qPCR detection of viable Bacillus cereus group cells in cosmetic products.

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 CT 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 CT 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.

Genomic characterization of Bacillus cereus sensu stricto 3A ES isolated from eye shadow cosmetic products.

BACKGROUND: The Bacillus cereus group, also known as B. cereus sensu lato (s.l.) contains ubiquitous spore-forming bacteria found in the environment including strains from the B. cereus sensu stricto (s.s.) species. They occur naturally in a wide range of raw materials and in consumer products. Characterizing isolates that have survived in consumer products allows us to better understand the mechanisms that permit spores to persist and potentially cause illness. Here we characterize the draft genome sequence of B. cereus s. s. 3A-ES, originally isolated from eye shadow and since investigated in several cosmetic studies and compared it to other top ten published complete genome sequences of B. cereus s.l. members. RESULTS: The draft genome sequence of B. cereus s.s. 3A ES consisted of an average of 90 contigs comprising approximately 5,335,727 bp and a GC content of 34,988%, and with 5509 predicted coding sequences. Based on the annotation statistics and comparison to other genomes within the same species archived in the Pathosystems Resource Integration Center (PATRIC), this genome "was of good quality. Annotation of B. cereus s.s. 3A ES revealed a variety of subsystem features, virulence factors and antibiotic resistant genes. The phylogenetic analysis of ten B. cereus group members showed B. cereus s.s. 3A-ES to be a closely related homolog of B. cereus s.s. ATCC 14,579, an established reference strain that is not adapted for cosmetic microbiological studies. Survival of 3A-ES in eye shadow could be linked to predicted stress-response genes and strengthened by additional stress-response genes such as VanB-type, VanRB, CAT15/16, BcrA, BcrB, Lsa(B), and recA that are lacking in B. cereus s.s. ATCC 14,579. CONCLUSION: Our genomic analysis of B. cereus s.s. 3A-ES revealed genes, which may allow this bacterium to withstand the action of preservatives and inhibitors in cosmetics, as well as virulence factors that could contribute to its pathogenicity. Having a well-characterized strain obtained from eye-shadow may be useful for establishing a reference strain for cosmetics testing.

Comparison of Different Culture Methods for the Detection of Bacillus cereus Group in Cosmetics.

BACKGROUND: The U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) reference culture method uses Modified Letheen Broth (MLB) for microbiological analyses for all types of cosmetic products. OBJECTIVE: This study evaluated the effectiveness of MLB and Tryptone Azolectin Tween (TAT) broths using BAM reference culture method for cosmetics. METHODS: Pure spore suspensions of B. cereus group members were experimentally spiked (McF: 0.5) into cosmetic products. After an aging period of 72 h, the products were analyzed using MLB and TAT broth. The enumeration of the cells was performed on B. cereus group selective plates Bacillus cereus rapid agar (BACARA) and Mannitol Yolk Polymyxin (MYP) plates. RESULTS: No statistical difference (p > 0.05) was found for the recovery of cells from the liquid products using either medium (MLB or TAT broth) and the selective plates. In solid/powder products, a combination of Tween 80 and MLB detected significantly more cells (p < 0.05) than combination of Tween 80 and TAT broth. The microbial counts on BACARA showed no significant differences (p > 0.05). However, when assessing cream/oil-based products, the number of cells detected by use of Tween 80/TAT broth was significantly higher than Tween 80/MLB, and MYP showed significantly higher counts than BACARA. CONCLUSIONS: This study showed that relative effectiveness of MLB vs. TAT for recovering of B. cereus group cells varied depending on the variety of formulation, and combination of preservatives of the tested cosmetic products. The findings suggest additional studies are needed to explore recovery of other relevant microorganisms that may contaminate cream/oil-based cosmetics.

Comparison of TEMPO® BC with Spiral Plating Methods for the Enumeration of Bacillus cereus in Cosmetic Products Either Naturally Preserved or Preserved with Phenoxyethanol.

Background: The U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM) uses Bacillus cereus rapid agar (BACARA) and Mannitol-yolk-polymyxin (MYP) agar for the enumeration of the members of B. cereus group. Objective: The automated TEMPO Most Probable Number system was compared with the FDA BAM method for the detection of B. cereus group members in cosmetic products. Methods: We inoculated a range of cosmetic products with pure B. cereus spore suspensions (density = 0.5 McFarland) at high (6 log CFU/mL), medium (5 log CFU/mL), and low (4 log CFU/mL) levels. Test portions were aged for 72 h. Five replicates per sample were analyzed; uninoculated test portions served as controls. We also evaluated whether TEMPO BC erroneously detected non-B. cereus or other adulterant organisms. Results: No significant differences (P > 0.05) were found among the TEMPO BC and the BAM spiral plating methods. Correlations between TEMPO BC - BACARA and TEMPO BC - MYP were 0.895 and 0.893 for powder type products, 0.834 and 0.846 for cream and oil-based products, and 0.929 and 0.923 for liquid products, respectively. Non-B. cereus strains were not detected by TEMPO BC. Conclusions: The TEMPO BC method can be used for the detection of B. cereus in cosmetic products without preservatives, or those preserved with either phenoxyethanol or other organic substances.

Antimicrobial activity of plant essential oils against Escherichia coli O157:H7 and Salmonella on lettuce.

Foodborne outbreaks associated with the consumption of fresh produce have increased. In an effort to identify natural antimicrobial agents as fresh produce-wash, the effect of essential oils in reducing enteric pathogens on iceberg and romaine lettuce was investigated. Lettuce pieces were inoculated with a five-strain cocktail of Escherichia coli O157:H7 or Salmonella enterica (5 log CFU/g) and then immersed in a treatment solution containing 5 ppm free chlorine, cinnamaldehyde, or Sporan(®) (800 and 1000 ppm) alone or in combination with 200 ppm acetic acid (20%) for 1 min. Treated leaves were spin-dried and stored at 4°C. Samples were taken to determine the surviving populations of E. coli O157:H7, Salmonella, total coliforms, mesophilic and psychrotrophic bacteria, and yeasts and molds during the 14-day storage period. The effect of treatments on lettuce color and texture was also determined. Cinnamaldehyde-Tween (800 ppm, 800T) reduced E. coli O157:H7 by 2.89 log CFU/g (p<0.05) on iceberg lettuce at day 0; Sporan(®)-acetic acid (1000SV) reduced E. coli O157:H7 and Salmonella on iceberg and romaine lettuce by 2.68 and 1.56 log CFU/g (p<0.05), respectively, at day 0. The effect of essential oils was comparable to that of 5 ppm free chlorine in reducing E. coli O157:H7 and Salmonella populations on iceberg and romaine lettuce throughout the storage time. The natural microbiota on treated lettuce leaves increased during the storage time, but remained similar (p>0.05) to those treated with chlorine and control (water). The texture and the color of iceberg and romaine lettuce treated with essential oils were not different from the control lettuce after 14 days of storage. This study demonstrates the potential of Sporan(®) and cinnamaldehyde as effective lettuce washes that do not affect lettuce color and texture.

Essential oils reduce Escherichia coli O157:H7 and Salmonella on spinach leaves.

The efficacy of cinnamaldehyde and Sporan for reducing Escherichia coli O157:H7 and Salmonella on spinach leaves was investigated. Spinach leaves were inoculated with a five-strain cocktail of Salmonella or E. coli O157:H7, air dried for ca. 30 min, and then immersed in a treatment solution containing 5 ppm of free chlorine, cinnamaldehyde, or Sporan (800 and 1,000 ppm) alone or in combination with 200 ppm of acetic acid (20%) for 1 min or with water (control). After spin drying, treated leaves were analyzed periodically during 14 days of storage at 4°C for Salmonella, E. coli O157:H7, total coliforms, mesophilic and psychrotrophic bacteria, and yeasts and molds. Treatment effects on color and texture of leaves also were determined. Sporan alone (1,000S), Sporan plus acetic acid (1,000SV), and cinnamaldehyde-Tween (800T) reduced E. coli O157:H7 by more than 3 log CFU/g (P < 0.05), and 1,000SV treatment reduced Salmonella by 2.5 log CFU/g on day 0. E. coli O157:H7 and Salmonella populations on treated spinach leaves declined during storage at 4°C. The 1,000SV treatment was superior to chlorine and other treatments for reducing E. coli O157:H7 during storage. Saprophytic microbiota on spinach leaves increased during storage at 4°C but remained lower on leaves treated with Sporan (800S) and Sporan plus acetic acid (1,000SV) than on control leaves. The color and texture of Sporan-treated leaves were not significantly different from those of the control leaves after 14 days. Sporan plus acetic acid (1,000SV) reduced E. coli O157:H7 and Salmonella on baby spinach leaves without adverse effects on leaf color and texture.

Inactivation of Salmonella in organic soil by cinnamaldehyde, eugenol, Ecotrol, and Sporan.

Salmonella can survive in soil for months to years; consequently, soil can be a preharvest source of contamination of produce. Elimination of Salmonella with natural products and processes such as essential oils is important to prevent infection among consumers. Essential oils (distilled extract from plants) have been mainly evaluated in liquid medium and foods in which minimum inhibitory concentration is determined. However, there are no reports describing the impact of essential oils in soil, especially organic soil. We evaluated essential oils for controlling Salmonella enterica serovars in organic soil. Two essential oils (cinnamaldehyde and eugenol), two bio-pesticides (Ecotrol and Sporan), and an organic acid (20% acetic acid) at 0.5%, 1.0%, 1.5%, and 2.0% were mixed with organic sandy soil and inoculated with six different serovars of S. enterica separately. Soils were incubated at room temperature, and samples obtained at 1, 7, and 28 days were enumerated to determine survival. The bactericidal effect of cinnamaldehyde was evident at 0.5%, 1.0%, 1.5%, and 2.0% and during all times of incubation. Overall, Salmonella Negev was the most sensitive strain to oils resulting in significant reductions compared with other strains. Increases in oil concentration resulted in further reduction of Salmonella with all oils used in the study. Up to six log reductions in Salmonella serovars Typhimurium, Negev, and Newport were found after 1 day when cinnamaldehyde, Ecotrol, eugenol, Sporan, or acetic acid was used at 2% level. This study shows the potential use of essential oils to effectively reduce Salmonella populations in soil. The significant reduction of Salmonella could greatly reduce potential contamination of fresh organic produce inadvertently contaminated by soil.