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.     

Development of a multiplexed PCR assay combined with propidium monoazide treatment for rapid and accurate detection and identification of three viable Salmonella enterica serovars

Salmonella is the leading pathogenic bacteria in food and contaminated water. The aim of this study was to develop a rapid and reliable technique for simultaneous detection of the main three serotypes (Salmonella enterica serovars Typhimurium, Paratyphi B and Typhi) of Salmonella. Primers were designed to amplify the genes specific to each of these three serotypes for simultaneous detection using polymerase chain reaction (PCR). To ensure the detection of only viable cells, propidium monoazide (PMA) was applied to selectively suppress the DNA signal from dead cells. Results showed that the PMA-multiplexed PCR (PMA-mPCR) assay always gave negative results for heat-killed Salmonella at concentrations up to 1 × 10⁶ CFU/ml in pure culture or 1 × 10⁶ CFU/g in spiked food products (tomato, chicken, beef and ham). Results showed that the detection limits of the PMA-mPCR assay were approximately 10² CFU/ml (4.3 × 10² CFU/ml for S. Typhimurium, 3.7 × 10² CFU/ml for S. Paratyphi B, 7.2 × 10² CFU/ml for S. Typhi) in pure culture and 10³ CFU/g (4.3 × 10³ CFU/g for S. Typhimurium, 3.7 × 10³ CFU/g for S. Paratyphi B, 7.2 × 10³ CFU/g for S. Typhi) in food produce. These results demonstrated that the PMA-mPCR assay can simultaneously detect and identify viable S. Typhimurium, Paratyphi B and Typhi in a short period of time, even in food produce.     

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.     

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.     

Development of an SD-PMA-mPCR assay with internal amplification control for rapid and sensitive detection of viable Salmonella spp., Shigella spp. and Staphylococcus aureus in food products

In this work, a specific, sensitive, and accurate technique was presented for simultaneous detection of Salmonella spp., Shigella spp., and Staphylococcus aureus in food products, three of the more frequent foodborne pathogens that were usually reported in a variety of food matrices. An internal amplification control (IAC) was added in a multiplex PCR (mPCR) reaction system as an indicator of false negative result that can come from the presence of PCR inhibitors in food products. In the presence of inhibitor, no signal would result for the target genes as well as the IAC which results in a positive signal, thereby, eliminating false negative results. To ensure detection of only the viable cells, the effects of sodium deoxycholate (SD) in combination with propidium monoazide (PMA) treatment in the presence of dead cells and viable cells were investigated. Results showed that PMA treatment alone could not effectively inhibit the detection of 10⁷ CFU/mL of dead Salmonella Typhimurium, Shigella sonnei, and S. aureus from PCR amplification. However, the SD in combination with PMA treatment gave negative results for PCR amplification of dead S. Typhimurium, S. sonnei, and S. aureus in pure culture and food products. When the developed SD-PMA-mPCR assay in combination with IAC was applied to detect the spiked food (milk, ground beef), the LOD of SD-PMA-mPCR assay for S. Typhimurium, S. sonnei, and S. aureus inoculated individually or inoculated simultaneously into milk or ground beef were 10¹ CFU/mL or 10¹ CFU/g after 15 h enrichment. The results suggested that the SD-PMA-mPCR assay in combination with IAC held promise for the detection of foodborne S. Typhimurium, S. sonnei, and S. aureus.     

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.     

Effectiveness of a peracetic acid-based disinfectant against spores of Bacillus cereus under different environmental conditions

A peracetic acid based disinfectant was tested for its efficacy against spores of different Bacillus cereus -strains (DSM 318, 4312, 4313 and 4384). To determine the influence of different factors like exposure-time, temperature and presence of protein quantitative and qualitative suspension tests were performed. Spore suspensions of B. cereus were treated with various concentrations of a representative peracetic acid based disinfectant at three temperatures (10, 15 and 20 °C), with protein load and with different exposure times (5, 30 and 60 min). Temperature, level of concentration and exposure-time had a significant influence on reduction of spores of B. cereus (p < 0.05). The susceptibility of spores of different strains greatly differed. A treatment of spores of DSM 4384 with 2.0% for 30 min even at 10 °C inactivated all present spores (initial number 6.18–6.71 log CFU/ml). Spores of B. cereus strain DSM 4313 had only reductions of 0.16–0.97 log CFU/ml at same treatment conditions. The presence of inactivated bovine serum as interfering substance had no significant influence on reduction (p > 0.05).     

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.     

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 potentials of Bacillus licheniformis strains for inhibition of B. cereus growth and reduction of biogenic amines in cheonggukjang (Korean fermented unsalted soybean paste)

The aim of this study was to control Bacillus cereus growth and biogenic amines formation by using a starter in cheonggukjang fermentation. We used the four selected B. licheniformis strains for cheonggukjang fermentation and investigated whether the strains are effective in reducing both B. cereus level and biogenic amines content. We confirmed that the selected strains are able to reduce B. cereus level to below l log CFU/g after cheonggukjang fermentation from the initial level of 3–4 log CFU/g (artificial contamination). Tryptamine, β-phenylethylamine, putrescine, cadaverine, tyramine, spermidine, and spermine were detected whereas histamine, serotonin, noradrenaline, and dopamine were not detected in the cheonggukjang samples. Most of biogenic amines contents were lower than those of previous studies, indicating that the strains produced the low level of biogenic amines in cheonggukjang fermentation. The acceptable levels of amino-type nitrogen, free amino acids, and isoflavones were contained in the cheonggukjang samples. These results demonstrate that the selected strains are not only effective for reduction of both B. cereus contamination and biogenic amines but also suitable for preparation of cheonggukjang with good quality.     

Development of a quantitative real-time PCR assay for viable Salmonella spp. without enrichment

Propidium monoazide (PMA) combined with molecular quantitative real-time PCR (qPCR) has been widely used for only detection of viable bacteria. However, recent studies indicated PMA did not fully inhibit the detection of dead Salmonella. In this study, we developed a more effective PMA Taqman-based qPCR than previous studies to quantify viable Salmonella spp. in raw shrimp. This method has high specificity by using 60 strains belonging to 23 species. The optimization of the PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10⁶ CFU/mL dead cells, while only 10³–10⁴ CFU/mL dead cells could be inhibited in previous reports. This assay could detect viable Salmonella spp. at as low as 36 CFU/mL in pure culture and 100 CFU/g in raw shrimp. By comparing with PMA-qPCR, qPCR and plate counting for quantifying Salmonella in samples, this PMA-qPCR was obviously superior to qPCR and had good agreement with plate counting. In conclusion, this effective method can be used as an available tool to quantify viable Salmonella spp. in raw shrimp.     

Short communication: Fate of major foodborne pathogens and Bacillus cereus spores in sterilized and non-sterilized Korean turbid rice wine (Makgeolli)

The objective of this study was to examine the fate of foodborne pathogens (Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus) and B. cereus spores in Korean turbid rice wine (Makgeolli). Samples of sterilized and non-sterilized turbid rice wine were inoculated with each of the vegetative bacteria or B. cereus spores at 3–4 log CFU/ml. The samples were stored at 5 °C or 22 °C, and bacterial survival was monitored over 28 days. Despite the harsh environment (alcohol content: 6–7% and pH: 3.43–3.98), long-term survival of pathogens was observed. Survival time was different depending on the type of beverage (pathogens survived longer in sterilized wine than in non-sterilized wine), cellular state (spores survived longer than vegetative cells), species (B. cereus survived longer than other species), and storage temperature (pathogens survived longer at 5 °C then at 22 °C). The number of B. cereus spores remained constant at both temperatures. The vegetative B. cereus population declined rapidly within 1 day, but then remained steady for up to 28 days (1.20–1.55 log CFU/ml in sterile wine). These results indicate that B. cereus formed spores that survived for a long time; therefore, it is possible that B. cereus may exist as spores in turbid rice wine. E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus survived for up to 28, 14, 14, and 14 days, respectively, in sterilized wine at 5 °C. Thus, the health implications of the long-term survival of pathogens in alcoholic beverages should be carefully considered. The results provide new information that may be useful in predicting the potential microbiological hazards associated with turbid rice wine.     

Investigation of the diversity and safety of the predominant Bacillus pumilus sensu lato and other Bacillus species involved in the alkaline fermentation of cassava leaves for the production of Ntoba Mbodi

The objective of the study was to investigate the identity, diversity, and safety of the Bacillus population involved in the fermentation of cassava (Manihot esculenta Crantz) leaves for the production of Ntoba Mbodi, a Congolese food. Ninety bacteria were identified by phenotyping and genotyping using ITS-PCR, rep-PCR, and sequencing of the 16S rRNA, gyrA, gyrB and rpoB genes. Moreover, the isolates were screened for the presence of genes coding for haemolytic (HblC, HblD) and non-haemolytic enterotoxins (NheA, NheB and NheC), cytotoxin K (CytK) and emetic toxin (EM1) as well as their ability to produce haemolysin.The investigations revealed the predominance (72.2%) of species of the Bacillus pumilus group i.e. B. safensis (48), B. pumilus (7), and B. pumilus sensu lato (10). Other species of Bacillus including B. cereus sensu lato (11), B. megaterium (4), B. subtilis (4), B. amyloliquefaciens (2), B. siamensis (2), B. licheniformis (1) and Lysinibacillus louembei (1) were also identified. Haemolytic, non-haemolytic and cytokin toxin genes were detected in the B. cereus strains which were also able to produce haemolysin. The emetic toxin gene was not detected in any isolates. The toxin genes screened were not detected in any of the non B. cereus species.     

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.     

Large-volume immunomagnetic separation combined with multiplex PCR assay for simultaneous detection of Listeria monocytogenes and Listeria ivanovii in lettuce

Multiplex polymerase chain reaction (mPCR) has been widely used for the simultaneous detection of various target bacteria in vegetables. However, an enrichment period is necessary to improve the sensitivity of the mPCR method. In this paper, large-volume (10 mL) immunomagnetic separation (IMS) combined with mPCR for the rapid detection of Listeria monocytogenes and Listeria ivanovii in lettuce without further enrichment process is reported for the first time. Various parameters that affected the capture efficiency (CE) of IMS, including the amounts of streptavidin and biotinylated anti-Listeria monoclonal antibodies on the surface of magnetic nanobeads, the amount of immunomagnetic beads, immunoreaction time, and magnetic separation time, were systematically investigated. Moreover, the concentrations of primers, PCR conditions, and genomic DNA isolation for mPCR assay were optimized. Under optimum conditions, the CE of large-volume IMS for L. monocytogenes and L. ivanovii was greater than 90% when the concentration of target bacteria was less than 10⁶ CFU/mL in pure culture, and was more than 80% when the concentration was below 10⁵ CFU/mL in lettuce samples. The limit of detection of IMS combined with mPCR assay reached as low as 1.0 CFU/mL in pure culture and 10 CFU/g in lettuce. The overall assay time, including sample preparation, large-volume IMS, and mPCR assay, took less than 7 h. In summary, the developed large-volume IMS-based mPCR system exhibits great potential for routine screening detection of foodborne pathogenic bacteria for safety monitoring.     

Assessment of microbiological quality and safety of marinated pork products from German retail during shelf life

Foodborne diseases are of major concern for public health. Here we assess the microbiological quality and safety of marinated pork steaks (n = 300) and marinades (n = 30) which were used for the production of marinated steaks by analyzing quantitative microbiological parameters and foodborne pathogens. Salmonella spp. and Listeria monocytogenes were isolated from about 2%, Staphylococcus aureus from 8% and Bacillus cereus from 21% of the steaks. One steak was MRSA-positive and one contained EHEC/STEC. B. cereus was the only pathogen detected in the marinades. Similar toxin patterns of B. cereus strains from meat and marinades suggested that a contamination of meat with B. cereus occurred via marinades.     

Whole soybean as probiotic lactic acid bacteria carrier food in solid-state fermentation

For the purpose of preparing lactic acid bacteria (LAB) carrier food, the solid-state fermentation of whole soybean was performed using Bifidobacterium animalis 937, Lactobacillus casei Zhang and Lactobacillus plantarum P-8 mixed with Bacillus subtilis natto, respectively. The physicochemical properties, the amino nitrogen content and peptide molecular weight distribution of the fermented whole soybean products were examined during this process. After 48 h of fermentation, the viable counts of the three samples were 1.41 × 10⁸ CFU/g (B. animalis 937), 1.74 × 10¹⁰ CFU/g (L. casei Zhang) and 2.19 × 10¹⁰ CFU/g (L. plantarum P-8), with the pH declined rapidly from 6.32 to 5.78, 5.60 and 5.44 at the early stage of the fermentation and increased to 6.71, 6.47 and 6.60 at the later stage of the fermentation. The fermentation caused a sharply increase in the content of the free amino nitrogen from 99.7 μmol/g to 301.9 μmol/g, 390.1 μmol/g and 529.1 μmol/g in the solid fermented soybean products, due to the multiplication of microorganism and the effect of enzyme system. Furthermore, the levels of soybean peptide with molecular weight less than 1000 Da increased 30.7%, 71.2% and 81.3% relative to that of the control group at 48 h. The result of the present work implied that whole soybean fermented by LAB can provide the different probiotics for the host, and there is potential to develope nutritious fermented soybean products.     

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.     

PMA based real-time fluorescent LAMP for detection of Vibrio parahaemolyticus in viable but nonculturable state

Vibrio parahaemolyticus is a crucial foodborne pathogen. The viable but non-culturable (VBNC) state of V. parahaemolyticus cannot be detected by traditional culture methods. The objective of this study was to develop and evaluate a method that combines propidium monoazide (PMA) treatment with real-time fluorescent loop-mediated isothermal amplification (RF-LAMP) to detect and quantify VBNC cells of V. parahaemolyticus. Different states of cells were treated with PMA in dark for 5 min and subsequently exposed to a 650 W halogen lamp for 5 min. The cells were collected and DNA was amplified by RF-LAMP. The primers which targeted six distinct regions in the tlh gene of V. parahaemolyticus were used for the PMA-RF-LAMP assay. The results indicated that the treatment with 4 μg/mL of PMA and a further exposure to light for 5 min was suitable for PMA-RF-LAMP to distinguish viable cells from dead cells of V. parahaemolyticus. The developed assay exhibited remarkably high specificity, sensitivity and rapidity, without any cross reaction with the tested non-V. parahaemolyticus strains. There was good linear correlation between Ct values and log copy/mL in the range of 6.8–6.8 × 10⁷ copy/mL, with the reaction endpoints less than 30 cycles (30 min). It could detect as low as 14 copy/g of V. parahaemolyticus in spiked seafood samples (pomfret, shrimp, scallop, oyster and salted fish) without any interference from food matrices, dead cells and other bacteria.     

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.