The potential presence of spore-forming bacteria related to the Bacillus cereus group in Mexican chili powder elaborated from Capsicum annuum L. is of commercial and clinical interest, because chili powder is an essential spice in the Mexican diet and in diets around the globe. To facilitate detection and isolation of members of this group of spore-forming bacteria from Mexican chili powder samples, we identified colonies that grew on agar medium selective for Bacillus cereus sensu lato, supplemented with polymyxin B (10 µg/mL) and ampicillin (10 to 100 µg/mL). The presumptive B. cereus (s.l.) isolates were tested using a tRNACys-PCR-based approach and the results identified species related phylogenetically to B. cereus, B. thuringiensis, and B. toyonensis. Their toxigenic potential was assessed by serological tests to detect enterotoxins (Nhe and Hbl) and by PCR targeting the hemolysin BL (hbl) component C (hblC) and non-hemolytic enterotoxin component A (nheA). The antibiotic profiles of the isolates showed a high resistance to β-lactams (100% of the isolates), trimethoprim-sulfamethoxazole (100%), tetracycline (90%), erythromycin (77%), clindamycin (74%), and chloramphenicol (42%). Our results indicate the presence of B. cereus s.l. with toxigenic characteristics in Mexican chili powder. Because of the potential for these organisms to cause disease through their production of various toxins, and resistance to antibiotics, we recommend that a microbiological risk assessment must be considered in the Mexican regulatory requirements.
Endospore-forming bacteria related to the Bacillus cereus group produce toxins that cause illnesses in organisms from invertebrates to mammals, including foodborne illnesses in humans. As commercial bee pollen can be contaminated with these bacteria, a comprehensive microbiological risk assessment of commercial bee pollen must be incorporated into the relevant regulatory requirements, including those that apply in Mexico. To facilitate detection of members of this group of bacteria, we have developed a PCR strategy that is based on the amplification of the single-copy tRNACys gene and specific genes associated with tRNACys to detect Bacillus cereus sensu lato (B. cereus s.l.). This tRNACys-PCR-based approach was used to examine commercial bee pollen for endospore-forming bacteria. Our analysis revealed that 3% of the endospore-forming colonies isolated from a commercial source of bee pollen were related to B. cereus s.l., and this result was corroborated by phylogenetic analysis, bacterial identification via MALDI-TOF MS, and detection of enterotoxin genes encoding the HBL and NHE complexes. The results show that the isolated colonies are closely related phylogenetically to B. cereus, B. thuringiensis, and B. bombysepticus. Our results indicate that the tRNACys-PCR, combined with other molecular tools, will be a useful approach for identifying B. cereus s.l. and will assist in controlling the spread of potential pathogens.
American foulbrood (AFB) caused by Paenibacillus larvae is the most destructive honeybee bacterial disease and its dissemination via commercial bee pollen is an important mechanism for the spread of this bacterium. Because Mexico imports bee pollen from several countries, we developed a tRNA Cys-PCR strategy and complemented that strategy with MALDI-TOF MS and amplicon-16S rRNA gene analysis to evaluate the presence of P. larvae in pollen samples. P. larvae was not detected when the tRNA Cys-PCR approach was applied to spore-forming bacterial colonies obtained from three different locations and this result was validated by bacterial identification via MALDI-TOF MS. The genera identified in the latter analysis were Bacillus (fourteen species) and Paenibacillus (six) species. However, amplicon-16S rRNA gene analysis for taxonomic composition revealed a low presence of Paenibacillaceae with 0.3 to 16.2% of relative abundance in the commercial pollen samples analyzed. Within this family, P. larvae accounted for 0.01% of the bacterial species present in one sample. Our results indicate that the tRNA Cys-PCR, combined with other molecular tools, will be a useful approach for identifying P. larvae in pollen samples and will assist in controlling the spread of the pathogen.
Chili powder is the most frequently consumed spice in Mexican diets. Thus, the dissemination of microorganisms associated with chili powder derived from Capsicum annuum L. is significant during microbial quality analysis, with special attention on detection of potential pathogens. The results presented here describe the initial characterization of bacterial community structure in commercial chili powder samples. Our results demonstrate that, within the domain Bacteria, the most abundant family was Bacillaceae, with a relative abundance of 99% in 71.4% of chili powder samples, while 28.6% of samples showed an average relative abundance of 60% for the Enterobacteriaceae family. Bacterial load for aerobic mesophilic bacteria (AMB) ranged from 104 to 106 cfu/g, while for sporulated mesophilic bacteria (SMB), the count ranged from 102 to 105 cfu/g. Bacillus cereus sensu lato (s.l.) was observed at ca. ˂600 cfu/g, while the count for Enterobacteriaceae ranged from 103 to 106 cfu/g, Escherichia coli and Salmonella were not detected. Fungal and yeast counts ranged from 102 to 105 cfu/g. Further analysis of the opportunistic pathogens isolated, such as B. cereus s.l. and Kosakonia cowanii, using antibiotic-resistance profiles and toxinogenic characteristics, revealed the presence of extended-spectrum β-lactamases (ESBLs) and Metallo-β-lactamases (MBLs) in these organisms. These results extend our knowledge of bacterial diversity and the presence of opportunistic pathogens associated with Mexican chili powder and highlight the potential health risks posed by its use through the spread of antibiotic-resistance and the production of various toxins. Our findings may be useful in developing procedures for microbial control during chili powder production.
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