Enterotoxigenic profiles and submerged and interface biofilms in Bacillus cereus group isolates from foods / Perfiles enterotoxigénicos y biopelículas de superficie y sumergidas en aislados del grupo Bacillus cereus de alimentos

Abstract Biofilm formation by Bacillus cereus strains is now recognized as a systematic contaminaron mechanism in foods; the aim of this study was to evaluate the production of submerged and interface biofilms in strains of B. cereus group in different materials, the effect of dex-trose, motility, the presence of genes related to biofilms and the enterotoxigenic profile of the strains. We determine biofilm production by safranin assay, motility on semi-solid medium, toxin gene profiling and genes related to biofilm production by PCR in B. cereus group iso-lated from food. In this study, we observe strains used a higher production of biofilms in PVC; in the BHI broth, no submerged biofilms were found compared to phenol red broth and phenol red broth supplemented with dextrose; no strains with the ces gene were found, the enterotoxin profile was the most common the profile that includes genes for the three enterotoxins. We observed a different distribution of tasA and sipW with the origin of isolation of the strain, being more frequent in the strains isolated from eggshell. The production and type of biofilms are differential according to the type of material and culture medium used.

Resumen La formación de biopelículas por cepas de Bacillus cereus es reconocida como un mecanismo de contaminación sistemática en alimentos; el objetivo del estudio fue evaluar la producción de biopelículas sumergidas y de superficie en cepas del grupo de Bacillus cereus en diferentes materiales, el efecto de la dextrosa, la motilidad, la presencia de genes relacionados a biopelículas y el perfil enterotoxigénico de las cepas. Determinamos la producción de biopelículas por el ensayo de safranina, motilidad en medio sólido, perfil enterotoxigénico y genes relacionados a producción de biopelículas por PCR en aislados del grupo de Bacillus cereus de alimentos. En este estudio, observamos en las cepas utilizadas una alta producción de biopelículas en PVC; en caldo BHI, no se encontraron biopelículas sumergidas en comparación con el caldo rojo de fenol y caldo rojo de fenol suplementando con dextrosa; no se encontraron cepas con el gen ces, el perfil de enterotoxinas más común fue el perfil que incluía los genes de las tres enterotoxinas, también observamos una distribución diferente de tasA y sipW con relación al origen de la cepa, siendo más frecuente estos genes en las cepas aisladas de huevos. La producción y el tipo de biopelículas es diferente de acuerdo con el tipo de material y el medio de cultivo utilizado.

Bacillus cereus in the Artisanal Cheese Production Chain in Southwestern Mexico.

BACKGROUND: Bacillus cereus is associated with milk, dairy product, and dairy farm contamination. The aim of this study was to characterize strains of B. cereus in the small-scale artisanal cheese production chain in southwestern Mexico. METHODS: 130 samples were collected. B. cereus isolation was performed on Mannitol Egg Yolk Polymyxin (MYP) agar. Genotyping, enterotoxigenic profile, and determination of genes involved in the formation of B. cereus biofilm were performed by PCR. An antimicrobial susceptibility test was made by broth microdilution assay. The phylogenetic analysis was performed by amplification and sequencing of 16s rRNA. RESULTS: B. cereus sensu lato was isolated and molecularly identified in 16 samples and B. cereus sensu stricto (B. cereus) was the most frequently isolated and identified species (81.25%). Of all the isolated B. cereus sensu lato strains, 93.75% presented at least one gene for some diarrheagenic toxins, 87.5% formed biofilms, and 18.75% were amylolytic. All B. cereus sensu lato strains were resistant to beta-lactams and folate inhibitors. A close phylogenetic relationship between isolates was found between the cheese isolates and the air isolates. CONCLUSIONS: Strains of B. cereus sensu lato were found in small-scale artisanal cheeses on a farm in southwestern Mexico.

Enterotoxigenic profiles and submerged and interface biofilms in Bacillus cereus group isolates from foods.

Biofilm formation by Bacillus cereus strains is now recognized as a systematic contamination mechanism in foods; the aim of this study was to evaluate the production of submerged and interface biofilms in strains of B. cereus group in different materials, the effect of dextrose, motility, the presence of genes related to biofilms and the enterotoxigenic profile of the strains. We determine biofilm production by safranin assay, motility on semi-solid medium, toxin gene profiling and genes related to biofilm production by PCR in B. cereus group isolated from food. In this study, we observe strains used a higher production of biofilms in PVC; in the BHI broth, no submerged biofilms were found compared to phenol red broth and phenol red broth supplemented with dextrose; no strains with the ces gene were found, the enterotoxin profile was the most common the profile that includes genes for the three enterotoxins. We observed a different distribution of tasA and sipW with the origin of isolation of the strain, being more frequent in the strains isolated from eggshell. The production and type of biofilms are differential according to the type of material and culture medium used.

Genetic diversity of enterotoxigenic Bacillus cereus strains in coriander in southwestern Mexico.

Background: Coriander, like other leafy green vegetables, is available all year round and is commonly consumed raw in Mexico as in other countries in the preparation of street or homemade food. Bacillus cereus (B. cereus) is a microorganism that can reach coriander because it is usually found in the soil and in some regions the vegetables are irrigated with polluted water. Therefore, the aim of this study was to determinate the presence of B. cereus in coriander used for human consumption in southwestern Mexico and determine the toxigenic profile, biofilm production, genes associated with the production of biofilms, sporulation rates, enzymatic profile, psychotropic properties, and genetic diversity of B. cereus. Methods: Fresh coriander samples were collected from several vegetable retailers in different markets, microbiological analysis was performed. Molecular identification, genes related to the production of biofilm, and toxin gene profiling of B. cereus isolates were determined by PCR. The biofilm formation was measured by performing a crystal violet assay. The genetic diversity of B. cereus strains was determined by PCR of repetitive elements using oligonucleotide (GTG) 5. Results: We found a frequency of B. cereus in vegetables was 20% (13/65). In this study, no strains with genes for the HBL toxin were found. In the case of genes related to biofilms, the frequency was low for sipW [5.8%, (1/17)] and tasA [11.7%, (2/17)]. B. cereus strains produce a low amount of biofilm with sporulation rates around 80%. As for genetic diversity, we observed that strains isolated from the same market, but different vegetable retailers are grouped into clusters. In the coriander marketed in southwestern Mexico, were found B. cereus strains with genes associated with the production of diarrheal toxins. Together, these results show actual information about the state of art of B. cereus strains circulating in the southwestern of Mexico.

Trichophyton species isolated from asymptomatic patients of the pet-owner pair in Mexico.

BACKGROUND AND PURPOSE: Superficial mycoses are the fourth most common cause of disease worldwide. It is not surprising that zoonotic transmission occurs to humans due to close contact with different animals, be it companion or farm animals. Therefore, the objective of this study was to determine the presence of asymptomatic dermatophyte carriers in the owner-pet pairs, identify the most common etiologic agents, and find the likely connection between the carrier status of an owner and the presence of dermatophytes in their pets. MATERIALS AND METHODS: From May 2019 to January 2020, 21 cats and 115 dogs with their respective owners were selected for dermatophyte culture. All the dogs and cats included in the study were from the communities of southeastern Mexico. The samples were taken with a cotton swab, which was vigorously rubbed and twisted on the scalp or body of the pet four times and grown on Mycosel Agar. The isolates were identified based on macroscopic and microscopic characteristics. The prevalence of the binomial ranged from 0.73% in pet skin and human hands to 2.2% in human scalp. In humans, the agents were Trichophyton tonsurans and Trichophyton verrucosum, while in pets, a strain of Trichophyton sp was found. CONCLUSION: Different species of dermatophytes were found in the owner/pet pairs, which denotes that coexistence is not related in asymptomatic cases.

Genetic Diversity and Virulence Factors of S. aureus Isolated from Food, Humans, and Animals.

Staphylococcus aureus is a commensal bacterium in humans and animals able to adapt to multiple environments. The aim of this study was to compare the genetic diversity and virulence profiles of strains of S. aureus isolated from food (29 strains), humans (43 strains), and animals (8 strains). 80 lipase-producing strains belonging to a biobank of 360 isolates, identified phenotypically as S. aureus, were selected. Confirmation of the species was made by amplifying the spA gene and 80% (64/80) of the strains were confirmed within this species. The virulence profile of each of the isolates was determined by PCR. The seA gene coding for enterotoxin A was found in 53.1% of the strains, the saK gene, which codes for Staphylokinase, was amplified in 57.8% of the strains, and, finally, the hlB gene coding for ß-Hemolysin was amplified in 17.2%. The profile of antimicrobial resistance was determined by the Kirby Bauer method showing that the strains from food presented greater resistance to erythromycin (40.7%) and ciprofloxacin (18.5%) while in strains isolated from humans were to erythromycin (48.4%) and clindamycin (21.2%). Also, in strains from animals, a high resistance to erythromycin was observed (75%). The frequency of MRSA was 12.5% due to the presence of the mec gene and resistance to cefoxitin. Of the total strains, 68.7% were typed by PCR-RFLP of the coa gene using the AluI enzyme; derived from this restriction, 17 profiles were generated. Profile 4 (490 bp, 300 bp) was the most frequent, containing a higher number of strains with a higher number of virulence factors and antimicrobial resistance, which is associated with greater adaptation to different environments. In this study, a wide genetic diversity of strains of S. aureus from different foods, humans, and animals was found. This demonstrates evolution, genetic versatility, and, therefore, the adaptation of this microorganism in different environments.

Biofilm Production by Enterotoxigenic Strains of Bacillus cereus in Different Materials and under Different Environmental Conditions.

Foodborne illnesses, such as infections or food poisoning, can be caused by bacterial biofilms present in food matrices or machinery. The production of biofilms by several strains of Bacillus cereus on different materials under different culture conditions was determined, as well as the relationship of biofilms with motility, in addition to the enterotoxigenic profile and candidate genes that participate in the production of biofilms. Biofilm production of B. cereus strains was determined on five materials: glass, polystyrene, polyethylene, polyvinylchloride (PVC), PVC/glass; in three culture media: Phenol red broth, tryptic soy broth, and brain heart infusion broth; in two different temperatures (37 °C and 25 °C), and in two different oxygen conditions (oxygen and CO2 tension). Furthermore, the strains were molecularly characterized by end-point polymerase chain reaction. Motility was determined on semi-solid agar. The B. cereus strains in this study were mainly characterized as enterotoxigenic strains; statistically significant differences were found in the PVC material and biofilm production. Motility was positively associated with the production of biofilm in glass/PVC. The sipW and tasA genes were found in two strains. The results of this study are important in the food industry because the strains carry at least one enterotoxin gene and produce biofilms on different materials.

Prevalence of the Strains of Bacillus cereus Group in Artisanal Mexican Cheese.

Bacillus cereus is a microorganism associated with food poisoning. It has been found in products, such as milk and dairy products. The aim of this study was to isolate and identify B. cereus group strains in artisan cheeses sold in southwestern Mexico, as well as its toxigenic profile, its psychrophilic ability, and its biofilm production. B. cereus isolation was performed on Mannitol Yolk Polymyxin (MYP) agar and this was molecularly confirmed by the amplification of the gyrB gene. Polymerase chain reaction was used to determine the toxigenic profile, amplifying conserved regions of hblABD and nheABC operons, which code for the subunits of Hbl and Nhe toxins, respectively, as well as ges and cytK genes, which code for toxin cereulide and cytotoxin K (Cytk). Frequency of B. cereus contamination in artisan cheeses was 29.48% (23/78), and the bacterium was isolated in similar quantities in all types of products. All strains were amylase positive, and 60.86% (14/23) were able to produce biofilm; 91.30% (21/23) of the strains were psychrophilic. In most of the strains, at least one gene related to enterotoxins was identified (21/23). B. cereus strains in this study have a high toxigenic potential, which increases the risk of food poisoning due to the consumption of artisan cheeses made in Mexico.

Prevalencia de cepas del grupo de Bacillus cereus productoras de biopelicula en helados comercializados en México / Prevalence of strains of the Bacillus cereus group biofilm producers in ice cream in Mexico

El helado es un vehículo para la transmisión de patógenos como Bacillus cereus. Por lo cual, se determinó la frecuencia de cepas del grupo Bacillus cereus en helados, perfil enterotoxigénico, psicrofilia y producción de biopelícula. Un total de 230 muestras de seis marcas de helado de producción y distribución nacional fueron colectadas en México. El análisis microbiológico incluyó aislamiento en agar manitol yema de huevo. Las cepas se identificaron molecularmente a partir de la amplificación del gen de la topoisomerasa (gyrB) y el perfil enterotoxigénico por la amplificación de regiones conservadas de los operones nheABC y hblABD y del gen cytK. Además, se determinó la producción de biopelícula en vidrio y policloruro de vinilo. La frecuencia de contaminación por cepas del grupo B. cereus fue de 3,6%, se encontró una cepa positiva para nheABC y cinco para cytK, el 87,5% de las cepas generó biopelícula en vidrio y todas en policloruro de vinilo, dos cepas fueron psicrofilicas. En conclusión, en el helado distribuido en México, se encontró una baja contaminación por cepas del grupo B. cereus con alta producción de biopelícula; sin embargo, no se debe subestimar el potencial enterotoxigénico de estas cepas

Ice cream is a medium for microbial growth due to its nutritional value and neutral pH. Therefore, the frequency of strains of the Bacillus cereus group in ice cream was determined, the enterotoxigenic profile, psychrophilic strains and biofilm production. A total of 230 samples of six brands of ice cream produced and distributed nationwide were collected in Mexico. The microbiological analysis was a cold pre-enrichment and isolation of the microorganism in egg yolk agar. The strains were identified molecularly from the amplification of the topoisomerase gene (gyrB) and the enterotoxigenic profile by the amplification of conserved regions of the nheABC and hblABD operons and of the cytK gene. In addition, the production of biofilm in glass and polyvinyl chloride and psychophilia was determined. The frequency of contamination by strains of the B. cereus group was 3.6%, a positive strain was found for nheABC and five for cytK, 87.5% of the strains generated biofilm in glass and all in polyvinyl chloride, two strains were psychrophilic. In the ice cream distributed in Mexico, a low contamination by strain of the B. cereus group was found, however, the enterotoxigenic potential of the strains should not be underestimated

Staphylococcus aureus resistentes a meticilina (SARM) y productores de enterotoxina A aislados de portadores nasales asintomáticos entre estudiantes universitarios de México / Methicillin resistant Staphylococcus aureus (MRSA) and enterotoxin A producer isolated from asymptomatic nasal carriage among undergraduate students of Mexico

El objetivo de este estudio fue determinar la frecuencia de S. aureus, incluyendo resistentes a meticilina y la producción de enterotoxina A en fosas nasales de estudiantes universitarios en México. Este fue un estudio transversal realizado en 471 estudiantes universitarios de una ciudad del suroeste de México. Las muestras nasales y los datos sociodemográficos fueron obtenidos de los pacientes. Las cepas fueron identificadas como S. aureus basándose en la morfología, tinción de Gram, prueba de catalasa, prueba de coagulasa y fermentación en agar manitol salado. Las cepas se biotipificaron, se determinó la resistencia a meticilina por difusión en agar y la producción de enterotoxina A por Dot- Blot. La frecuencia de portadores nasales de S. aureus fue 10,40 %; 73,46 % resistentes a meticilina; 36,73 % producen enterotoxina A. En un análisis bivariado, se encontraron diferencias estadísticamente significativas en pacientes que viven cerca de aguas residuales y granjas con el estado de portador de S. aureus, (p=0,01, OR 2,59 [1,06-5,81]; p=0,01, OR 3,18, [1,07- 8,33]). Los portadores nasales muestran una diversidad de cepas de S. aureus, mayormente resistentes a meticilina, pero no todas producen enterotoxina A.

The aim at this study was determine the frequency of S. aureus, including methicillin-resistant and enterotoxin A production in nostrils of university students in Mexico. This was a cross-sectional study conducted in 471 university students from a city in southwestern Mexico. Nasal samples and sociodemographic data were obtained from the patients. Strains were identified as S. aureus based on morphology, Gram stain, catalase test, coagulase test and fermentation on salted mannitol agar. Isolated strains were subjected to biotyping, their methicillin resistance was analyzed using the agar diffusion method and examined their enterotoxin A (SEA) production by a Dot-blot analysis. The nasal carriage rate of S. aureus was 10.40%; 73.46% of the isolates were resistant to methicillin; 36.73% of the strains produced enterotoxin A. In the bivariate analysis, a statistically significant difference was found in patients who lived near sewage and farms with S. aureus carriage (p=0.012, odds ratio 2.59, [ 1.06-5.81]; p=0.009, odds ratio 3.18, [1.07- 8.33]) and the first group also associated with methicillin resistant S. aureus carriage (p=0.020, odds ratio 3.38, [1.30-8.06]). Nasal carriers show a wide variety of strains of S. aureus, mostly MRSA strains, but not all produce enterotoxin A.

Methicillin-Resistant Staphylococcus aureus (MRSA) in Artisanal Cheeses in México.

Milk and dairy foods have frequently been implicated in staphylococcal food poisoning, and contaminated raw milk is often involved. The aim of the study was to determine the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in raw cow milk cheese produced in Mexico. A total of 78 unpasteurized cow milk cheese samples were screened for S. aureus. The isolates were identified as S. aureus based on morphology, Gram stain, catalase test, coagulase test, and mannitol salt agar fermentation. Isolates were subjected to biotyping, the methicillin resistance was analyzed using the disk diffusion, and the Staphylococcus enterotoxin A (SEA) production was examined by a dot-blot analysis. From a total of 78 samples of unpasteurized cheeses analyzed in this study, 44 cheeses were positive for S. aureus; however, a differential contamination between the different types of cheeses was observed, with high risk of contamination in adobero cheese (12, 95% CI 1.75 to 94.20; p=0.002). In this study, the frequency of methicillin-resistant Staphylococcus aureus (MRSA) was 18.1% (8/44) and of enterotoxin A producers was 18.1% (8/44). When classified by biotypes, MRSA only belongs to the human ecovar biotype (2/8, 25%) and the D biotype (4/8, 50%). S. aureus producers of enterotoxin A were distributed in specific nonhost biotypes.