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Detection of Viable <i>Bacillus Cereus</i> Group Cells in Cosmetic Products Using qPCR

2022; RELX Group (Netherlands); Linguagem: Inglês

10.2139/ssrn.4098431

1556-5068

Irene Tchagou, Son Minh Hoang, Travis Canida, Rachel Binet, Dumitru Macarisin, Rebecca Bell, Sandra M. Tallent, Eric W. Brown, Thomas S. Hammack,

Identification and Quantification in Food

Reference methods for microbiological safety assessments of cosmetics rely on culture methods that reveal colonies of live microorganisms on growth media. Rapid molecular technologies, such as qPCR, detects the presence of target DNA in samples, but the DNA may be derived from dead cells which may cause false positive results. DNA intercalating dyes, propidium monoazide (PMAxx TM), are capable of restricting PCR amplification to viable microbial cells by preferentially penetrating the damaged cell membranes of non-viable bacteria and preventing PCR amplification. DNA intercalating dyes have not yet been used for assessing cosmetics. Here we developed singleplex and multiplex real time (qPCR) assays for the detection of Bacillus cereus (B. cereus) using previously established 16S rRNA and phosphatidylcholine-specific phospholipase C (PLC) gene specific sequences and PMAxx TM to reduce the amplification of signal from dead cells. The limit of detection (LOD) was determined to be ~ 1 log CFU/ml for 16S rRNA and 3 log CFU/ml for PLC detection in pure culture using an eye shadow isolate, B. cereus 3A. We assessed the inclusivity and exclusivity of our qPCR assays using 212 strains, including 143 members of B. cereus, 38 non- B. cereus. and 31 non- Bacillus species; inclusivity was 100% for the 16S rRNA and 97.9% for the PLC targets; the exclusivity was 100% for 16S rRNA and 98.6 % for PLC targets. These qPCR assays were then used to assess samples of commercial cosmetics for the presence of viable B. cereus cells: one set of liquid face toners (N = 3), artificially contaminated with B. cereus 3A , and one set of powdered cosmetics (N = 8), which were previously determined to be contaminated with B. cereus. For some samples, test portions were analyzed by qPCR in parallel, with and without PMAxx TM treatment. All test portions were simultaneously streaked on BACARA plates to confirm viable cells of B. cereus, according to the culture method. We found no difference in sensitivity between the singleplex and the multiplex qPCR assays (P > 0.05). Inoculated samples that did not recover B. cereus on plates still showed amplification of the DNA targets. However, that amplification was significantly delayed in PMAxxTM –treated samples (P < 0.0001) with C T value differences of 7.82 for 16S rRNA and 7.22 for PLC. Likewise, amplification delay was significant (P < 0.0001) with inoculated samples that recovered B. cereus on plates with C T value differences of 2.96 and 2.36 for 16S rRNA and PLC, respectively, demonstrating the presence of dead cells in the samples. All our qPCR results correlated with detection on BACARA plates (kappa, k = 0.99), independently of the presence of PMAxxTM in the PCR assays. Nevertheless, the amplification threshold with PMAxxTM dyes was significantly higher than the non-PMAxxTM dyes. Our findings confirm qPCR can be used for more rapid detection of microorganisms in cosmetics, including B. cereus and that selective detection of viable cells can be improved using PMAxxTM dyes.