qPCR detection of viable Bacillus cereus group cells in cosmetic products

Abstract

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 from dead and viable cells. DNA intercalating dyes, such as propidium monoazide (PMAxx), are capable of restricting PCR amplification to viable microbial cells. Here we developed singleplex and multiplex real time (qPCR) assays for the detection of Bacillus cereus (B. cereus) using 16S rRNA and phosphatidylcholine-specific phospholipase C (PLC) gene specific sequences coupled with PMAxx. The limit of detection 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: one set of liquid face toners (N = 3), artificially contaminated with B. cereus 3A, and one set of powdered cosmetics (N = 8), previously determined to be contaminated with B. cereus. For some samples, test portions were analyzed by qPCR in parallel, with and without PMAxx 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 PMAxx -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 PMAxx in the PCR assays. Nevertheless, the amplification threshold with PMAxx dyes was significantly higher than the non-PMAxx dyes. Our findings confirm qPCR can be used for more rapid detection of microorganisms in cosmetics, including B. cereus, and selective detection of viable cells can be improved using PMAxx dyes.

Figure 1

Overview of the workflow of the PMAxx-treated vs non-treated samples. MLB modified letheen broth, PMAxx modified propidium monoazide.

Figure 2

Tenfold serial dilution multiplex amplification of 16S rRNA and PLC of B. cereus 3A The Blue line for the reporter FAM represents 16S rRNA from d0 to d6 corresponding C_T values of 21.28, 24.95, 27.99, 31.99, 35.09, 36.23 and undetermined. The Fuchsia line tracks the passive reference dye, Mustang Purple providing an internal reference for normalizing the reporter dye signal during analysis. The Pink line for the reporter JUN represents PLC from d0 to d4 with C_T values of 26.29, 29.90, 32.90, 36.98; dilutions 5 and 6 were undetermined. The Green line for the reporter VIC represents the Internal Positive Control amplification used to ensure the quality of the reagents and the instrument. The Yellow line represents the quencher TAMRA. PLC phosphatidylcholine-specific phospholipase C.

Figure 3

Laser Scanning Confocal microscopy imaging of High-level inoculated B. cereus cells in the liquid-type cosmetic aged for 14 days and then stained with Live/Dead dye. Live cells are green and dead/injured cells fluoresce red. (A): DIC, Differential interference contrast; (B): green channel; (C): red channel; (D): all channels.

Figure 4

Detection of B. cereus on BACARA plates in powder-type cosmetics. 100 μl of the initial dilution of products (C1, C-2, C-3, C-4, C-5, RP, O-1, 0–2, and GC) spread on MLA and BACARA plates in duplicate (top 4 plates in each image), and a loop streaked on a plate of BACARA after 24 h enrichment (fifth plate at the bottom of each set). (A) positive (B). cereus result on BACARA shows as orange colonies with a white halo.