In vitro evaluation of antibacterial and antioxidant activities of the essential oil and methanol extract of endemic Zataria multiflora Boiss

The present study was conducted to evaluate in vitro antibacterial and antioxidant properties of essential oil and methanol extracts from a unique and endemic plant, Zataria multiflora Boiss. The antibacterial test results showed that the essential oil of the plant strongly inhibited the growth of all of the microorganisms studied especially the Gram-negative strains. The polar fraction of methanol extract has been effective against Gram-positive strains, while the non-polar fraction has shown activity similar to essential oil. The antioxidant potential of the samples was evaluated using two separate methods, inhibition of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ammonium thiocyanate systems. Sub fractions of the methanol extract were able to reduce the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) with an IC₅₀ of 11.7 ± 1.58 and 16.2 ± 1.61 μg/ml, respectively for non-polar and polar ones, which the activity of the latter almost is equal to synthetic antioxidant BHA (18.2 ± 1.94 μg/ml). Inhibition values of linoleic oxidation were calculated to be 82.4% and 80.3% for the polar and non-polar fractions, respectively. The essential oil to be showed more inhibition (89.7 ± 2.5), similar to the synthetic antioxidants BHA (97.8 ± 2.94) and ascorbic acid (93.2 ± 2.1). The chemical composition of hydrodistilled essential oils of Z. multiflora was analyzed by GC/MS. A total of 25 compounds representing 99.78% of the oil were identified: thymol (37.59%), carvacrol (33.65%); para-cymene (7.72%), γ-terpinene (3.88%) and β-caryophyllene (2.06%) were the main components comprising 84.9% of the oil. Results here show that the essential oil and methanol extract of Z. multiflora possess antioxidant and antibacterial activity, and therefore it could be used as a natural preservative ingredient in food and/or pharmaceutical industries.     

Evaluation of Zataria multiflora Boiss. essential oil activity against Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes by propidium monoazide quantitative PCR in vegetables

Essential oils (EOs) have long been applied as flavoring agents in foods, and due to their content in antimicrobial compounds, they have potential as natural agents for food preservation. Recently, real-time PCR in combination with PMA has successfully been applied to discriminate between live Escherichia coli O157:H7 and dead bacteria killed by cumin, clove, oregano and cinnamon EOs. In this study, initial experiments were performed in order to elucidate the minimum bactericidal concentration of Zataria multiflora EOs on E. coli O157:H7, Salmonella enterica and Listeria monocytogenes. Thereafter PMA-qPCR was applied in order to selectively quantify life cells within a bacterial population treated with Z. multiflora EO. Inactivation was obtained at EO concentrations of 0.02, 0.035, 0.045 for L. monocytogenes, E. coli O157:H7 and S. enterica, respectively. L. monocytogenes were totally killed in 30 min while it took 1 h 30 min for the gram negative pathogens. As a conclusion Z. multiflora EO has potential as natural food additive or biopreservative since it was able to irreversibly inactivate the three pathogens tested, at lower concentrations than other EOs and short exposition times. In addition, the PMA-qPCR approach proved efficient to selectively detect live pathogenic bacteria in vegetables following inactivation with Z. multiflora EO.     

Antimicrobial activities of Iranian sumac and avishan-e shirazi (Zataria multiflora) against some food-borne bacteria

Food poisoning originating from contaminated foods by both Gram-positive and Gram-negative bacteria causes concern to society and to the industry. Spices have been used safely since ancient times as food flavoring agents and also as herbal medicines and are now mainly considered “generally regarded as safe” (GRAS). Antimicrobial effects of two spices used in Iranian traditional medicine were investigated against some pathogenic food-borne bacteria. Hydroalcoholic extracts of Rhus coriaria L. (sumac) and Zataria multiflora Boiss. (avishan-e shirazi or zaatar) obtained from Tehran botanicals market were prepared by cool percolation method using 80% (v/v) aqueous alcohol. Antimicrobial activities of the extracts were tested against several Gram-positive and Gram-negative bacteria including Staphylococcus aureus, Bacillus cereus, Escherichia coli, Salmonella typhi, Proteus vulgaris, and Shigella flexneri. All the extracts were primarily screened for their possible antimicrobial effects using disc and well diffusion methods. Both R. coriaria L. and Z. multiflora Boiss. showed considerable antibacterial effects. The potential antibacterial activities of the two herbal extracts were further investigated at concentrations of 0.05%, 0.1%, 0.2%, 0.4%, 0.8% and 1.6% (w/v) and minimum inhibitory concentration (MICs) as well as minimum bactericidal concentrations (MBCs) of the extracts were elucidated. Sumac showed better activity against the tested bacteria compared to avishan-e shirazi inhibiting Bacillus cereus and Staphylococcus aureus at concentrations of 0.05% and 0.1%, respectively while Gram-negative bacteria were affected by higher concentrations of sumac ranging 0.1–2%. The MICs of avishan-e shirazi against the tested bacteria ranged from 0.4% to 0.8%. Salmonella typhi was found to be the most resistant showing the MICs of 0.2% and 0.8% with sumac and avishan-e shirazi, respectively. Both popular Iranian spices which are traditionally used as astringent agents have promising inhibitory effects on food-borne bacteria and could be considered as natural food preservatives.     

Intelligent release of cinnamon oil from engineered proteoliposome via stimulation of Bacillus cereus protease

Inspired by the hydrolysis of casein by protease, a new approach to deliver antimicrobials against bacterial infections was developed in this study. As a natural antibacterial agent, cinnamon oil was encapsulated into engineered liposomes inlaid with casein. The average particle size of proteoliposomes was 615.0 nm and their entrapment efficiency (EE) was 40.0%. In this work, Bacillus cereus (B. cereus) was chosen as model bacterium. The controlled release of liposome-encapsulated cinnamon oil was realized via bacterial protease secreted from B. cereus. As a result, 99.99% of the bacteria could be efficiently inhibited in rice and wheat flour.     

Radiation tolerance of Bacillus cereus pre-treated with carvacrol alone or in combination with nisin after exposure to single and multiple sub-lethal radiation treatment

Currently little information exists on the response of Bacillus cereus after repetitive exposure to combined mild treatments involving antimicrobials and gamma irradiation. Therefore, the aim of this present study was to evaluate the radiation stress on growth and physiology of B. cereus LSPQ 2872 vegetative cells at stationary phase, following exposure to single and repetitive sub-lethal γ-radiation treatment at 1 kGy simultaneously with carvacrol alone or combined with nisin at sub-inhibitory concentrations. Results indicated that the combination of carvacrol and nisin significantly enhance the radiation sensitivity of B. cereus since lower D₁₀ values were recorded after both single and repetitive irradiation treatments. Flow cytometric analysis of radiation-stressed B. cereus cells following repetitive treatment revealed the heterogenic behaviour of the bacterium leading to the induction of a radiation tolerance response. When compared to carvacrol alone, the combination of carvacrol and nisin developed also increased radioresistance if repetitively processed with γ-radiation at 1 kGy, since the decrease percentage of dead cells was accompanied by an increase in the number of injured cells. However, good agreement was not found between classical plate counting (log cfu reductions) and flow cytometry method. For both antimicrobials, the increase of radioresistance after repetitive mild treatment was not accompanied by augmentations of lag phase or growth rate. The structural changes of the outer membranes were assessed by TEM analysis and results revealed that radioresistance might be related to changes in the cell wall.     

Survival and growth of Bacillus cereus during Gouda cheese manufacturing

Survival and growth of Bacillus cereus was investigated during manufacturing of Gouda type cheese. The cheese was prepared in the pilot plant from pasteurised milk artificially contaminated with spores to give a final concentration of approximately 10² B. cereus spores per millilitre of cheese milk. B. cereus was enumerated by surface plating on B. cereus selective media and lactic acid bacteria were enumerated on lactic agar and MRS agar (de Man-Rogosa-Sharpe). Samples were taken for microbiological analysis of the milk before renneting, curd at cutting, at half whey removal, at final whey removal, at hooping of the curd, the cheese after pressing, after brining, after 1 week, after 2 weeks, after 4 weeks and after 6 weeks. The spores germinated into vegetative cells, which grew and reached a maximum of approximately 10⁴ CFU per gram of cheese at hooping (about 4 h after renneting). After pressing (approximately 16 h after renneting ) the viable cells were reduced to less than 10² CFU per gram. After brining (about 40 h after renneting) B. cereus was not detected in the cheese curd. At this stage the conditions of the cheese, particularly lower moisture content and a_w, lower Eh, high salt content, depleted lactose content combined with high acidity may have inhibited the growth of B. cereus. B. cereus did not affect the growth of lactic acid bacteria during cheese manufacturing. Lactic acid bacteria grew from 10⁷ to 10⁹ CFU per gram of curd during cheese manufacturing and stayed fairly constant at about 10⁹ for 6 weeks.     

Application of MPN-PCR in biosafety of Bacillus cereus s.l. for ready-to-eat cereals

Since Bacillus cereus is one of the important foodborne pathogens, it is interesting to investigate the biosafety of Bacillus spp. and B. cereus in ready-to-eat cereals marketed local supermarkets. For this investigation, the prevalence and enumeration of Bacillus spp. and B. cereus were assayed using MPN-PCR method. Results showed that 78% of the processed cereal products intended for direct consumption were positive for the presence of B. cereus with concentrations ranging from as low as 30 MPN/g to more than 24,000 MPN/g. The concentration obtained from this study also reflects on the differences in the contamination level between the infant food, raw cereals, cereal bars, ready-to-eat breakfast cereals and pre-mixed drinks examined. Hence, application of the MPN-PCR method was found to be useful to address the biosafety concerns of B. cereus in ready-to-eat cereals.     

Occurrence and diversity of Bacillus cereus and moulds in spices and herbs

Spices and herbs can contain toxin-producing bacteria and moulds, which can cause health problems for consumers and contribute to food spoilage and shelf-life reduction. The aims of the present work were (i) to determine the occurence and levels of B. cereus and moulds; (ii) to charactize the incidence and diversity of B. cereus emetic toxin (ces, CER), and diarrhoeal toxin-encoding genes (entFM, nheA, hblC, cytK) and toxigenic potential of Hbl toxin-producing B. cereus strains. Black ground pepper samples showed the most contamination with the highest concentration of B. cereus 2.49 log₁₀ CFU/g. Moreover, cumin contained the most prominent mould concentration level of 3.6 log₁₀ CFU/g. The most common moulds were Aspergillus and Penicillium spp. Compared to packaging type, all products acquired from the local market, except curry for B. cereus, exchibited high concentrations of B. cereus and moulds. Four genes were identified – 96% of B. cereus strains contained entFM, 94% nheA, 56% hblC, 42% cytK. None of the samples contained emetic toxin-encoding genes (ces, CER). Toxigenic potential of Hbl toxin was found in 72% of B. cereus strains. Different temperature, moisture levels and hygiene practices were observed at places of sale in local markets thus facilitating contamination and development of moulds. Moreover, the presence of B. cereus and its ability to produce toxins in spices and herbs, may suggest the need to establish microbiological criteria for mould and spore-forming bacteria in spices and herbs.     

The antimicrobial effect of wine on Bacillus cereus in simulated gastro-intestinal conditions

This study aimed to evaluate the antimicrobial activity of wine against Bacillus cereus vegetative cells and spores. The results clearly show that wine exerts a strong inactivation effect against vegetative cells of B. cereus. The red wine tested inactivated stationary phase cultures to undetectable numbers in less than 10 s. Thus, further inactivation assays were carried out with wine diluted with water (1:4 and 1:8). Diluted wine 1:4 caused a reduction of approximately 5 log cycles on viable cell counts in 20 s. On the other hand, B. cereus spores were found to be highly resistant to wine exposure. The influence of wine components (organic acids, ethanol and phenolic compounds) was investigated on vegetative cells. The wine organic acids tested exhibited a strong inactivation effect, and, when combined with ethanol, a slight synergistic effect was observed. The wine phenolic compounds assayed displayed no activity against the vegetative cells at the concentrations tested. At the simulated gastric conditions studied (in the presence of food), wine diminished considerably the number of B. cereus viable cells in addition to the effect of the synthetic gastric fluid. The behaviour of B. cereus spores under gastro-intestinal conditions was also evaluated. In a consumption-like scenario, the addition of wine led to lower total counts (vegetative cells + spores) of B. cereus in the simulated intestine conditions, showing that wine inhibits the proliferation of the vegetative cells obtained from the germination of spores. This work provides evidence that consumption of wine during a meal may diminish the number of viable cells of B. cereus and reduces the impact of the germination of spores that may occur in the small intestine, thus lowering the risk of toxi-infection that may be caused by this pathogen.     

Antibacterial activity of plant essential oils and extracts: The role of thyme essential oil,nisin, and their combination to control Listeria monocytogenes inoculated in minced fish meat

Food poisoning caused by Listeria monocytogenes leads to a 30% rate of mortality among patients. The antibacterial activity of cinnamon, thyme, and rosemary essential oils (EOs) and shallot and turmeric extracts was tested against L. monocytogenes using agar well and disc diffusion techniques. Results showed that thyme EO had the highest antimicrobial activity, followed by cinnamon and rosemary EOs, respectively. The antilisterial activity of thyme EO at 0.4%, 0.8%, and 1.2% levels, nisin at 500 or 1000 IU/g level, and their combination against L. monocytogenes was examined in minced fish samples. The antilisterial properties of nisin were also investigated in cooked minced fish treatments. Nisin at 500 or 1000 IU/g in the minced fish meat demonstrated bacteriostatic activity against L. monocytogenes. The use of thyme EO at 0.8% and 1.2% reduced the L. monocytogenes viable count below 2 log cfu/g after 6 days. Furthermore, simultaneous use of thyme EO at 0.8% and 1.2%, and nisin at 500 or 1000 IU/g level, reduced the L. monocytogenes viable count below 2 log cfu/g after the second day of storage. The antilisterial activity of nisin in the cooked minced fish samples was slightly stronger than that of the raw group.     

Tenacity of Bacillus cereus and Staphylococcus aureus in dried spices and herbs

Numerous studies examined the antimicrobial effects of spice and herb extracts, whereas little is known about the effects of dry condiments on the survival of microorganisms. This study investigated the impact of dried condiments on the survival of Bacillus cereus and B. thuringiensis spores as well as Staphylococcus aureus cells. In addition, the survival variability between different strains was evaluated. Condiments (allspice, basil, cinnamon, nutmeg, oregano, paprika, parsley and pepper) were artificially contaminated by a dry spiking method using sand as carrier matrix and as control. The results show that counts of B. cereus and B. thuringiensis spores (initial spore count 5.6 ± 0.2 log₁₀ cfu/g and 6.7 ± 0.1 log₁₀ cfu/g, respectively) did not decline significantly in all condiments over a period of 50 weeks. In contrast, in some of the spiked test materials, cell counts of S. aureus (initial cell count 8.1 ± 0.5 log₁₀ cfu/g) were reduced below the detection limit of 10 cfu/g within 10 weeks of storage. D values for S. aureus ranged between 5 and 31 days depending on the strain, condiment and initial contamination level. In conclusion, dried condiments may not affect the survival of spores but can significantly affect the survival of non-spore forming bacteria. As strain variability can occur, tenacity studies should be conducted including a variety of strains.     

Extracellular enzyme production and enterotoxigenic gene profiles of Bacillus cereus and Bacillus thuringiensis strains isolated from cheese in Turkey

The aim of the present study was to investigate the biochemical characteristics, extracellular enzyme production and enterotoxigenic genes contents of 6 Bacillus cereus and 22 Bacillus thuringiensis strains, isolated from 100 cheese samples in Turkey. Crystal morphologies of B. thuringiensis strains were found either spherical (n = 12) or spherical and irregular-shaped (n = 10) by phase contrast microscopy. B. cereus and B. thuringiensis strains were found to produce extracellular enzymes, respectively: gelatinase (83% and 91%), DNase (83% and 77%), lecithinase (83% and 95%), protease on skim milk agar (100% and 100%), protease on milk agar (100% and 91%), protease on casein agar (83% and 77%), xylanase (100% and 45%), and cellulase (0% and 41%), and amylase (83% and 27%). All of the strains, except for Bt-D1, hydrolyzed Tween 20 (96%), but not Tween 80 or tributyrin. Pectinolytic activity was obtained to be the least frequent (4%). PCR analysis showed that all strains contained nheA, nheB, nheC and hblD genes. The hblA and hblC genes were present in 2 and 4 of B. thuringiensis strains, respectively. The bceT gene was detected in 1 B. cereus and 9 B. thuringiensis strains. The entFM gene was detected more frequently in B. thuringiensis (82%) than in B. cereus strains (50%). To our knowledge, this is the first report about the isolation and identification of enterotoxigenic B. cereus and B. thuringiensis strains from cheese samples in Turkey.     

Phylogenetic and toxinogenic characteristics of Bacillus cereus group members isolated from spices and herbs

The foodborne pathogen Bacillus (B.) cereus is a common contaminant in spices and herbs. To further characterise B. cereus and its closely related group members present in spices and herbs, we analysed presumptive B. cereus strains isolated from six different condiments with view to B. cereus group species, phylogenetic affiliation and toxinogenic potential.Of a total of 59 isolates 44 were identified as B. cereus sensu stricto (s.s.), four as B. toyonensis-like, five as B. thuringiensis, one as B. weihenstephanensis, two as B. pseudomycoides/B. mycoides and three as undefined B. cereus group species. A maximum of three different species occurred simultaneously in the same spice sample. The isolates comprised 33 multilocus (ML) sequence types (STs), which can be assigned to three different phylogenetic groups. Except two B. pseudomycoides/B. mycoides strains, all isolates were able to produce enterotoxins and one strain the emetic toxin cereulide as detected by an immunoassay and LC-MS, respectively. The prevalence of toxin genes was 96.6% for nheA, 94.9% for hblD, 50.8% for cytK-2 and 1.7% for ces. The emetic strain was characterised by ST 869, which for the first time was assigned to an emetic B. cereus (s.s.) strain and is not part of the previously known two emetic MLST clusters.Our results demonstrate that not only B. cereus (s.s.) but also toxin producing B. thuringiensis, B. weihenstephanensis and B. toyonensis-like strains could be detected in condiments. For some isolates MLST revealed disagreements between phylogenetic relationship and the classification as B. weihenstephanensis and B. mycoides based on previously described species markers.     

Sporulation dynamics and spore heat resistance in wet and dry biofilms of Bacillus cereus

Environmental conditions and growth history can affect the sporulation process as well as subsequent properties of formed spores. The sporulation dynamics was studied in wet and air-dried biofilms formed on stainless steel (SS) and polystyrene (PS) for Bacillus cereus ATCC 10987 and the undomesticated food isolate B. cereus NIZO 4080. After harvesting and maturation, the wet heat resistance of spores obtained from these biofilms was tested and compared to planktonic and agar plate-derived spores. Drying/air exposure of the preformed 24 h old biofilms accelerated spore formation for both strains and resulted in higher final spore percentages. Prolonged dry incubation of more than three days triggered germination of spores in the biofilms of ATCC 10987. Spores harvested from wet biofilms on SS displayed the highest heat resistance compared to liquid, agar plate and PS biofilm derived spores. The D_95 °C values for these spores were 17 and 22 min for NIZO 4080 and ATCC 10987, respectively, which was 2 and 1.2 fold higher compared to planktonic spores of these strains. Spores obtained from dried biofilms of ATCC 10987 displayed reduced heat resistance compared to wet biofilm spores. The results indicate that environmental conditions encountered by biofilms affect sporulation dynamics and spore heat resistance, thus affecting subsequent quality issues and safety risks related to these biofilms.     

Quantification and differentiation of Bacillus cereus group species in spices and herbs by real-time PCR

Spores of Bacillus (B.) cereus group species are frequent contaminants in foodstuffs including spices and herbs. However, the distribution of individual B. cereus group species is unknown as standard cultural methods are insufficient for differentiation. Real-time PCR is an alternative method to detect, differentiate and quantify B. cereus group species in food.In our study we applied a combination of previously described real-time PCR assays to detect and quantify the B. cereus group (excluding B. cytotoxicus) with simultaneous discrimination of B. pseudomycoides and cry1-positive B. thuringiensis as well as differentiation of B. weihenstephanensis from B. cereus group species with non-rhizoid colony morphology. For testing food matrices, which can also include PCR inhibiting substances, an internal amplification control was included. In total, five DNA extraction kits were tested on pure spore suspensions to select the one with the best recovery rate when coupled to real-time PCR. The Qiagen DNeasy Blood & Tissue Kit performed best with a limit of detection (LOD) of approximately 100 cfu/ml for spores of each B. cereus, B. weihenstephanensis, B. thuringiensis and B. pseudomycoides strain. However, applied to allspice, paprika, pepper and oregano artificially contaminated with B. cereus spores the LOD was ≥10⁵ cfu/g. In contrast, using the open-formula cetyltrimethylammonium bromide (CTAB) method LODs of 10² to 10³ cfu/g were achieved for paprika, pepper and oregano. For allspice, the LOD was 10⁶ cfu/g.Our quantitative multiplex real-time PCR coupled to DNA extraction by the CTAB method provides a sensitive culture independent technique with the potential to quantitatively detect and differentiate B. cereus group species in several spices and herbs.     

The effects of high pressure processing and slightly acidic electrolysed water on the structure of Bacillus cereus spores

The aim of this study was to investigate the effects of high pressure processing (HPP) and slightly acidic electrolysed water (SAEW) on the structure of B. cereus spores in order to understand the inactivation mechanism. The physiological response of spores was studied by scanning electronic microscopy (SEM), transmission electronic microscopy (TEM), dipicolinic acid (DPA) release, su­per-res­o­lu­tion mul­ti­pho­ton con­fo­cal mi­cro­scope (SMCM), and flow cytometry (FCM). The morphology of B. cereus spores treated with HPP-SAEW exhibited a large fraction damage, and also an incomplete exosporium. Furthermore, spores treated with HPP-SAEW had an irregular surface bumps, and even a critical distortion. Approximately 90% of DPA was released in the groups of treated by HPP-SAEW, and 80% of DPA was released in single HPP treatment groups. The SMCM and FCM results indicated that HPP-SAEW methods was not mainly depended on germination, but directly killed. The mechanism of HPP-SAEW possibly was that one of the proteins was particularly susceptible to SAEW under HPP conditions.     

Inhibitory activity of yarrow essential oil on Listeria planktonic cells and biofilms

The inhibitory effects of the essential oil obtained from yarrow (Achillea millefolium) were tested against planktonic cells and biofilms of Listeria monocytogenes and Listeria innocua isolates obtained from food processing environments. The plant essential oil was found to have strong bactericidal activity against planktonic cells which was found to be equivalent to that of tea tree oil (Melaleuca alternifolia). Inhibition of biofilm formation and growth after incubation with different concentrations of yarrow essential oil was assessed by the crystal violet and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide reduction assay. Yarrow essential oil significantly inhibited the initial cell attachment of the Listeria cells on polystyrene, stainless steel and high density polyethylene surfaces but was less inhibitory towards 6 h preformed biofilms formed on polystyrene surface. Metabolic activity of the biofilms decreased considerably after incubation with the oil. Yarrow essential oil, or its constituents, may be useful additives for the development of new disinfectant and sanitizer formulations for application in the food industry.     

Effect of high pressure homogenization applied individually or in combination with other mild physical or chemical stresses on Bacillus cereus and Bacillus subtilis spore viability

High pressure homogenization (HPH) has been proposed as an effective alternative to high hydrostatic pressure in the continuous sanitization of fluid food systems. In this study, we evaluated the influence of HPH treatment, applied individually (one, two or three cycles) or in combination with other mild physical or chemical stresses (mild heat treatment H₂O₂ and low pH), on the capability of Bacillus cereus and Bacillus subtilis spore, suspended in sterilized double distilled water, to form colonies. Although plate count only slightly decreased in all the strains when one cycle of HPH at 150 MPa was applied alone, the spores released significant levels of dipicolinic acid (up to 28%) that could indicate a possible disruption of spore layers. Three consecutive cycles of HPH determined high reduction of colony count (about 5 log CFU/ml) and high DPA release (52%). Among the stress conditions applied, it was observed that only the thermal shock after one HPH cycle reduced the colony count of 2.3 log CFU/ml and induced a DPA release up to 57%.These results suggested HPH as a novel application for B. cereus and B. subtilis control in fluid foods.     

Growth and inhibition by spices of growth from spores of enterotoxigenic Bacillus cereus in cooked rice

Bacillus cereus is a pathogenic spore-forming bacterium implicated in cases of diarrheal and emetic type of foodborne illness. We previously found that enterotoxigenic B. cereus is widely present in retail spices. Here we analyzed the spore heat resistance of nine diarrheal strains isolated from spices. Seven had D_95°C values ranging from 0.64 to 3.53 min while two emetic strains had D_95°C values of 7.04 min and 6.64 min. The ability of selected strains to grow in cooked rice at temperatures 20 °C, 17 °C and 12 °C was determined as well as their toxin expression capability. After 48 h, B. cereus levels of 1.26 × 10⁷ and 3.8 × 10⁷ CFU/g were obtained in cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 10⁴ CFU/g even after 48 h of incubation. The increase in the aerobic, mesophilic bacterial population (APC) and B. cereus population naturally present in 0.1% crushed pepper added to cooked rice was determined over a period of 48 h at 20 °C and 17 °C. Levels of B. cereus in pepper/rice samples reached a maximum of 1600 MPN/g at 20 °C while the APC was 2.4 × 10⁸/g at this temperature. When thyme, containing the same initial natural level of B. cereus, was added to rice in place of pepper, more than a five-fold greater level of B. cereus was detected at 20 °C. Since thyme contained initial APC of 2.5 log₁₀ less than pepper we conclude that the high APC functions in a competitive-exclusion manner, minimizing the growth of B. cereus and potentially other agents of foodborne illness. This is particularly relevant in instances of temperature abuse of foods and may explain the low incidence of foodborne illness due to B. cereus despite its widespread presence shown in previous surveys of market spices.