Bacillus cereus hazard and control in industrial dairy processing environment

Bacillus cereus is one of the most important spoilage microorganisms in dairy environment and its growth may result in various dairy defects. Moreover, it is a great safety concern for dairy industry as it is associated with incidences of food poisoning by producing enterotoxin. Because of its outstanding ability to adhere to stainless steel surfaces of dairy plant and form biofilm, B. cereus can lead to serious hygiene problems and economic loss due to spoilage of dairy products and equipment impairment. Biofilms are more resistant to antimicrobials and cleaning regimes compared to planktonic cells, and this makes their elimination from dairy industry a big challenge. B. cereus biofilms may develop particularly in storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. These biofilms in pasteurizer and storage tanks can be a source of post-pasteurization recurrent contamination. Cleaning-in-place (CIP) regimes, commonly used in dairy industry, showed a varied effectiveness in eliminating B. cereus biofilms. Optimization of alkali-based CIP significantly increases B. cereus biofilm cell removal, as compared to the reference CIP usually used in dairy industry. Thus, optimization of the existing cleaning processes and development of novel and effective strategies as well are of utmost importance to the dairy industry, as these may lead to quality improvement of products and processes. This review discusses the characteristics, and spoilage and toxigenic potential of B. cereus in dairy industry, with an emphasis on biofilm development and emerging control strategies.     

Isolation, identification and monitoring of contaminant bacteria in Iranian Kefir type drink by 16S rDNA sequencing

Iranian Kefir type drink (IKTD) is a highly consumed, traditional Iranian, fermented milk product. To improve monitoring procedures for food safety 32 industrial Kefir type drinks from 4 brands and 8 different production dates as well as 32 samples from pasteurized milk of the same Kefir manufacturers and air of the production sites were analyzed for contaminations. 16S rDNA extraction from Kefir samples as well as 16S rDNA obtained from samples incubated on Columbia agar were analyzed using PCR/DGGE, cloning, sequencing and phylogenetic classification. Already DGGE analysis indicated contaminations including Bacillus strains. Subsequently analysis of cultured clones indicated contaminations with Bacillus sp. including Bacillus cereus, Bacillus thuringiensis and Paenibacillus sp. in 9 (28%) from all analyzed samples. Also 38% of pasteurized milk samples were contaminated with B. cereus. The average count of B. cereus was 74 ± 19 cfu/ml. B. cereus and B. thuringiensis were found as contaminant bacteria in the air of the all manufacturing sites. These results suggest that milk is one of the most important sources of contamination with Bacillus sp., especially B. cereus for Kefir products in Iran. But bacterial contamination in Kefir samples might also originate from the air of the production sites. 16S rDNA analysis accelerates monitoring strategies.     

Prevalence and antimicrobial resistance of Listeria species isolated from milk and dairy products in Iran

The objective of this study was to determine the prevalence of Listeria spp. in milk and dairy products in Isfahan province, Iran. From March 2007 to September 2009, a total of 594 samples of various milk and dairy products were obtained from randomly selected retail stores. Using conventional bacteriologic method, 55 samples (9.3%) were positive for Listeria spp. The highest prevalence of Listeria was found in raw sheep milk samples (22.6%), followed by cheese samples (18.9%). The most species recovered was Listeria innocua (58.2%); the remaining isolates were Listeria monocytogenes (32.7%) and Listeria seeligari (9.1%). Overall, 54 Listeria isolates (98.2%) were resistant to one or more antimicrobial agents. Resistance to nalidixic acid was the most common finding (96.4%). All Listeria isolates were susceptible to vancomycin. The results of this study indicate the potential risk of infection with Listeria in people consuming raw and unpasteurized milk and dairy products.     

Prevalence and antimicrobial resistance of Staphylococcus aureus isolated from raw milk and dairy products

The objectives of this study were to determine the prevalence and antimicrobial resistance of Staphylococcus aureus isolated from raw milk (cow and sheep) and dairy products (traditional cheese and kashk) in Mazandaran Province, Iran. A total of 2650 samples, including 1930 raw milk and 720 dairy products were purchased from retail stores. Out of 2650 samples, S. aureus was detected in 328 samples (12.4%) in which 53 (16.2%) were positive for methicillin-resistant S. aureus. The S. aureus isolates showed resistance to tetracycline (56.1%), followed by penicillin G (47.3%), oxacillin (16.2%), lincomycin (11.9%), clindamycin (11.3%), erythromycin (7.9%), streptomycin (5.8%), cefoxitin (5.5%), kanamycin (4%), chloramphenicol (3.7%), and gentamicin (2.1%). A high frequency of blaZ (46%) and tetM (34.8%) resistance genes was found in S. aureus isolates. The findings of this study revealed consumption of raw milk and dairy products as a potential risk of foodborne infection in this region.     

Incidence and genetic variability of Listeria species from three milk processing plants

The presence of Listeria in three milk processing environments as a potential source of milk contamination was assessed. Swab samples (n = 210) taken from milk processing plants were examined. Sample sites included the milk processing equipment, besides areas handling raw and pasteurized milk. The USDA Listeria-selective enrichment procedure was used to process the samples. Forty one (19.52%) Listeria isolates were recovered. The isolates were further subjected to biochemical and genotypic characterization. Out of 41 isolates, 16 (7.62%) were confirmed as Listeria monocytogenes, 2 (0.95%) as L. ivanovii, 19 (9.05%) as L. innocua. 1 (0.48%) as L. seeligeri and 3 (1.43%) as L. grayi. All the L. monocytogenes isolates were positive for the hlyA gene. PCR based serotyping revealed all L. monocytogenes to be of 1/2a, 1/2c, 3a and 3c serovar group. AscI and ApaI restriction analysis yielded four PFGE clusters for 16 L. monocytogenes isolates obtained from raw milk collector, milk silos, buttermilk mixer, cheese and other milk product processor. No predominant PFGE cluster was observed among these L. monocytogenes isolates. The main sources of L. monocytogenes were found to be raw milk collector and milk silos. In the present study L. monocytogenes was isolated from milk and milk products processing plants which could cross-contaminate the processed products and may possess a potential threat to public health.     

Substratum attachment location and biofilm formation by Bacillus cereus strains isolated from different sources: Effect on total biomass production and sporulation in different growth conditions

Bacillus cereus, an endospore forming human pathogen associated with foodborne diseases, can form biofilms and attach to surfaces of processing equipment in the food industry. It is a consistent source of contamination and/or cross contamination of processed food products. The objective of this study was to understand substratum attachment location and biofilm formation behavior of B. cereus strains under different growth conditions. A total of 60 strains isolated from food, human, or farm and a number of reference strains were used in this study. Substratum attachment locations of these strains in 96-well microtiter plates were highly diversified among these strains. Strains isolated from food showed higher preference to attach at the air-liquid interface during early stage of biofilm formation. To the best of our knowledge, this is the first report showing the level of substratum attachment location and biofilm formation of B. cereus strains isolated from different sources. Substratum properties did not affect biofilm formation location when a number of selected strains were grown on stainless steel coupon, indicating that biofilm formation location might be independent of the type of substratum. Substratum attachment location and biofilm formation related phenotypes such as total biomass production, number of sessile cells, and sporulation were closely correlated. Substratum attachment location and sporulation behavior were strongly affected during biofilm formation under nutrient stress condition. The number of spores was significantly increased in biofilms grown under nutrient stress condition even though total biomass formation was lower. Our results on substratum attachment location and related biofilm formation behavior are substantially important for food industries where different surfaces are prone to B. cereus attachment, particularly for setting up and implementing clean in place (CIP) protocols.     

Surface conditioning of stainless steel coupons with skim milk,buttermilk, and butter serum solutions and its effect on bacterial adherence

In the dairy industry, dairy by-products such as skim milk, buttermilk and butter serum which possess different specific compositions, could contact with processing surfaces to form conditioning layers and subsequently alter bacterial attachment behavior of the surfaces. In order to simulate and elucidate this process, stainless steel coupons were conditioned with skim milk, buttermilk and butter serum solutions. Formed conditioning layers were examined under a confocal laser scanning microscope (CLSM) and the influence of surface conditioning on bacterial adherence was investigated. The results showed that different conditioning layers were formed by different dairy by-products. The layer formed by skim milk, buttermilk and butter serum was the thinnest, medium and the thickest, respectively. The treatment of stainless steel surfaces with skim milk, buttermilk and butter serum could reduce the adherence of dairy-related bacteria (Lactococcus lactis subsp. lactis NBRC 100933, Leuconostoc mesenteroides subsp. cremoris NBRC 107766 and Lactobacillus casei FIRI 108) at different levels. In the majority of cases, the adherence-reducing ability of buttermilk and butter serum was found better than skim milk. While skim milk could reduce bacterial adherence during shorter exposure time (almost of 30 min), buttermilk and butter serum could act during the longer period (up to 720 min). The result suggested that, bacterial adherence-reducing effect of buttermilk and butter serum may correlate to their substances associated with milk fat globule membrane. In order to decrease bacterial adherence, surface conditioning with skim milk, buttermilk and butter serum is recommended. Surface conditioning with skim milk is suitable for short bacterial exposure time (30 min), for a longer period of time (more than 180 min), only surface conditioning with buttermilk and with butter serum is advisable.     

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.     

Factors impacting microbial load of food refrigeration equipment

Our aim was to determine factors that have an impact on the bacterial load of inner surfaces of food refrigeration equipment to develop recommendations that should be made to consumers. We investigated 23 domestic refrigerators (DRs) and, for comparison, six serve-over counters (SOCs). Several zones were studied for aerobic mesophilic counts (AMC) presumptive Bacillus cereus and coagulase-positive staphylococci. In addition, for each DR sample, we collected data on the condition of the sampled surface and refrigeration practices. In DRs, there was no correlation between AMC and temperature, relative humidity, pH or cleaning frequency. AMC counts in SOCs, which are cleaned and disinfected weekly, were similar to the figures from the less frequently cleaned DRs, but B. cereus and coagulase-positive Staphylococus were less frequently found in SOCs. In DRs, the highest AMC counts were reached when both condensation and food traces were visible, i.e. when growth conditions were met, resulting in a mean of 10⁴ CFU/cm² against of mean of 32 CFU/cm² on clean surfaces and dry surfaces with food traces. Consequently, two recommendations for consumers are (1) to avoid condensation and (2) to clean up food spills as soon as possible.     

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.     

Occurrence and distribution of species of Enterobacteriaceae in selected Ethiopian traditional dairy products: A contribution to epidemiology

A total of 316 samples of traditional milk and milk products (66 milk, 52 Ergo – naturally fermented milk, 66 butter, 66 buttermilk and 66 Ayib, Ethiopian cottage cheese); 20 samples of cleaning water and 20 samples of udder swabs were collected in the central highlands of Ethiopia to assess the occurrence and distribution of enteric bacteria. A total of 534 isolates distributed among 10 genera and 20 species were identified. Klebsiella, Escherichia and Enterobacter were the dominant genera in their order of abundance with Escherichia coli, the most prevalent species. Erwinia, Klyuvera and Providentia were the least abundant genera. Most of the genera/species identified were isolated predominantly from milk samples followed by butter and buttermilk during the dry season and from milk, buttermilk and butter during the wet season in order of their abundance. The overall mean aerobic mesophilic, coliform and enterobacterial counts of dairy products were 8.3, 4.5 and 5.2 log cfu mL⁻¹ or g⁻¹, respectively. Ergo samples had 0.7 more log cfu of aerobic mesophilic count than butter samples. Coliform and enterobacterial counts were generally lower in samples of fermented milk products than milk with the largest difference being 1.2 log cfu mL⁻¹ for coliform and 1 log cfu mL⁻¹ for enterobacteria between milk and Ergo samples. The highest and lowest coliform counts were 5.2 and 3.9 log cfu mL⁻¹ or g⁻¹ in samples collected from cooperative centers and smallholder producers, respectively. The knowledge of the enteric bacterial properties of traditional dairy products is essential for the improvement of quality and preservation of the products thereby provide a wholesome product to the consumer with minimum health risk.     

Distribution and expression of the enterotoxin genes of Bacillus cereus in food products from Jiangxi Province,China

Bacillus cereus is a major foodborne pathogen that can cause a type of diarrhea. Diarrheal syndrome results from the ingestion of food products contaminated with enterotoxin-producing B. cereus. This study investigated the presence of four enterotoxins genes in 130 B. cereus isolated from various food products from Jiangxi, China. The expression levels of the enterotoxin genes in three B. cereus strains of different origins were subsequently analyzed. All of the B. cereus strains harbor at least 1 enterotoxin gene, whereas 34 strains harbor 2 enterotoxin genes, 44 strains harbor 3 enterotoxin genes, and 47 strains carry all of the four enterotoxin genes. The detection rates of the cytK, hblD, nheA, and entFM enterotoxin genes in all B. cereus isolates were 71%, 43%, 92%, and 99%, respectively. Moreover, the food matrix significantly affected the expression of these enterotoxin genes. The majority of the enterotoxin genes were also downregulated in four food matrices, indicating that the relative expression of certain enterotoxin genes, especially the entFM gene, was lower in a real food matrix than in laboratory broths. Hence, these data revealed that B. cereus is a serious health hazard and that the food matrix may influence the virulence expression of B. cereus. Our results provide better insights into the physiology of the microorganism grown in food products.     

Quality of raw milk intended for direct consumption in Estonia

The main aim of the present study was to estimate the occurrence of zoonotic bacteria in raw milk intended for sale directly to consumers in Estonia. In-line milk filters, bulk milk samples and milk samples from selling points were collected from a total of 14 dairy farms and respective retail selling points. The somatic cell counts, total bacterial counts and the presence of Salmonella spp and Listeria monocytogenes were studied from bulk milk samples. Campylobacter spp., Salmonella spp., L. monoscytogenes and Shiga-toxin producing Escherichia coli (STEC) were studied in farms in-line milk filters. The total bacterial counts exceeded 100,000 cfu/ml in three (21.4%) bulk milk samples and in 10 samples (71.4%) collected at the retail level. STEC genes were detected in 64.3% of the in-line milk filter samples. More than one STEC serogroup-specific gene was detected in four dairy farms. L. monocytogenes was found in 36% of the in-line milk filters. Neither Salmonella spp. nor Campylobacter spp. were found in any samples.     

Occurrence of Listeria spp. in dairy plants in Southern Italy and molecular subtyping of isolates using AFLP

We report the detection of Listeria spp. and Listeria monocytogenes in 34 dairy plants. In total, 547 of food, product contact surface and floor drain samples were collected along the product lines. Nineteen cheese factories (55.8%) were contaminated by Listeria spp. Of these 20.6% were L. monocytogenes positive. Listeria spp. was found in 6.8% of food samples, 11.3% of product contact surfaces and 40.6% of floor drains. L. monocytogenes was found in 2.4% of food samples, 4.9% of product contact surfaces and 18.8% of floor drains. Twentyfive L. monocytogenes isolates were serotyped using commercial specific antisera and genotyped using Amplified Fragment Length Polymorphism (AFLP) analysis. AFLP genotyping discriminated the four species of Listeria isolated and different genotypes for each species, moreover it could identify persistent genotypes in some dairy facilities. Listeria spp. and L. monocytogenes are widely spread in the dairy sector and probably contaminate foods during the production process. Facility-based monitoring can identify possible routes of transmission and thus allow establishment of more effective strategies to prevent food contamination.     

In vitro control of multiplication of some food-associated bacteria by thyme, rosemary and sage isolates

Antibacterial activity of thyme, rosemary and sage isolates obtained by supercritical fluid extraction and hydrodistillation was investigated on Geobacillus stearotermophillus, Bacillus cereus, Bacillus subtilis var. niger, Enterococcus faecium, Salmonella enteritidis and Escherichia coli strains. Bacillus species were the most susceptible to all tested isolates. The thyme isolates showed the strongest antibacterial activity against tested bacteria with MIC values of 40-640 μg/ml, followed by rosemary (MIC = 320-1280 μg/ml) and sage (MIC = 160-2560 μg/ml) isolates. Therefore, the antibacterial activity of the most abundant components found in the thyme isolates, thymol, p-cymene and their mixture was investigated as well. The thyme isolates, especially supercritical extract, showed stronger antibacterial activity against Bacillus strains compared to the single components and their mixture, which indicated synergetic effect of the other components. Results of this study indicated thyme as a valuable source of natural antibacterial agents and supercritical fluid extraction as an efficient isolation method.     

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.     

Bioflocculation of (Iron oxide – Silica) system using Bacillus cereus bacteria isolated from Egyptian iron ore surface

In mineral bio-beneficiation, it is very important to understand the microbial surface characteristics and its behaviour onto the mineral surface. Bacillus cereus bacterium has never been used before as a bio-reagent for separation of different mineral systems. In this work, complete characterization of such type of bacteria, isolated from Egyptian iron ore surface, including gram stain, growth curve, Biolog microbial identification, Zeta potential characterization, Fourier Transform Infrared Spectrometer, FTIR characterization as well as protein and polysaccharide analysis have been studied. The results confirmed that Bacillus cereus is a gram positive bacterium, rod shaped, smooth and circular with different types of by-products as polysaccharides, carboxylic acids and amino acids that gives an amphoteric behaviour on the cell surface. The results of zeta potential showed that the iso-electric points (IEP) of iron oxide (≈6.3) and silica (≈1.8) were significantly displaced to low values (≈2.2) and (≈1) respectively after treatment with the bacterial isolates. The results obtained showed a better affinity of Bacillus cereus to hematite mineral surface rather than silica surface and could be used in separation of such mineral from its associated gangue minerals. On applying B. cereus bacterial strain as a sole flocculating agent, to selectively separate hematite from its mixture with silica, succeeded in the removal of 80% of SiO₂ as a concentrate containing about 2% SiO₂ and 98% Fe₂O₃ with 82% flocculated by Wt. and a good recovery of 89.20%.     

Detection of verocytotoxin-producing Escherichia coli (VTEC) in minced beef and raw milk by colony blot hybridization

Verocytotoxin-producing Escherichia coli (VTEC) are foodborne pathogens that cause outbreaks linked to consumption of meat and raw milk. In this note the authors report results obtained from a survey conducted on minced beef and raw bovine milk samples using a Multiplex PCR (M-PCR) for the detection of eae, stx₁, stx₂ and hlyA genes as a screening step followed by a colony blot hybridization (CBH) technique for the isolation of the VTEC. Of 100 minced beef and 123 raw milk samples, 13 (13%) and 7 (5.7%) were positive in the M-PCR and among these 9 and 3 strains were isolated using CBH, respectively. All isolates showed the presence of the stx₂ gene, single or in association with the other investigated genes. None of the isolates belonged to the O157, O26, O91, O103, O111 and O145 serogroups. The study showed that the use of M-PCR for the screening of samples coupled with a sensitive and specific detection technique, could improve the possibility of detection of VTEC strains in foods. Moreover, the presence of VTEC in minced beef and in raw milk confirms their important role as putative vehicles of infection to humans. Stringent control of these foodstuffs is essential for food safety purposes.     

A survey on occurrence of thermophilic bacilli in commercial milk powders in China

Twenty-two milk powders including infant formula milk powders and whole milk powders collected from different manufacturers in China were examined for the presence of contaminated thermophilic bacilli. The thermophilic isolates were grouped by whole cell protein profiles, and representative strains of each group were further identified by analyzing partial 16S rDNA. 80.5% of all 801 isolates were identified as Bacillus licheniformis, Anoxybacillus flavithermus and Geobacillus stearothermophilus. The most dominant strain was B. licheniformis L1, which accounted for 27.8% of all isolates and appeared in eighteen of twenty-two milk powders. Furthermore, the inactivation effect of conventional heat treatments that were widely used in industry for milk powder was tested on ten representative strains. The three pasteurization processes did not show inactivation effect on most strains, but in some cases even activated thermophilic spores. The UHT process efficiently inactivated both thermophilic vegetative cells and spores.     

Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control

Bacillus cereus is an etiological agent of food-borne disease that can cause a type of emesis. To develop a sensitive and reliable diagnosis technique for detecting all the species of the B. cereus group, specific primers were designed to target a recently discovered part of the cereulide synthetase gene (cesB) for emetic B. cereus and 16S rRNA for non-emetic B. cereus. To detect PCR signals only from viable cells, propidium monoazide (PMA) was selected to eliminate the false-positive results. In addition, an internal amplification control (IAC) was applied to meet diagnostic multiplex PCR requirements that will prevent the occurrence of false-negative results. The inclusivity and exclusivity of the mPCR assay were estimated using a panel of 100 strains, including 2 emetic B. cereus, 77 non-emetic B. cereus and 21 non-Bacillus strains. The limit of detection (LOD) for dead B. cereus without PMA treatment in pure bacteria culture was 4.0 × 10² CFU/mL, as low as 7.5 × 10⁰ CFU/mL for viable B. cereus without PMA treatment, and 7.5 × 10¹ CFU/mL for viable B. cereus with PMA treatment. B. cereus in spiked food produce was detected specifically and sensitively at 1.0 × 10³ CFU/g which was the lowest concentration detected. This novel PMA-mPCR-IAC assay is rapid and reliable, providing an efficient diagnostic tool with promising application in monitoring food samples.