qPCR detection of viable Bacillus cereus group cells in cosmetic products

Bacterial strains and preparation

The 212 bacterial strains used in this study were obtained from cosmetics, food, environmental sources, as well as from the American Type Culture Collection (Table 1).

All 212 strains used in this study were maintained at − 80ºC in broth supplemented with 20% glycerol. Each strain was aseptically streaked onto Tryptic soy agar (TSA) (Difco™, Franklin Lakes, NJ) and incubated for 24 h at 30ºC, from which an isolated colony was sub-cultured in Nutrient Broth (NB, pH 7.2) (Difco™), then incubated at 30ºC for 24 h.

Inclusivity testing was performed using 143 members of the B. cereus group. One of these strains, B. cereus “3A”, previously obtained from eye shadow (Yossa and Jo Huang, personal communication), was used to establish the limit of detection (LOD) for our assays. Exclusivity testing was performed using a panel of 69 strains: 38 non-cereus strains of Bacillus, and 31 strains of non-Bacillus bacteria.

Cosmetic products

There were two types of cosmetics used in this study: liquid and powder. To assess how well our assays could detect the presence of B. cereus 3A in artificially contaminated samples of liquid-type cosmetics, we purchased facial toner products [N = 3], all of which were labeled “alcohol free”. These facial toners were primarily composed of water and plant-derived compounds and preserved with phenoxyethanol, an antibacterial agent.

To assess the performance of our assays on powder-type cosmetics, we selected 8 cosmetic products: Green Clay (GC), Pink Clay (C1–4), Rice Powder (RP), and Tattoo Powder (O1–2), which had been purchased from a retail establishment online. The two clay products contained no preservatives, the rice powder product contained phenoxyethanol, and no information was available about any preservative in the tattoo product. Prior research had determined 6 powder-based products to be contaminated with B. cereus (Yossa and Jo Huang, personal communication); therefore, we classified these as “naturally-contaminated” and did not add any additional bacteria to these powders.

DNA extractions

Inclusivity, exclusivity, limits of detection

We isolated DNA from overnight cultures of each pure bacterial strain using the MagMAX™ Express 96 Magnetic Particle Processor (ThermoFisher) with PrepSEQ Nucleic Acid Extraction Kit for Food and Environment (ThermoFisher, P/N 4,428,176) using protocol PrepSeq_ResDNA_20011 (Life Technologies, Carlsbad, CA). Total DNA extracted from these pure cultures were used to evaluate the inclusivity (n = 143) and exclusivity (n = 69) of the qPCR assays.

To determine the limit of detection of our qPCR assays, we used DNA extracted from the B. cereus 3A strain, which was grown overnight in NB and adjusted to a density of 0.5 ± 0.05 MacFarland (McF; ~ 6.7 log CFU/ml).

DNA extractions from cosmetics

Two different extraction procedures were used to perform our qPCR assays on the sets of contaminated cosmetics, due to differences in the matrices. The MagMAX™ and PrepSeq kits were only appropriate for liquid cosmetics. Effective DNA extraction from the powder products required using the DNeasy® PowerPlant® Pro kit (Qiagen, Catalog Number 69204) instead, because powder products become thick and dense in the presence of the proteinase/proteinase DNA extraction buffer used in the MagMAX and PrepSeq kits²⁷.

Primers/probes

Table 2 shows the primers we used and the associated publications documenting their first use. These were purchased from Life Technologies (ThermoFisher).

The 16S rRNA sequences (P/N 4,331,348, Life Technologies) used here had previously been determined by De Clerck, et al.; their team targeted the 5’ hypervariable fragment, which can be amplified using a universal forward and a B. cereus-specific reverse primer²⁸. Our TaqMan probe was labeled with 6 -carboxyfluorescein (FAM) reporter dye at the 5’ end and labeled at the 3’-end with a Black Hole quencher, BHQ1, to reduce background fluorescence²⁹. We ordered the PLC primer probe (CCU001SNR, Life Technologies) labeled with a JUN reporter dye at the 5’-end and a non-fluorescent quencher at the 3’-end, to facilitate multiplex qPCR using 4 dyes. QSY PLC- JUN probe is a custom probe, compatible with the TaqMan Multiplex Master Mix (P/N 446,188; Applied Biosystems, Life Technologies) and ordered through Life Technologies.

The TaqMan Internal Positive Control (IPC) from Life Technologies (P/N 4,308,323) was included to monitor the PCR progress and ensure that a negative result is not caused by failed PCR in the sample³⁰ and used with the TaqMan Multiplex Master Mix to amplify both the single target and the multiple target reactions.

qPCR reactions of non-treated and PMAxx -treated samples

PMAxx -PCR is an innovative technology that allows differentiation between live and dead microorganisms, based on the loss of cell membrane integrity in dead cells³¹. This system uses a DNA-intercalating dye, PMAxx, that disrupts DNA transcription only in dead cells, as their damaged cell membranes permit entry of the dye. After photoactivation with a defined wavelength, PMAxx intercalates and binds covalently to DNA. Subsequent amplification of that modified DNA is inhibited, thereby reducing the amplification signal from dead/damaged cells in comparison to that from live cells.

For each qPCR reaction, a 20 μl volume consisting of 10 μl Multiplex Master Mix, 2 μl 10X Exo IPC Mix, 0.4 μl 50X EXO IPC DNA, 1 μl of primer assay, 2 μl of sample/template DNA, and 4.6 μl of sterile deionized water was placed into a 96 well fast plate. Both primers for the multiplex PCR were combined to reach a final concentration representing 5% of the total reaction volume. These qPCR runs were performed on a 7500 Fast qPCR System (Applied Biosystems) under the following conditions: 2 min at 50 °C, then 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C.

B. cereus 3A testing in artificially contaminated liquid products

Here we used 3 different cosmetic toner products, which we artificially contaminated with B. cereus 3A. For each trial (N = 3), 3 sterile Wheaton bottles (125 ml) were filled with 30 ml of facial toner and were repeatedly spiked with 300 μl of microbial suspension, containing either a High (~ 4 or 3 log CFU/ml) or Low (~ 3 or 2 log CFU/ml) level of B. cereus 3A vegetative cells (optical density of 0.5 ± 0.05 McF), over three consecutive days, and the 3rd bottle of sample remained un-spiked for the negative controls. These repeated inoculations were necessary to enable recovery of B. cereus from products containing preservatives³². To mimic a product contamination event more closely, these inoculated toner samples, along with the un-spiked negative control, were aged for 14 days at room temperature. After 14 days of aging, the un-inoculated and inoculated samples from the High- and Low-level inoculation were gently separately mixed, then 1 ml aliquots were diluted with 9 ml MLB (Difco).

Our first goal was to achieve fractional results, which in this case would consist of 5 replicates at the high level of inoculation all yielding positive results, while 20 replicates at the low level of inoculation would yield only 50 ± 25% positive results, and 5 replicates of un-inoculated sample all yielding negative results³³. Based on this, 5 replicates of toner from the High level, 20 replicates from the Low level, and 5 replicates from the uninoculated samples (negative controls) were pre-enriched for 24 h at 30 °C in MLB, streaked onto BACARA plates and processed simultaneously for molecular analysis. Two equal sets of test portions were taken at the same time in all the replicates and placed into vials, and the first set of test portions proceeded to genomic DNAs extraction without any treatment. From the second set, 1 ml of each test portion was supplemented with 25 µM PMAxx, mixed through tap-spin, and allowed to incubate at room temperature for 10 min in the dark before exposure on the PMA-Lite (Biotium) for 15 min to crosslink free DNA³⁴. Immediately following PMAxx treatment, genomic DNAs were extracted, and all genomics (non-treated and treated) were analyzed independently in duplicate using singleplex and multiplex qPCR assays. Figure 1 underlines an overview of the workflow of this portion of our study.

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

Microscopic observation of live/dead B. cereus cells aged for 14 days in toner-samples using BacLight kit

To provide a microscopic perspective on the ratio of live/dead cells in the inoculated toner samples, 1 ml from the 14-day aged samples inoculated with B. cereus at the high level was centrifuged at 10,000 × g for 10 min. The supernatant was removed, and the pellet was resuspended in 1 ml of 0.85% sodium chloride (NaCl) and kept at room temperature for 1 h, mixing every 15 min. After centrifugation at 10 000 × g for 10 min, the cells were resuspended in NaCl and stained with the Live/Dead™ Bacterial Viability kit (Cat: L7007, Invitrogen by ThermoFisher Scientific) according to manufacturer’s protocols³⁵. Stained cells were viewed under a Zeiss 880 Laser Scanning Microscope (LSM, Carl Zeiss Microscopy, LLC, White Plains, NY). The cells were observed using a Zeiss Axio Observer inverted microscope with × 63 1.4 NA oil immersion plan apochromatic objective. Differential interference contrast (DIC) and confocal fluorescence images were acquired simultaneously. A photomultiplier tube captured the light emitted from a 488-nm argon laser with a 3.7-m pin hole passing through an MBS 488 filter with limits set between 472 and 562 nm for detection of SITO 9 stains for green fluorescence of live cells and between 597 and 669 nm for detection of propidium iodide stains for red fluorescence of bacterial cells with damaged membrane. Zeiss Zen Black software was used to obtain the images with 1024 × 1024-pixel resolution.

B. cereus testing in powder-type cosmetic products

Each of the 8 powder-types cosmetic products (GC, C-1, C-2, C-3, C-4, O-1, O-2 and RP) was aseptically homogenized, and 1 g was added to 1 ml of Tween 80, before mixing with 8 ml of MLB broth in presence of 10 sterile glass beads to ameliorate homogenization. These samples were diluted, and the 10⁻¹–10⁻² dilutions were spread on MLA and BACARA plates, before enrichment at 30 °C for 24 h. Spread plating was used instead of spiral plating, to prevent powder particles from clogging the stylus of the spiral plater. All these enriched samples were streaked onto BACARA plates to screen for B. cereus presence, according to BAM Chapter 23¹ and processed simultaneously to extract bacterial DNA for B. cereus qPCR analysis, in duplicate. This will permit cross-method comparisons.

Statistical analysis

Data were analyzed using a linear mixed effects model and p-values were adjusted to account for multiple comparisons. Cutoff points were established using a logistic regression model, such that the cutoff Ct value gives a > 50% chance of a sample being negative. Inclusivity and exclusivity cutoff values were established using the upper bound of the 95% confidence interval of the 95th quantile estimate³⁶. The linear mixed effects model was fit using the nlme package version 3.1–145³⁷. Quantile regression was done using the quantreg package version 5.55. All other analyses were done using R version 3.6.0³⁸.

Ethics declarations

This research does not involve human, animal, seed or plant samples.