Journal of biological and health sciences http://biotecnia.unison.mx
Universidad de Sonora
ISSN: 1665-1456
Artículo Original
Perfil enterotoxigénico, producción de biopelícula y resistencia antimicrobiana de Bacillus cereus
aislado de arroz comercializado al sur de México
1 Laboratorio de Investigación en Patometabolismo Microbiano, Universidad Autónoma de Guerrero. México.
2 Unidad de Investigación en Virología y Cáncer, Hospital Infantil de México Federico Gómez. México.
3 Laboratorio de Investigación en Inmunología y Microbiología, Universidad Autónoma de Guerrero. México.
4 Laboratorio de Investigación en Biomedicina Molecular, Universidad Autónoma de Guerrero. México.
Bacillus cereus is responsible for food poisoning worldwide, thus, the characterization of strains isolated from food, in this case rice, is essential. Therefore, the objective of this study was to identify the toxigenic profile, lytic enzymes, antimi- crobial resistance, and biofilm production of B. cereus strains isolated from rice. The genetic profile of toxins and biofilm- related genes of strains was determined by endpoint PCR. Biofilm was visualized by safranin staining. Evaluation of lytic enzymes was determined in culture media. Psychrophiles were monitored by the growth of the strains at refrigeration temperature. The GTG5 technique was used to determine the genetic diversity of the strains. Antimicrobial resistance of the strains was validated by minimum inhibitory concentration. The strains of B. cereus s.l. from rice contained genes for ente- rotoxins and genes associated with biofilm production. The strains did not have the cereulide gene. The strain isolated from fried rice was the only one that contained the Hbl toxin gene and the Eps2 operon. The same strain was the only one that did not produce biofilm, had intermediate sensitivity to erythromycin, was amylase positive, had high lecithinase activity, and grew at refrigeration temperature.
Bacillus cereus es responsable de intoxicaciones alimentarias a nivel mundial, siendo importante la caracterización de ce- pas aisladas de alimentos, en este caso, de arroz. Por lo tanto, el objetivo de este estudio fue identificar el perfil toxigénico, enzimas líticas, resistencia antimicrobiana y producción de biopelículas en cepas de B. cereus aisladas de arroz. El perfil genético de toxinas y los relacionados a biopelículas fue determinado por PCR en punto final. La biopelícula fue vi- sualizada por tinción con safranina. La evaluación de enzimas líticas fue determinada en medios de cultivo. Los psicrófilos fueron monitoreados por crecimiento de cepas en tempe- ratura de refrigeración. La técnica de GTG5 fue usada para determinar la diversidad genética de las cepas. La resistencia
*Author for correspondence: Arturo Ramírez-Peralta e-mail: ramirezperaltauagro@gmail.com
Received: Augst 30, 2024
Accepted: November 11, 2024
Published: Decembre 17, 2024
a antimicrobianos fue validada por concentración mínima inhibitoria. Las cepas de B. cereus s.l. de arroz contenían los genes de enteroxinas y genes asociados a producción de biopelículas. Las cepas no contienen el gen de la cereulida. La cepa aislada de arroz frito es la única que contiene el gen de la toxina Hbl y el operón eps2. Esta misma cepa no produce biopelícula, tiene sensibilidad intermedia a eritromicina, es amilasa positiva, tiene alta actividad lecitinolítica y crece en temperaturas de refrigeración.
The Bacillus cereus group, or B. cereus sensu lato (B. cereus s.l.), are Gram-positive bacteria with a low GC content that belong to the Firmicutes phylum. It is a group that includes bacillus- shaped, spore-forming, aerobic to facultative anaerobic bacteria, with peritrichous flagella involved in locomotion, including at least eight highly related species: B. anthracis,
B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, B. weihenstephanensis, B. cytotoxicus and B. toyonensis (Enosi Tuipulotu et al., 2021; Liu et al., 2015).
The species of the B. cereus group are endemic soil bacteria that occupy diverse ecological habitats due to the formation of endospores resistant to heat, UV radiation, acids, and desiccation, for which the bacteria can persist in a dormant state (Ehling-Schulz et al., 2019). In addition to spo- re production, B. cereus resists adverse environmental condi- tions due to the formation of biofilms on abiotic surfaces and living tissues (Duport et al., 2016). In this sense, it has been reported that B. cereus can produce biofilms in pipes and food storage systems, favoring the systematic contamination of food products (Majed et al., 2016; Shemesh and Ostrov, 2020; Wijman et al., 2007).
In addition to soil, species have been isolated from fresh and frozen foods, invertebrates, and plants (Ehling-Schulz et al., 2019). In this sense, it is estimated that B. cereus is responsible for 1.4%-12% of all food poisonings worldwide (Grutsch et al., 2018). B. cereus is responsible for two types
Volume XXVI
DOI: 10.18633/biotecnia.v26.2429
of gastrointestinal syndromes. The emetic type is mainly characterized by nausea and emesis, which appears one hour after consumption of contaminated food and is clinically indistinguishable from Staphylococcus aureus enterotoxin poisoning (Stenfors Arnesen et al., 2008). The diarrheal type of food poisoning is also associated with various foods. The disease mainly manifests in diarrhea and abdominal cramps, like Clostridium perfringes food poisoning, and occurs after approximately 8 to 16 h after consumption (Jessberger et al., 2020; Stenfors Arnesen et al., 2008).
The diarrhoeal syndrome is associated with different enterotoxins produced after germination of spores and growth of vegetative cells, ingested through contaminated food and activated in the small intestine (Schoeni and Wong, 2005, Stenfors Arnesen et al., 2008). B. cereus produces three pore-forming enterotoxins: Nhe (a three-component non- haemolytic enterotoxin), Hbl (a three-component haemolytic enterotoxin) and, the single-component cytotoxin CytK. The emetic syndrome results from poisoning caused by a toxin called cereulide, which is found in food (Schoeni and Wong, 2005). Cereulide is a dodecapeptide composed of alpha amino and alpha hydroxy acids, structurally related to the potassium ionophore valinomycin. Cereulide is produced by a peptide synthetase called ces, which represents a new type of non-ribosomal peptide synthetase (NRPS) (Ehling-Schulz et al., 2004).
Epidemiological data show that rice, pasta, cakes, and
noodles are associated with emetic syndrome, whereas vegetables, meat, and dairy products have been associated with diarrheal syndrome (Enosi Tuipulotu et al., 2021). In Mexico, there is currently no health legislation related to the presence of B. cereus in foods, but the presence of B. cereus in vegetables (Castulo-Arcos et al., 2022; Flores-Urbán et al., 2014), ice cream (Adame-Gomez et al., 2019), artisanal chee- ses and eggs (Adame-Gómez et al., 2020b; Cruz-Facundo et al., 2022; 2023) has been reported. Rice is a product consu- med in Mexico in different preparations, including fried rice and milk dessert with rice. In a preliminary study, B. cereus was isolated from 5% of rice samples, with a higher frequency in rice desserts (8.5%). However, not only the isolation of the microorganism is essential, but also the molecular characte- rization of different virulence factors, are needed. Therefore, the objective of this study is to identify the toxigenic profile, lytic enzymes, antimicrobial resistance, and biofilm produc- tion of B. cereus strains isolated from rice.
In this study, six strains previously characterized as B. cereus
s.l. (Cano- Ponce and Ramirez- Peralta, 2023) are included, based on the isolation and presumptive identification in MYP agar and the amplification of the gyrB gene as a confirmatory test (Wei et al., 2018). The strains are named according to uni- que laboratory strain codes such as B629, B630, B631, B632, B633, B634. The B629 strain was isolated from fried rice and the rest of the strains were from rice-based desserts.
For DNA extraction, 1 mL of bacterial liquid culture was centrifuged for 10 minutes at 10,000 rpm. The pellet was re- suspended in 200 μL of lysis solution (10 mM Tris-HCl, 1 mM EDTA pH 8.0, and 1 mg/mL lysozyme) and incubated for 30 minutes at 37°C. Afterwards, 250 μL of phenol-chloroform- isoamyl alcohol (ratio 25:24:1) were added and homogenized by inversion. Then, it was centrifuged at 10,000 rpm for 5 min, 200 μL of the aqueous phase was recovered to avoid contamination with organic phase (chloroform/ phenol phase) and mixed with 1 mL of cold absolute ethanol. Finally, the solution was centrifuged at 10,000 rpm for 10 min. The supernatant was completely removed, and the DNA was resuspended in 20 μL of TE buffer (Cruz-Facundo et al., 2023).
Detection of the B. cereus toxin genes was performed by PCR using conserved regions of the nheABC (NA2-F AAGCI- GCTCTTCGIATTC, NB1-R ITIGTTGAAATAAGCTGTGG), hblACD (HD2-F GTAAATTAIGATGAICAATTTC, HA4-R AGAATAGGCATT-
CATAGATT) operons, which encode non-hemolytic ente- rotoxins and hemolysin BL, respectively; and the genes ces (CesF1- GGTGACACATTATCATATAAGGTG, CesR2- GTAAGCGA- ACCTGTCTG-TAACAACA) and cytK (P1-cytK CAAAACTCATC- TATGCAATTATGCAT, P3-cytK ACCAGTTGTATTAA-TAACGG-
CAATC), which encode for emetic toxin and cytotoxin, respectively (Ehling-Schulz et al., 2006; Ołtuszak-Walczak et al., 2006). For each PCR, the mixture contained the following: 25 μL of REDTaq DNA Polymerase Ready Mix (Sigma-Aldrich, St. Louis, MO, USA), 11 μL of sterile MiliQ water, 10 to 20 ng of genomic DNA, and 0.02 μM of each oligonucleotide. The conditions for the nhe, hbl, and ces genes were 1 cycle at 94°C for 5 minutes, 25 cycles at 94°C for 30s, 49°C for 1 minute, 72°C for 1 minute, and one cycle at 72°C for 5 minutes. For the cytK gene it was one cycle at 94°C for 2 minutes, 35 cycles at 94°C for 30 seconds, 52°C for 1 minute, 72°C for 30 seconds, and one cycle at 72°C for 10 minutes. The B. cereus strains used as positive controls, were ATCC14579 for hbl and cytK genes, and BC133 for the nhe gene. The latter strain was previously isolated and characterized in the laboratory from dairy formula (Adame-Gómez et al., 2020a).
Biofilm determination was performed in glass and polyethyle- ne tubes, as well as 96-well polystyrene plates. The tubes and plates were filled with 200 μL of Brain Heart Infusion (BHI) broth supplemented with 1% dextrose and inoculated with 20 μL of 24 h liquid cultures of the strains (6x106 UFC/mL). The tubes and plate were incubated at 37°C for 48 h. Later, the cultures were removed from the tubes and plates, which were then washed three times with PBS 1X (137 mM NaCl, 2.7
mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.2). Biofilms
were stained with 200 μL of safranin for 30 min. Then, tubes
and plate were washed three times with PBS 1X and distai- ned with absolute alcohol for 10 minutes. The absorbance of the alcohol from safranin staining was determined at 550 nm (Adame-Gómez et al., 2020a).
The genes involved in biofilm formation were amplified by endpoint PCR using the following oligonucleotides for each of the genes of interest: sipW (sipW- F AGATAATTAGCAACGC- GATCTC, sipW-R AGAAATAGCGGAATAACCAAGC), tasA (tasA- F AGCAGCTTTAGTTGGTGGAG, tasA- R GTAACTTATCGCCTT- GGAATTG), calY (calY-F AGGTATCGGGAGTTCATCAG, calY R CAGCTTCTTGGTTGGCATTG), and eps2 (eps2-F TGTTTTGA- GCGGATTTGTTTTGT, eps2-R GATTGCTCTGCCAATGTCTTT)
(Caro-Astorga et al., 2014; 2020). For each PCR, the mixture contained the following: 25 μL of REDTaq DNA Polymerase Ready Mix (Sigma-Aldrich, St. Louis, MO, USA), 11 μL of sterile MiliQ water, 10 to 20 ng of genomic DNA, and 0.02 μM of each oligonucleotide. The conditions for the sipW, tasA, and calY genes were 1 cycle at 94°C for 5 minutes, 35 cycles at 94°C for 30 seconds, 61°C for 45 seconds, 72°C for 45 seconds, and a cycle of 72°C for 5 minutes. For the eps2 operon, it was one cycle at 94°C for 2 minutes, 35 cycles at 94°C for 30 seconds, 52°C for 30 seconds, 72°C for 90 seconds, and one cycle at 72°C for 10 minutes. The B. cereus ATCC14579 strain was used as a positive control (Cruz-Facundo et al., 2023).
The determination of extracellular enzymes was carried out by inoculating 2 μL of a 24-h culture on different agar plates: BHI with 1% starch agar, 5% casein agar, 5% gelose blood agar, and 10% MYP agar of egg yolk emulsion. The agar pla- tes were incubated at 30°C for 24 h, and the 5% blood agar plates were incubated at 10°C for 10 days. After incubation, 30 μL of lugol were added to demonstrate amylolytic activity on 1% starch agar. For proteolytic and lecitinolytic activity, the presence or absence of hydrolysis zones was observed in 5% casein agar and 10% egg yolk emulsion agar (Claus and Berkeley, 1986). To estimate the lecithinase activity, the ratio was estimated by zone diameter of colony, and zone dia- meter of halo. In the case of psychotropic capacity, growth/ non-growth at 10°C of the strains was monitored for ten days by visual observation, considering the day on which growth was observed as the detection time (DT) (Berthold-Pluta et al., 2019; Cruz-Facundo et al., 2023).
The phylogenetic relationship between the strains of the B. cereus group was determined using the repetitive palindro- mic element PCR technique (rep-PCR) using GTG5 primers (GTGGTGGTGGTGGTG) with the following reaction condi- tions: initial denaturation at 95°C for 2 minutes, 30 cycles of 94°C for 30 seconds, 40°C for 2 minutes, 72°C for 4 minutes and a final extension of 72°C for 5 minutes (De Jonghe et al., 2008). Electrophoresis was performed in 2% agarose gels at 90V for 120 minutes. The gels were stained with Midori Green (Nippon Genetics, Germany) and visualized with LED light.
The DICE similarity coefficient was calculated to establish the genetic distances of the profiles. The genetic distance
matrix was analyzed by the UPGMA method. A dendrogram was made with the analyzed data using NTSYS 2.0 software.
Broth microdilution testing was performed for each strain using Mueller-Hinton, broth in accordance with CLSI gui- deline M45: ED3 (Hindler and Richter, 2016) . A total of ten antibiotics (Oxoid, UK) were tested: ampicillin (0.12 - 16 μg/ mL), ciprofloxacin (0.5 - 4 μg/mL), clindamycin (0.2 - 4 μg/
mL), gentamicin (2 -500 μg/mL), tetracycline (2-16 μg/mL),
trimethoprim (0.5 - 9.5 μg/mL), kanamycin (2-500 μg/mL),
vancomycin (1-64 μg/mL), erythromycin (0.5 - 8 μg/mL) and chloramphenicol (0.5 - 32 μg/mL). The B. cereus s.l. inoculum was prepared from a 24 h culture by first adjusting the cultu- re concentration to 0.5 McFarland of the standard turbidity scale (1 x 108 CFU/mL). The final inoculum concentration in the microplate was 5 x 105 CFU/mL. Each plate included a positive control (MH broth with inoculum without antibiotic) and a negative control (MH broth without inoculum with antibiotic). The microplates were incubated at 30°C for 24 h. Ten μL of a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide) solution were added to verify growth and incubated for 30 min at 30°C in the dark. Formazan pro- duction indicated the presence of viable cells.
In Mexico, the annual per capita consumption of rice is 8.5 kg, considered the second crop with the highest expenditure among mexican families (SAGARPA, 2017). However, B. cereus, a pathogen associated with foodborne diseases worldwide, has been isolated in rice (Dietrich et al., 2021; Enosi Tuipulotu et al., 2021). Rice and its derivatives serve as an ideal growth medium for bacteria, particularly B. cereus, due to their com- positional and chemical characteristics (pH close to 7, appro- ximately 79% carbohydrates, 7% protein, and 2% fat). These properties, combined with the resilience of B. cereus spores, which can survive in dehydrated grains and withstand the temperatures typically applied during processing, enable the bacterium to proliferate under a variety of conditions (Jaquette and Beuchat, 1998). Therefore, in this study, strains of B. cereus s.l. were characterized and isolated from different rice-based preparations.
In the different studies of B. cereus in rice, strains with the same toxigenic profiles have not been isolated, and even the frequencies change depending on the type of rice and country (Ankolekar et al., 2009; Kim et al., 2009; 2014; Park et al., 2009), which is why studying the diversity of B. cereus stra- ins in Mexico is essential. A total of six strains of the B. cereus
s.l. group previously isolated from rice were characterized. All these strains were positive for the non-hemolytic toxin gene (nhe) and negative for the cereulide toxin gene (ces). Only one strain (B629) was positive for the hemolytic toxin BL gene (hbl) and negative for the cytotoxin K gene (cytK) (Table 1) (Figure 1).
In this study, a remarkable occurrence of the non- hemolytic toxin gene (nheABC) with a 100% frequency was
Table 1. Toxin-coding genes identified in the B. cereus s.l. strains from this study.
Tabla 1. Genes de las toxinas identificadas en las cepas de B. cereus s.l. de este estudio.
Strain | hbl | nhe | cytK | ces |
B629 | + | + | - | - |
B630 | - | + | + | - |
B631 | - | + | + | - |
B632 | - | + | + | - |
B633 | - | + | + | - |
B634 | - | + | + | - |
identified in the analyzed strains. Globally, non-hemolytic toxin genes are frequently reported, often reaching values of up to 100%. In Mexico, several food products, including cheese, ice cream, eggs, and vegetables, have shown a high prevalence of non-hemolytic toxin genes (Adame-Gomez et al., 2019; 2020b; Castulo-Arcos et al., 2022; Cruz-Facundo et al., 2022; 2023); it is important to note that these findings do not exclude the possibility that these strains are circulating in rice, in which, the frequency of non-hemolytic toxin genes show a range from 47.3% to 100%. The variability in these results is suggested to be related to the specific type of rice analyzed. For example, in ready-to-eat rice-based foods, a frequency of 47.3% was observed (Chen et al., 2022) while frequencies approaching 100% were identified in different types of rice, including white rice, brown rice, black rice, hard rice, and glutinous rice (Ankolekar et al., 2009; Kim et al., 2009; 2014; Park et al., 2009).
The frequencies of Hbl toxin genes were low (16.6%) compared to the global reported frequencies for these toxin genes (40-70%) (Jessberger et al., 2020). As for Mexico, the frequencies are low (Adame-Gomez et al., 2019; 2020b; Castulo-Arcos et al., 2022; Cruz-Facundo et al., 2022; 2023) as in this study, which does not rule out a regional circulation of B. cereus strains in different food products. Regarding rice,
the reported frequencies of Hbl toxin genes are high compa- red to this study. The lowest frequencies for the Hbl toxin are found in strains isolated from rice-based ready-to-eat foods (36.3 %), and the highest frequencies are found in white and brown rice (83-100%) in China and Korea. In the United States, even when it was isolated from white and brown rice, the frequency of the Hbl toxin was low (56.6%), so it is not surprising that not only the type of product, but also the geo- graphic region, is important in the frequency of enterotoxins. In this sense, the regionalization of diarrheal and emetic syndromes with the dietary habits of each country has even been described (Enosi Tuipulotu et al., 2021; Kotiranta et al., 2000).
Another virulence factor, in addition to spore formation and toxin production by B. cereus, is the ability to form bio- films. Biofilms are bacterial communities that, in the case of B. cereus, have been described in food production areas, beco- ming a problem because, on one hand, they favor systematic contamination of food. On the other hand, the formation of biofilms prevents the eradication of the microorganisms trapped in it due to the characteristics of resistance to disin- fectant agents (Majed et al., 2016).
Regarding biofilm formation, strains B631, B632, B633, and B634 exhibit substantial biofilm production on glass material in contrast to strain B629 (p = 0.0211, p = 0.0445, p = 0.0211, p = 0.0216). Notably, three strains (B632, B633, B634) show the ability to form biofilms on polystyrene. It is noteworthy that none of the strains form biofilms on po- lyethylene as shown in Figure 2.
In this investigation, prolific biofilm production was observed exclusively on glass material, in contrast to mi- nimal production on polyethylene and, to a lesser extent, polystyrene. The variation in biofilm production on different materials is consistent with previous literature which attri- bute such differences to the hydrophobic properties and
Figure 1. Gel electrophoresis of PCR products from toxin profile of B. cereus s.l. A) nheABC, B) hblABD, C) plcr- cytK. D) ces. Lane 1. B634, Lane 2. B633, Lane 3. B632, Lane 4. B631, Lane 5. B630, Lane 6. B629, Lane 7. Negative control, Lane 8. Positive control B. cereus ATCC 14579 (hbl+, plcr- cytK+).
Figura 1. Electroforesis en gel de los productos de PCR del perfil de toxinas de B. cereus s.l. A) nheABC, B) hblABD, C) plcr- cytK. D) ces. Carril 1. B634, Carril 2. B633, Carril 3. B632, Carril 4. B631, Carril 5. B630, Carril 6. B629, Carril 7. Control negativo, Carril 8. Control positivo B. cereus ATCC 14579 (hbl+, plcr- cytK+).
Figure 2. Biofilm production of B. cereus s.l. strains in different materials to 37°C for 48h. Safranin absorbance data from two independent assays are shown, represented as mean and standard deviation. The p values were cal- culated from the ANOVA statistical test with Tukey posthoc.
Figura 2. Producción de biofilm de las cepas de B. cereus s.l. en diferentes materiales a 37°C por 48h. Se muestran los datos de absorbancia de safra- nina de dos ensayos diferentes, representados como media y desviación es- tándar. El valor de p fue calculado por la prueba estadística de ANOVA con posthoc de Tukey.
surface roughness of the materials(Adame-Gómez et al., 2020a; De-la-Pinta et al., 2019). In addition, the presence of trace elements, particularly iron, has been implicated in bio- film production. Hayrapetyan et al. (2015) reported increased biofilm production on stainless steel, attributed to iron ions, compared to polystyrene. Within our research group, previous studies have indicated higher biofilm production on PVC compared to other materials (Adame-Gómez et al., 2020a; Cruz-Facundo et al., 2022). It’s worth noting that dis- crepancies between our results and those of this study may
be related to the different origins of the strains. Hayrapetyan et al. (2016) relate the origin of the isolation to the adaptation capacity of the strains to environments with different iron conditions.
Regarding the genes related to biofilm production, only strain B629 has the eps2 operon. All strains have the sipW- tasA-calY operon genes (Figure 3). In B. cereus, the participa- tion of different genes in the production of biofilms has been described; these genes are grouped in the sipW-tasA-calY and eps2 operons (Caro-Astorga et al., 2014; 2020). SipW is a peptidase that participates in the maturation of the tasA pro- tein, which functions in the formation of amyloid-type fibers, which are part of a mature biofilm (Caro-Astorga et al., 2014). CalY fulfills a dual function during biofilm production, par- ticipating as an adhesin on glass but not polystyrene in the early stages of biofilm formation and forming fibers during the late phases (Candela et al., 2019). Eps2 is an operon that include genes related to the extracellular matrix, the main component of biofilms (Caro-Astorga et al., 2020). The diverse functions these proteins fulfill explain why most of the stra- ins in this study produce abundant biofilms on glass, except for strain B629, which, despite having all the genes related to biofilms, visually produces a biofilm that easily detaches from the air-liquid interface and therefore is not evident by safranin staining. Dogsa et al. (2013) showed that the width of the biofilm is related to the culture medium and to the presence of the tasA and epsA genes. The absence of one of these genes drastically reduces the thickness of the biofilm, and in the absence of both, the strain does not produce a biofilm. For B629, the production of a thin biofilm could be explained by presence of a non-functional tasA gene, which is reflected in a thin biofilm that easily detaches from the tube. Despite the detailed information about the participation of
Figure 3. Genes related to biofilm production. A) sipW, B) tasA, C) calY, D) eps2. Lane 1. B634, Lane 2. B633, Lane 3. B632, Lane 4. B631, Lane 5. B630, Lane 6. B629, Lane 7. Negative control, Lane 8. Positive B. cereus ATCC 14579 control.
Figura 3. Genes relacionados a la producción de biopelículas. A) sipW, B) tasA, C) calY, D) eps2. Carril 1. B634, Carril 2. B633, Carril 3. B632, Carril 4. B631, Carril 5. B630, Carril 6. B629, Carril 7. Control negativo, Carril 8. Control positivo B. cereus ATCC 14579.
the sipW-tasA-calY and eps2 operons in biofilm production (Caro-Astorga et al., 2020; 2014), few epidemiological studies include them in molecular characterization (Cruz-Facundo et al., 2023). Since biofilm production is part of the resistance mechanisms and systematic contamination of food products (Enosi Tuipulotu et al., 2021), not only enterotoxigenic and enzymatic profiles should be considered, but also those ge- netic profiles associated with biofilm production, which are included in this study.
B. cereus not only produces enterotoxins, but also a significant number of extracellular enzymes with degrada- tive activity, such as phospholipases, proteases, chitinases, and amylases (Ivanova et al., 2003). This enzymatic profile is related to the ability of B. cereus to spoilage food and as a nutrient assimilation mechanism (Arslan et al., 2014). In this sense, the B629 strain is the only amylolytic strain in the study, and coincides with being the only strain isolated from fried rice. Strains isolated from sweet rice are capable of pro- ducing proteases, which can have a significant impact on the decomposition of the product, since it has been described that proteases are capable of gelatinizing milk (Chen et al., 2003). All strains produce proteases and strains in different proportions produce lecithinase. Only one strain can grow at refrigeration temperatures (B629) (Table 2). In this sense,
Table 2. Lytic enzymes and cold tolerance of B. cereus s.l. strains.
Strain Amylase Protease
B629 + +
B630 - +
B631 - +
B632 - +
B633 - +
Lecithinase
0.71
0.52
0.60
0.50
0.50
Growth in low
t T)
emperatures (D
5
-
-
-
-
B634 - + 0.60 -
Tabla 2. Enzimas líticas y tolerancia al frío de las cepas de B. cereus s.l.
Detection time (DT): the day on which growth was observed Tiempo de detección (DT): El día en donde se observa crecimiento
strains of B. cereus are divided into two groups: psychotro- phic and mesophilic. In this study, it is reported that strain B629 is a psychotrophic strain capable of growing in 5 days at a temperature of 10°C, and that the other strains, since they cannot grow at refrigeration temperatures, could be consi- dered mesophilic strains. The presence of psychrotrophic strains, such as B629, impact the food safety of food products that are preserved in the cold, including rice.
Genetic diversity techniques, such as GTG’5, have allowed strains to be grouped according to different characte- ristics, including growth temperature. Regarding the genetic diversity of the B. cereus s.l. group, strains were grouped into three clusters. In the first cluster, two clones were identified; the first clone includes strain B629 isolated from fried rice, the only strain that grows at refrigeration temperatures and is amylase positive, and is also the only one positive for the toxin BL gene (hbl); the second clone includes strains B632 and B633, which share most of the characteristics with the clones belonging to the other clusters (Figure 4).
The GTG’5 technique allowed the identification of two clones, one with tolerance to refrigeration temperatures, amylolytic, not a biofilm producer, and isolated from fried rice. Furthermore, the technique allowed us to separate this clone from three remaining clones isolated from sweet rice with similar genotypic characteristics, including the profile of toxins and genes associated with biofilm production. This is evidence that the strains circulating in fried rice are different from the strains found in sweet rice, which allows us to esti- mate that the risk of poisoning also depends on the product consumed. For example, emetic strains have been linked to the consumption of products such as pasta and rice, while the consumption of meat and vegetables has been linked to enterotoxigenic strains (Enosi Tuipulotu et al., 2021).
Regarding antimicrobial resistance, it was found that all strains are resistant to penicillin, ceftriaxone, clindamycin, and trimethoprim. The strains were susceptible to kanamycin,
Figure 4. Dendrogram obtained by GTG-5’ of B. cereus s.l. strains. Six gyrB-positive rice strains were grouped, three clusters and four clones. The DICE test was used, and the strains were grouped by hierarchical cluster. A DICE coefficient >0.8 was used to cluster the strains. FR: Fried rice. SR: Sweet rice.
Figura 4. Dendrograma obtenido por GTG-5’ de las cepas de B. cereus s.l. Se agruparon seis cepas positivas para gyrB, tres grupos y
cuatro clones. Se utilizó la prueba DICE y las cepas fueron agrupados por grupo jerárquico. Un coeficiente DICE >0.8 fue usado para agrupar las cepas. FR: Arroz frito. SR: Arroz dulce.
gentamicin, vancomycin, tetracycline, chloramphenicol, and ciprofloxacin. It is important to note that only B629 has inter- mediate sensitivity to erythromycin (Table 3).
Regarding antibiotic resistance, strains of B. cereus s.l. from this study were resistant to beta-lactams, which has been reported in other studies (Chen et al., 2022; Fraccal- vieri et al., 2022; Park et al., 2009; Perera and Ranasinghe, 2012). Fraccalvieri et al. (2022) demonstrated that the main mechanism of resistance to beta-lactams is mediated by the beta-lactamases BLA-1 and BLA-2, since all their beta-lactam resistant strains presented these genes. In addition to resis- tance to beta-lactams, high resistance to trimethoprim has also been reported in other studies in strains of B. cereus s.l. isolated from rice and cheese (Chen et al., 2022; Cruz-Facundo et al., 2023). It is important to mention the reports related to the high susceptibility to different groups of antimicrobials such as aminoglycosides, glycopeptides, tetracyclines, and phenicol’s (Chen et al., 2022; Cruz-Facundo et al., 2023; Park et al., 2009) which is also consistent with what was reported in these strains. However, the presence of a strain with interme- diate sensitivity to erythromycin in this study (B629) should not be underestimated.
Table 3. Antimicrobial resistance of B. cereus s.l. strains from rice.
Tabla 3. Resistencia a antimicrobianos de las cepas de B. cereus s.l. obtenidas de arroz.
Antibiotic | Sensible (≤ 2g/ mL) | Intermediate (>2- <16 g/ mL) | Resistant (≥ 16 g/ mL) |
Penicillin | 6 (100%) | ||
Ceftriaxone | 6 (100%) | ||
Erythromycin | 5 (83.4%) | 1 (16.6%) | |
Clindamycin | 6 (100%) | ||
Ciprofloxacin | 6 (100%) | ||
Tetracycline | 6 (100%) | ||
Chloramphenicol | 6 (100%) | ||
Vancomycin | 6 (100%) | ||
Gentamycin | 6 (100%) | ||
Kanamycin Trimethoprim | 6 (100%) | 6 (100%) |
The Bacillus cereus sensu lato (B. cereus s.l.) strains isolated from rice exhibit enterotoxigenic profiles, indicating their potential to cause food poisoning. Of particular note is the finding that strains isolated from sweet rice have a propensity to form biofilms on glass, and exhibit a distinct enterotoxige- nic profile compared to the strain isolated from fried rice. The unique characteristics identified in the strain isolated from fried rice underscore the importance of further investigation and warrant an expansion of the sample size, specifically within this rice type. This expansion aims to systematically explore the presence of strains with similar metabolic and virulence characteristics.
We are grateful to the General Directorate of Postgraduate and Research of the Universidad Autónoma of Guerrero,
particularly to PhD. Berenice Illades Aguiar for promoting the summers of scientific research and facilitating the incorpo- ration of Mariela Cano Ponce to the laboratory. Luis Daniel Sánchez Arcos for graphical abstract author.
The author(s) declare(s) that there is no conflict of interest regarding the publication of this paper
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