The safranin assays were repeated in three independent experiments. The culture medium without inoculum was used as a negative control. To determine the specific biofilm formation (SBF) the formula proposed by Niu & Gilbert (2016) was used.

Production of biofilm in eggshells

White eggshells were disinfected and carefully cut into 1cm2 coupons and immersed in tubes with 1mL of sterile BHI broth, determining the biofilm production in the same way as the PVC material. In this case, it was not discolored with ethanol and only the differences in staining between the negative control (eggshell not inoculated, but stained with safranin) and the samples were visually checked.

Statistical analysis

Relative proportions were performed with STATA v program. 12 using the chi- square test or Fisher’s exact test. The effects of the material on biofilm formation by B. cereus strains were compared using Kruskal Wallis test with Dunn’s post hoc test. Statistical significance was considered when the p value was less than <0.05.

RESULTS

Determined by microbiological analysis and molecular identification, the frequency of B. cereus was 5.5% (55/1000). The frequency was compared with the commercial brand of the egg, the color of the eggshell, and the source of commercialization. There is a higher frequency of B. cereus in brand III of 11.6% (p=0.0001) (Table 2).

Table 2
Frequency of B. cereus in eggshells by brand.

Also, when comparing by the color of the eggshell, with 500 eggs processed for each color, we found a higher frequency of B. cereus in white eggshell with 7.6% (p=0.003) (Table 3).

Table 3
B. cereus frequency in eggshell by eggshell color.

At the same time, the B. cereus frequency was compared by the origin of commercialization; finding similar frequencies between market (5.2% / 26/500) and supermarket (5.8%, 29/500).

Of the 55 strains, 8 enterotoxigenic profiles were determined (Figure 1, table 4), the most common being the toxigenic profile A (23/55) which includes genes of the three enterotoxins, only two strains were classified in the H profile (2 / 55,) which refers to strains without any gene encoding toxins.

Figure 1
Molecular identification of toxins genes of B. cereus. In a) amplification of hemolitic BL gene (hbl, 1091bp), 1: negative control (B. subtilis), 2: positive control (B. cereus ATCC14579), 4,5,7: positive strain, 3,6: negative strains, 8: molecular weight market of 100 bp. In b) amplification of no-hemolitic enterotoxin gene (nhe,766bp) 1: Control negative (B. subtilis), 2: positive control (B. cereus ATCC14579), 3: molecular weight market of 100 bp, 4,5, 7: Positive strains, 6: negative strain. In c) amplification of cytotoxin K gene (cytk-PlcR, 320bp) 1: molecular weight market of 100 bp, 2: positive control (B. cereus ATCC14579), 3: negative control (B. subtilis), 4 to 11 positive strains.

Table 4
Toxigenic profiles of B. cereus strains isolated from eggshells.

Biofilm production by B. cereus was determined in different materials, observing a higher production in PVC in relation to other materials such as glass (p<0.0001), polystyrene (p<0.0001), and polyethylene (p<0.0001) (Figure 2)

Figure 2
Biofilm production of B cereus strains from eggshell isolation in different materials. light blue circles, glass; red circles, PVC; purple circles, PVC/glass; pink circles polystyrene; green circles, polyethylene * p≤0.05, **** p≤0.0001. In the X axis are the materials and, in the Y, the specific biofilm formation (SBF). Kruskal Wallis post hoc Dunn’s.

A high frequency of genes related to biofilm production was observed, 72.7% for tasA and 40% for sipW (Figure 3). The production of biofilms in different materials was related to the presence of these two genes; tasA positive strains were found to produce a higher number of biofilms compared to negative strains in PVC (p=0.017), polystyrene (p=0.03), and polyethylene (p=0.002).

Figure 3
Biofilm production of B. cereus according to the presence of the tasA and sipW genes. In a) Agarose gel electrophoresis of the PCR products of tasA, 7: molecular weight market of 100 bp, 6: positive control (B. cereus ATCC14579), 5: negative control, 4: negative strain, 3 to 1: positive strains. In b) Agarose gel electrophoresis of the PCR products of sipW, 2: molecular weight market of 100 bp, 5: positive control, 1: negative control, 3,4: negative strains, 6,7: positive strains. In c) biofilm production of B. cereus strains according to tasA + and tasA - gene in different materials. Black circles are positive strains to tasA and gray circles are negative strains tasA. In d) biofilm production of B. cereus strains according to sipW + and sipW - gene in different materials. Black circles are positive strains to sipW and gray circles are negative strains sipW. *p≤0.05, **** p≤0.0001. In the X axis are the materials and, in the Y, the specific biofilm formation (SBF). Sidak´s multiple comparisons test.

It was observed that most of the 54/55 strains are capable of producing biofilms on the surface of the eggshell (Figure 4).

Figure 4
Production biofilm of B. cereus strains on eggshell. 1: negative control, 2 to 5: B. cereus strains from eggshell isolation. The biofilm producing strains were observed when safranin's red color was kept on the egg's surface. The experiments were made to 30°C on Brain heart infusion broth.

DISCUSSION

Eggs are important in human nutrition, due to their low cost, nutritional content and because of their incorporation in a large number of products (Howard et al., 2012Howard ZR, O'Bryan CA, Crandall PG, Ricke SC. Salmonella Enteritidis in shell eggs: current issues and prospects for control. Food Research International 2012;45(2):755-764.; Kone et al., 2013Kone AZ, Jan S, Le Marechal C, Grosset N, Gautier M, Puterflam J, Baron F. Identifying risk factors for eggshell contamination by Bacillus cereus group bacteria in French laying farms. British Poultry Science 2013;54(3):298-305.; Salfinger & Tortorello, 2015Salfinger Y, Tortorello ML. Compendium of methods for the microbiological examination of foods. Washington: American Public Health Association; 2015. Available from: https://doi.org/10.2105/MBEF.0222). Therefore, the egg in all its presentations must be monitored regarding the sanitary quality of the product because natural contamination by pathogens such as Salmonella has been reported (Howard et al., 2012), contamination in laying by microorganisms of the gastrointestinal and environmental tract, as well as contamination associated with poor hygienic practices has been reported (Quarles et al.,1970Quarles CL, Gentry RF, Bressler GO. Bacterial contamination in poultry houses and its relationship to egg hatchability. Poultry Science 1970;49(1):60-66., Gentry & Quarles, 1972Gentry RF, Quarles CL. The measurement of bacterial contamination on egg shells. Poultry Science 1972;51(3):930-933.).

B. cereus is a widely distributed microorganism in nature, whose presence has been detected in poultry feed (Mahami et al., 2019Mahami T, Togby-Tetteh W, Kottoh DI, Amoakoah-Twum L, Gasu E, Annan SNY, et al. Microbial food safety risk to humans associated with poultry feed: the role of irradiation. International Journal of Food Science 2019;6915736.), poultry slaughter facilities (Lues et al., 2007Lues JFR, Theron MM, Venter P, Rasephei MHR. Microbial composition in bioaerosols of a high-throughput chicken-slaughtering facility. Poultry Science 2007;86(1):142-149.; Liang et al., 2013Liang R, Tian J, She R, Meng H, Xiao P, Chang L. Airborne microbial composition in a high-throughput poultry slaughtering facility. Journal of Food Protection 2013;76(3):413-419.) and in poultry meat (Smith et al., 2004Smith DP, Berrang ME, Feldner PW, Phillips RW, Meinersmann RJ. Detection of Bacillus cereus on selected retail chicken products. Journal of Food Protection 2004;67(8):1770-1773.; López et al., 2015López AC, Minnaard J, Pérez PF, Alippi AM. A case of intoxication due to a highly cytotoxic Bacillus cereus strain isolated from cooked chicken. Food Microbiology 2015;46:195-199.; Osman et al., 2018Osman KM, Kappell AD, Orabi A, Al-Maary KS, Mubarak AS, Dawoud TM, et al. Poultry and beef meat as potential seedbeds for antimicrobial resistant enterotoxigenic Bacillus species: A materializing epidemiological and potential severe health hazard. Scientific Reports 2018;8(1):11600.); therefore, it is not surprising that it can be found in products such as the egg. This study did not survey farms, but different egg brands that are marketed in Mexico, isolating this microorganism in all the brands analyzed. A higher frequency of the microorganism in the product could be explained by contamination deriving from the farm, and its association with different risk factors such as the lack of cleaning and draining of the drinking fountains, the hygiene of the sanitary fence, the type of disinfection and the concentration of dust (Kone et al., 2013Kone AZ, Jan S, Le Marechal C, Grosset N, Gautier M, Puterflam J, Baron F. Identifying risk factors for eggshell contamination by Bacillus cereus group bacteria in French laying farms. British Poultry Science 2013;54(3):298-305.). In the case of other microorganisms, the time that the egg spends in contact with other eggs as well as with the hen, which contributes to the content of the shell microbiome (Trudeau et al., 2020Trudeau S, Thibodeau A, Côté JC, Gaucher ML, Fravalo P. Contribution of the broiler breeders' fecal microbiota to the establishment of the eggshell microbiota. Frontiers in Microbiology 2020;11:666.) or the diet of chickens, which affects the gut microbiome and therefore that of the shell (Smith et al., 2000) is associated to contamination. For the total count of microorganisms in the eggshell, the cage system used has also been analyzed; theeggs produced by hens in a free system had a higher CFU count than the eggs obtained from hens in conventional systems (Gentry & Quarles, 1972Gentry RF, Quarles CL. The measurement of bacterial contamination on egg shells. Poultry Science 1972;51(3):930-933.; De Reu et al., 2005De Reu K, Grijspeerdt K, Heyndrickx M, Zoons J, De Baere K, Uyttendaele M, et al. Bacterial eggshell contamination in conventional cages, furnished cages and aviary housing systems for laying hens. British Poultry Science 2005;46(2):149-155.; Samiullah & Chousalkar, 2014). Together, these data could explain the abundance of B. cereus in all brands and, on the other hand, the differences in the percentage of contaminated eggs between brands in this study.

Also, there are differences in contamination by eggshell color, being B. cereus more frequent in eggs with white hells, in this sense, it has been reported that the brown pigment of the egg has antibacterial activity against microorganisms such as Staphylococcus aureus and B. cereus (Ishikawa et al., 2010Ishikawa S, Suzuki K, Fukuda E, Arihara K, Yamamoto Y, Mukai T, Itoh M. Photodynamic antimicrobial activity of avian eggshell pigments. FEBS Letters 2010;584(4):770-774.), which could explain the low prevalence in brown eggs hells in our study. Finally, no differences were found regarding the final point of sale, noting that at this point they arrive in containers, which are generally covered and protected from environmental pollutants. Therefore, we deduce that the product contamination occurs before reaching the markets and supermarkets; Because contamination should not only be considered during the laying of the eggs but also in transport or preparation for sale; in this sense, the egg can be contaminated by any surface on which the egg comes in contact with. Water, packaging material, insects, hands, broken shells, dust, are the main sources of contamination (Board & Tranter, 1995Board R, Tranter H. Egg science and technology. 4th ed. Boca Raton: Food Products Press; 1995.). Even egg washing before hatching is performed to reduce the number of bacteria present in the eggs, which is considered routine practice in the United States, Australia, and Japan (Hutchison et al., 2004Hutchison ML, Gittins J, Sparks AWN, Humphrey TJ, Burton C, Moore A. An assessment of the microbiological risks involved with egg washing under commercial conditions. Journal of Food Protection 2004;67(1):4-11.) although it has also been described that it favors the penetration of microorganisms (Gole et al., 2014Gole VC, Chousalkar KK, Roberts JR, Sexton M, May D, Tan J, et al. Effect of egg washing and correlation between eggshell characteristics and egg penetration by various Salmonella Typhimurium Strains. PLoS ONE 2014;9(3):e90987.).

A key characteristic of our study is that it was not only based on the isolation of the microorganism, but the molecular characterization of virulence factors was carried out. In this study, we show the high toxigenic potential of the strains because 53/55 strains contain at least one gene for a B. cereus enterotoxin; Furthermore, the A profile that contains the genes of the three enterotoxins was the most frequent in the population studied, similar to previous studies of B. cereus in food (Park et al., 2009Park YB, Kim JB, Shin SW, Kim JC, Cho SH, Lee BK, et al. Prevalence, genetic diversity, and antibiotic susceptibility of Bacillus cereus strains isolated from rice and cereals collected in Korea. Journal of Food Protection 2009;72(3):612-617.; Chon et al., 2012Chon JW, Kim JH, Lee S-J, Hyeon JY, Seo KH. Toxin profile, antibiotic resistance, and phenotypic and molecular characterization of Bacillus cereus in Sunsik. Food Microbiology 2012;32(1):217-222.; Hwang & Park, 2015Hwang JY, Park JH. Characteristics of enterotoxin distribution, hemolysis, lecithinase, and starch hydrolysis of Bacillus cereus isolated from infant formulas and ready-to-eat foods. Journal of Dairy Science 2015;98(3):1652-1660.; Adame-Gómez et al., 2018Adame-Gómez R, Guzman-Guzman K del A, Vences-Velazquez A, Leyva-Vazquez MA, Muñoz-Barrios S, Ramirez-Peralta A. Prevalence and genetic diversity of the strains of Bacillus cereus groups in food for infants and young children in Mexico. African Journal of Microbiology Research 2018;12(30):730-735.; Adame-Gómez et al., 2020; Adame-Gómez et al., 2020).

The enterotoxin gene nhe was reported more frequently in our study, which has been previously reported in strains isolated from food, including chicken samples (Gaviria et al., 2000; Hansen & Hendriksen, 2001Hansen BM, Hendriksen NB. Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis. Applied and Environmental Microbiology 2001;67(1):185-189.; Guinebretière et al., 2002Guinebretière MH, Broussolle V, Nguyen-The C. Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus strains. Journal of Clinical Microbiology 2002;40(8):3053-3056.; Smith et al., 2004Smith DP, Berrang ME, Feldner PW, Phillips RW, Meinersmann RJ. Detection of Bacillus cereus on selected retail chicken products. Journal of Food Protection 2004;67(8):1770-1773.; Ankolekar et al., 2009Ankolekar C, Rahmati T, Labbé RG. Detection of toxigenic Bacillus cereus and Bacillus thuringiensis spores in U.S. rice. International Journal of Food Microbiology 2009;128(3):460-466.; Chaves et al., 2012Chaves JQ, Cavados C de FG, Vivoni A. M. Molecular and toxigenic characterization of Bacillus cereus and Bacillus thuringiensis strains isolated from commercial ground roasted coffee. Journal of Food Protection 2012;75(3):518-522.; Chon et al., 2012Chon JW, Kim JH, Lee S-J, Hyeon JY, Seo KH. Toxin profile, antibiotic resistance, and phenotypic and molecular characterization of Bacillus cereus in Sunsik. Food Microbiology 2012;32(1):217-222.; Ouoba et al., 2008Ouoba LII, Thorsen L, Varnam AH. Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermented food condiments. International Journal of Food Microbiology 2008;124(3):224-230.) and silo tanks (Ehling-Schulz et al., 2006Ehling-Schulz M, Guinebretiere MH, Monthan A, Berge O, Fricker M, Svensson B. Toxin gene profiling of enterotoxic and emetic Bacillus cereus. FEMS Microbiology Letters 2006;260(2):232-240.). In this study, no strains with the cereulide gene (ces) were found, which coincides with different studies that found a low frequency of emetic strains (Altayar & Sutherland, 2006Altayar M, Sutherland AD. Bacillus cereus is common in the environment but emetic toxin producing isolates are rare. Journal of Applied Microbiology 2006;100(1):7-14.; Ouoba et al., 2008; Chon et al., 2012; Jessberger et al., 2020Jessberger N, Dietrich R, Granum PE, Märtlbauer E. The Bacillus cereus Food Infection as Multifactorial Process. Toxins 2020;12(11):701.), in addition, a higher frequency of emetic syndrome has been reported in countries such as Japan and the United Kingdom (Kramer et al., 1989Kramer J, Gilbert R, Doyle M. Foodborne bacterial pathogens. New York: M. Dekker; 1989. p.22-70.). In turn, negative emetic strains have been described as containing the three enterotoxin genes (Ehling-Schulz et al., 2005), which is also observed in this study. Also, the characterization of emetic strains can not only be carried out from ces gene, which is important for the search for other genes such as bceT or EM1 (Guinebretière et al., 2002; Ouoba et al., 2008; Chon et al., 2012). It is important to note that B. cereus is not only a toxigenic microorganism; B. cereus has been described as having an enzyme profile that gives it the ability deteriorate food products such as milk. Benahmed et al (2020Benahmed M, Leguerinel I, Moussa-Boudjemaa B. Biodiversity, spoilage capacity and heat resistance of mesophilic aerobic spores isolated from milk powders marketed in Algeria. International Journal of Dairy Technology 2020;73: 771-780.) described that 78% of their strains can produce lecithinase; while Mehta et al (2019Mehta DS, Metzger LE, Hassan AN, Nelson BK, Patel HA. The ability of spore-formers to degrade milk proteins, fat, phospholipids, common stabilizers, and exopolysaccharides. Journal of Dairy Science 2019;102:10799-10813.) show that Bacillus strains can produce different levels of lipase. Both enzymes capable of degrading major components of the egg yolk; Therefore, it is not surprising that this microorganism can deteriorate the egg yolk if it is able to adhere to the eggshell and colonize the egg yolk.

The persistence of B. cereus in the eggshell may be related to the adhesive characteristics and environmental resistance of the spores (Drobniewski, 1993Drobniewski FA. Bacillus cereus and related species. Clinical Microbiology Reviews 1993;6(4):324-338.); However, in recent years, particular emphasis has been placed on the study of biofilm production by B. cereus as a mechanism of persistence and contamination of various food products (Rajkovic et al., 2008Rajkovic A, Uyttendaele M, Dierick K, Samapundo S, Botteldoorn N, Mahillon J, et al. Risk profile of the Bacillus cereus group implicated in food poisoning. Brussels: Superior Health Council Belgium; 2008. p.80. Available from: https://www.health.belgium.be/sites/default/files/uploads/fields/fpshealth_theme_file/19060475/Risico-profiel%20voor%20Bacillus%20cereus%20Groep%20in%20voedsel%20toxi-infecties%3A%20situatie%20in%20België%20en%20aanbevelingen%20%5BBijlage%5D%20%28januari%202010%29%20%28HGR%208316%29.pdf.; Majed et al., 2016Majed R, Faille C, Kallassy M, Gohar M. Bacillus cereus biofilms-same, only different. Frontiers in Microbiology 2016;7.) affecting the shelf life of the products and their safety. The production of biofilms has even been reported under conditions like the production of food associated with poultry (Iñiguez-Moreno et al., 2019Iñiguez-Moreno M, Gutiérrez-Lomelí M, Avila-Novoa MG. Kinetics of biofilm formation by pathogenic and spoilage microorganisms under conditions that mimic the poultry, meat, and egg processing industries. International Journal of Food Microbiology 2019;303:32-41.) and milk (Alonso & Kabuki, 2019Alonso VPP, Kabuki DY. Formation and dispersal of biofilms in dairy substrates. International Journal of Dairy Technology 2019;72:472-478.). In this study, we determined the production of biofilms by B. cereus strains in different materials; the included materials are commonly used in the food industry as part of conveyor belts (PVC) or as packaging (CFR, 2020); furthermore, we decided to include different materials since it has previously been reported that biofilm production is differential according to the type of material used (Wijman et al., 2007Wijman JGE, de Leeuw PPLA, Moezelaar R, Zwietering MH, Abee T. Air-liquid interface biofilms of bacillus cereus: formation, sporulation, and dispersion. Applied and Environmental Microbiology 2007;73(5):1481-1488.; Hayrapetyan et al., 2015Hayrapetyan H, Muller L, Tempelaars M, Abee T, Nierop Groot M. Comparative analysis of biofilm formation by Bacillus cereus reference strains and undomesticated food isolates and the effect of free iron. International Journal of Food Microbiology 2015;200, 72-79.).

In this study, high biofilm production on PVC was found, which has previously been reported by our group with strains isolated from other food sources (Adame-Gómez et al., 2020Adame-Gómez R, Muñoz-Barrios S, Castro-Alarcón N, Leyva-Vázquez M-A, Toribio-Jiménez J, Ramírez-Peralta A. Prevalence of the Strains of Bacillus cereus Group in Artisanal Mexican Cheese. Foodborne Pathogens and Disease 2020;17(1):8-14.); These production differences by the material may be related to the physicochemical characteristics of the materials (De-la-Pinta et al., 2019De-la-Pinta I, Cobos M, Ibarretxe J, Montoya E, Eraso E, Guraya T, et al. Effect of biomaterials hydrophobicity and roughness on biofilm development. Journal of Materials Science. Materials in Medicine 2019;30(7):77.) or to the own requirements or the genetic profile of the microorganisms to generate biofilm (Hayrapetyan et al., 2016Hayrapetyan H, Siezen R, Abee T, Nierop Groot M. Comparative genomics of iron-transporting systems in bacillus cereus strains and impact of iron sources on growth and biofilm formation. Frontiers in Microbiology 2016;7, 842.; Caro-Astorga et al., 2020Caro-Astorga J, Álvarez-Mena A, Hierrezuelo J, Guadix JA, Heredia-Ponce Z, Arboleda-Estudillo Y, et al. Two genomic regions encoding exopolysaccharide production systems have complementary functions in B. cereus multicellularity and host interaction. Scientific Reports 2020;10(1):1000.). For example, in glass and in polystyrene, the presence of extracellular DNA has been described as a requirement to favor the production of biofilms (Vilain et al., 2009Vilain S, Pretorius JM, Theron J, Brozel VS. DNA as an Adhesin: Bacillus cereus requires extracellular DNA to form biofilms. Applied and Environmental Microbiology 2009;75(9):2861-2868.). On the other hand, we found that strains positive for tasA produce a greater quantity of biofilms in PVC, polystyrene, and polyethylene, grouped as positive or negative for tasA. This gene is paralogue to tasA in Bacillus subtilis (Caro-Astorga et al., 2015), which codes for an amyloid-type protein of the same name and has been reported as the most abundant structural component of B. subtilis floating biofilms (Romero et al., 2011Romero D, Vlamakis H, Losick R, Kolter R. An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms. Molecular Microbiology 2011;80(5):1155-1168.); which could support the theory that tasA-positive B. cereus strains are indeed producing a greater number of biofilms.

In this study, we report for the first time that the strains of B. cereus produce biofilms in the eggshell, which reaffirms that this could be the persistence mechanism in the product. The production of biofilms in the eggshell has been reported for bacteria of the Salmonella genus (Pande et al., 2016Pande VV, Devon RL, Sharma P, McWhorter AR, Chousalkar KK. Study of Salmonella typhimurium infection in laying hens. Frontiers in microbiology, 2016;7(203).), which with other physical and nutritional factors of the shell favor its permanence and gradually the invasion towards the yolk (Gole et al., 2014Gole VC, Chousalkar KK, Roberts JR, Sexton M, May D, Tan J, et al. Effect of egg washing and correlation between eggshell characteristics and egg penetration by various Salmonella Typhimurium Strains. PLoS ONE 2014;9(3):e90987.). Therefore, it is important to define the molecular characteristics for this process to be carried out and to show that the microorganism can pass through the shell and the mechanisms by which it does so.

This study reaffirms the circulation of B. cereus strains in the product, their toxigenic potential and their persistence characteristics in the product; which highlights the need for a sanitary regulation of this microorganism in Mexico in this type of product.

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  • FUNDING

    PFCE 2018. Secretaria de Educación Pública

Publication Dates

  • Publication in this collection
    22 Apr 2022
  • Date of issue
    2022

History

  • Received
    26 June 2021
  • Accepted
    02 Dec 2021
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