qPCR detection of viable Bacillus cereus group cells in beauty merchandise

Bacterial strains and preparation

The 212 bacterial strains used on this examine had been obtained from cosmetics, meals, environmental sources, in addition to from the American Kind Tradition Assortment (Desk 1).

All 212 strains used on this examine had been maintained at − 80ºC in broth supplemented with 20% glycerol. Every pressure was aseptically streaked onto Tryptic soy agar (TSA) (Difco™, Franklin Lakes, NJ) and incubated for twenty-four h at 30ºC, from which an remoted colony was sub-cultured in Nutrient Broth (NB, pH 7.2) (Difco™), then incubated at 30ºC for twenty-four h.

Inclusivity testing was carried out utilizing 143 members of the B. cereus group. Considered one of these strains, B. cereus “3A”, beforehand obtained from eye shadow (Yossa and Jo Huang, private communication), was used to ascertain the restrict of detection (LOD) for our assays. Exclusivity testing was carried out utilizing a panel of 69 strains: 38 non-cereus strains of Bacillus, and 31 strains of non-Bacillus micro organism.

Beauty merchandise

There have been two kinds of cosmetics used on this examine: liquid and powder. To evaluate how effectively our assays may detect the presence of B. cereus 3A in artificially contaminated samples of liquid-type cosmetics, we bought facial toner merchandise [N = 3], all of which had been labeled “alcohol free”. These facial toners had been primarily composed of water and plant-derived compounds and preserved with phenoxyethanol, an antibacterial agent.

To evaluate the efficiency of our assays on powder-type cosmetics, we chosen 8 beauty merchandise: Inexperienced Clay (GC), Pink Clay (C1–4), Rice Powder (RP), and Tattoo Powder (O1–2), which had been bought from a retail institution on-line. The 2 clay merchandise contained no preservatives, the rice powder product contained phenoxyethanol, and no info was obtainable about any preservative within the tattoo product. Prior analysis had decided 6 powder-based merchandise to be contaminated with B. cereus (Yossa and Jo Huang, private communication); subsequently, we categorized these as “naturally-contaminated” and didn’t add any further micro organism to those powders.

DNA extractions

Inclusivity, exclusivity, limits of detection

We remoted DNA from in a single day cultures of every pure bacterial pressure utilizing the MagMAX™ Specific 96 Magnetic Particle Processor (ThermoFisher) with PrepSEQ Nucleic Acid Extraction Equipment for Meals and Atmosphere (ThermoFisher, P/N 4,428,176) utilizing protocol PrepSeq_ResDNA_20011 (Life Applied sciences, Carlsbad, CA). Whole DNA extracted from these pure cultures had been used to judge the inclusivity (n = 143) and exclusivity (n = 69) of the qPCR assays.

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

DNA extractions from cosmetics

Two completely different extraction procedures had been used to carry out our qPCR assays on the units of contaminated cosmetics, as a result of variations within the matrices. The MagMAX™ and PrepSeq kits had been solely applicable for liquid cosmetics. Efficient DNA extraction from the powder merchandise required utilizing the DNeasy® PowerPlant® Professional package (Qiagen, Catalog Quantity 69204) as a substitute, as a result of powder merchandise develop into thick and dense within the presence of the proteinase/proteinase DNA extraction buffer used within the MagMAX and PrepSeq kits²⁷.

Primers/probes

Desk 2 exhibits the primers we used and the related publications documenting their first use. These had been bought from Life Applied sciences (ThermoFisher).

The 16S rRNA sequences (P/N 4,331,348, Life Applied sciences) used right here had beforehand been decided by De Clerck, et al.; their staff focused the 5’ hypervariable fragment, which might be amplified utilizing a common ahead and a B. cereus-specific reverse primer²⁸. Our TaqMan probe was labeled with 6 -carboxyfluorescein (FAM) reporter dye on the 5’ finish and labeled on the 3’-end with a Black Gap quencher, BHQ1, to scale back background fluorescence²⁹. We ordered the PLC primer probe (CCU001SNR, Life Applied sciences) labeled with a JUN reporter dye on the 5’-end and a non-fluorescent quencher on the 3’-end, to facilitate multiplex qPCR utilizing 4 dyes. QSY PLC- JUN probe is a customized probe, appropriate with the TaqMan Multiplex Grasp Combine (P/N 446,188; Utilized Biosystems, Life Applied sciences) and ordered by way of Life Applied sciences.

The TaqMan Inside Optimistic Management (IPC) from Life Applied sciences (P/N 4,308,323) was included to watch the PCR progress and be sure that a unfavorable outcome will not be attributable to failed PCR within the pattern³⁰ and used with the TaqMan Multiplex Grasp Combine to amplify each the one goal and the a number of goal reactions.

qPCR reactions of non-treated and PMAxx -treated samples

PMAxx -PCR is an progressive know-how that permits differentiation between reside and useless microorganisms, primarily based on the lack of cell membrane integrity in useless cells³¹. This technique makes use of a DNA-intercalating dye, PMAxx, that disrupts DNA transcription solely in useless cells, as their broken cell membranes allow entry of the dye. After photoactivation with an outlined wavelength, PMAxx intercalates and binds covalently to DNA. Subsequent amplification of that changed DNA is inhibited, thereby lowering the amplification sign from useless/broken cells compared to that from reside cells.

For every qPCR response, a 20 μl quantity consisting of 10 μl Multiplex Grasp Combine, 2 μl 10X Exo IPC Combine, 0.4 μl 50X EXO IPC DNA, 1 μl of primer assay, 2 μl of pattern/template DNA, and 4.6 μl of sterile deionized water was positioned right into a 96 effectively quick plate. Each primers for the multiplex PCR had been mixed to achieve a closing focus representing 5% of the overall response quantity. These qPCR runs had been carried out on a 7500 Quick qPCR System (Utilized Biosystems) below the next circumstances: 2 min at 50 °C, then 10 min at 95 °C, adopted by 40 cycles of 15 s at 95 °C and 1 min at 60 °C.

B. cereus 3A testing in artificially contaminated liquid merchandise

Right here we used 3 completely different beauty toner merchandise, which we artificially contaminated with B. cereus 3A. For every trial (N = 3), 3 sterile Wheaton bottles (125 ml) had been stuffed with 30 ml of facial toner and had been repeatedly spiked with 300 μl of microbial suspension, containing both a Excessive (~ 4 or 3 log CFU/ml) or Low (~ 3 or 2 log CFU/ml) degree of B. cereus 3A vegetative cells (optical density of 0.5 ± 0.05 McF), over three consecutive days, and the third bottle of pattern remained un-spiked for the unfavorable controls. These repeated inoculations had been essential to allow restoration of B. cereus from merchandise containing preservatives³². To imitate a product contamination occasion extra intently, these inoculated toner samples, together with the un-spiked unfavorable management, had been aged for 14 days at room temperature. After 14 days of getting old, the un-inoculated and inoculated samples from the Excessive- and Low-level inoculation had been gently individually combined, then 1 ml aliquots had been diluted with 9 ml MLB (Difco).

Our first purpose was to realize fractional outcomes, which on this case would include 5 replicates on the excessive degree of inoculation all yielding constructive outcomes, whereas 20 replicates on the low degree of inoculation would yield solely 50 ± 25% constructive outcomes, and 5 replicates of un-inoculated pattern all yielding unfavorable outcomes³³. Based mostly on this, 5 replicates of toner from the Excessive degree, 20 replicates from the Low degree, and 5 replicates from the uninoculated samples (unfavorable controls) had been pre-enriched for twenty-four h at 30 °C in MLB, streaked onto BACARA plates and processed concurrently for molecular evaluation. Two equal units of take a look at parts had been taken on the similar time in all of the replicates and positioned into vials, and the primary set of take a look at parts proceeded to genomic DNAs extraction with none therapy. From the second set, 1 ml of every take a look at portion was supplemented with 25 µM PMAxx, combined by way of tap-spin, and allowed to incubate at room temperature for 10 min at nighttime earlier than publicity on the PMA-Lite (Biotium) for 15 min to crosslink free DNA³⁴. Instantly following PMAxx therapy, genomic DNAs had been extracted, and all genomics (non-treated and handled) had been analyzed independently in duplicate utilizing singleplex and multiplex qPCR assays. Determine 1 underlines an summary of the workflow of this portion of our examine.

Microscopic statement of reside/useless B. cereus cells aged for 14 days in toner-samples utilizing BacLight package

To offer a microscopic perspective on the ratio of reside/useless cells within the inoculated toner samples, 1 ml from the 14-day aged samples inoculated with B. cereus on the excessive degree was centrifuged at 10,000 × g for 10 min. The supernatant was eliminated, and the pellet was resuspended in 1 ml of 0.85% sodium chloride (NaCl) and stored at room temperature for 1 h, mixing each 15 min. After centrifugation at 10 000 × g for 10 min, the cells had been resuspended in NaCl and stained with the Reside/Lifeless™ Bacterial Viability package (Cat: L7007, Invitrogen by ThermoFisher Scientific) based on producer’s protocols³⁵. Stained cells had been seen below a Zeiss 880 Laser Scanning Microscope (LSM, Carl Zeiss Microscopy, LLC, White Plains, NY). The cells had been noticed utilizing a Zeiss Axio Observer inverted microscope with × 63 1.4 NA oil immersion plan apochromatic goal. Differential interference distinction (DIC) and confocal fluorescence pictures had been acquired concurrently. A photomultiplier tube captured the sunshine emitted from a 488-nm argon laser with a 3.7-m pin gap passing by way of an MBS 488 filter with limits set between 472 and 562 nm for detection of SITO 9 stains for inexperienced fluorescence of reside cells and between 597 and 669 nm for detection of propidium iodide stains for pink fluorescence of bacterial cells with broken membrane. Zeiss Zen Black software program was used to acquire the pictures with 1024 × 1024-pixel decision.

B. cereus testing in powder-type beauty merchandise

Every of the 8 powder-types beauty merchandise (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, earlier than mixing with 8 ml of MLB broth in presence of 10 sterile glass beads to ameliorate homogenization. These samples had been diluted, and the ten⁻¹–10⁻² dilutions had been unfold on MLA and BACARA plates, earlier than enrichment at 30 °C for twenty-four h. Unfold plating was used as a substitute of spiral plating, to forestall powder particles from clogging the stylus of the spiral plater. All these enriched samples had been streaked onto BACARA plates to display for B. cereus presence, based on BAM Chapter 23¹ and processed concurrently to extract bacterial DNA for B. cereus qPCR evaluation, in duplicate. This can allow cross-method comparisons.

Statistical evaluation

Information had been analyzed utilizing a linear combined results mannequin and p-values had been adjusted to account for a number of comparisons. Cutoff factors had been established utilizing a logistic regression mannequin, such that the cutoff Ct worth offers a > 50% likelihood of a pattern being unfavorable. Inclusivity and exclusivity cutoff values had been established utilizing the higher sure of the 95% confidence interval of the ninety fifth quantile estimate³⁶. The linear combined results mannequin was match utilizing the nlme package deal model 3.1–145³⁷. Quantile regression was completed utilizing the quantreg package deal model 5.55. All different analyses had been completed utilizing R model 3.6.0³⁸.

Ethics declarations

This analysis doesn’t contain human, animal, seed or plant samples.