Andreja Rajkovic

Heat resistance of Bacillus cereus emetic toxin, cereulide

Aims:  The study describes the effects of heating temperature and exposure time on the thermal stability of cereulide under different conditions (pH, presence/absence of oil phase and cereulide concentration).

Regulation of toxin production by Bacillus cereus and its food safety implications

Toxin expression is of utmost importance for the food-borne pathogen B. cereus, both in food poisoning and non-gastrointestinal host infections as well as in interbacterial competition. Therefore it is no surprise that the toxin gene expression is tightly regulated by various internal and environmental signals. An overview of the current knowledge regarding emetic and diarrheal toxin transcription and expression is presented in this review. The food safety aspects and management tools such as temperature control, food preservatives and modified atmosphere packaging are discussed specifically for B. cereus emetic and diarrheal toxin production.

Evaluation of a challenge testing protocol to assess the stability of ready-to-eat cooked meat products against growth of Listeria monocytogenes

Challenge testing of ready-to-eat (RTE) foods with Listeria monocytogenes is recommended to assess the potential for growth. The present study was undertaken to evaluate a protocol for challenge testing applied to RTE cooked meat products. In order to choose L. monocytogenes strains with a representative behaviour, initially, the variability of the response of multiple L. monocytogenes strains of human and food origin to different stress and growth conditions was established. The strains were not inhibited in their growth at moderate acid pH (5.25) and the four strains tested in particular showed a similar acid-adaptive response. Growth of the various strains under four different combined stress conditions indicated that no L. monocytogenes strain had consistently significant longer or shorter lag phase or higher or lower maximum specific growth rates. The effect of choice of strain and history (pre-incubation temperature 7 or 30 degrees C) on growth of L. monocytogenes under optimum conditions (Brain Heart Infusion, BHI) and modified BHI simulating conditions of cooked ham and paté was studied. In general, all four L. monocytogenes strains behaved similarly. In BHI, no difference in lag phase was observed for the cold-adapted and standard inoculum, whereas in BHI adjusted to ham and pâté conditions, a ca. 40-h reduction of the lag phase was noted for the cold-adapted inoculum. Subsequently, microbial challenge testing of L. monocytogenes in modified atmosphere packaged sliced cooked ham and paté was performed. A mixed inoculum of four L. monocytogenes strains and an inoculum level of ca. 1-10 cfu/g was used. On vacuum packed sliced cooked ham, the concentration of 100 cfu/g, the safety limit considered as low risk for causing listeriosis, was exceeded after 5 days whereas ca. 10(5) cfu/g were obtained after 14 days when also LAB spoilers reached unacceptable numbers (ca. 10(7) cfu/g) whether standard or cold-adapted inoculum was used. The concentration of sodium lactate determined the opportunities for growth of L. monocytogenes in pâté. If growth of L. monocytogenes in pâté was noticed, the threshold of 100 cfu/ml was crossed earlier for the cold-adapted inoculum compared to the standard inoculum.

Characterization of Escherichia coli from raw poultry in Belgium and impact on the detection of Campylobacter jejuni using Bolton broth.

A comparative study examining Bolton broth and Preston broth for enrichment and reliable detection of Campylobacter jejuni (both healthy and freeze stressed cells) was performed. Tested as pure cultures, Bolton broth enabled faster resuscitation and growth of C. jejuni compared to Preston broth. When C. jejuni was co-incubated with extended-spectrum-beta-lactamase (ESBL) producing Escherichia coli isolated from Belgian poultry meat preparations, the latter dominated in the Bolton enrichment broth and crowded the mCCDA plates. This resulted in the inability to recover C. jejuni by ISO 10272-1:2006 standard method. Preston broth did not support the growth of the ESBL E. coli isolates, but showed longer detection time of C. jejuni compared to Bolton broth. The use of the same antibiotic (sodium cefoperazone) in Bolton broth and in mCCDA plates may explain the problems encountered for detection of C. jejuni, as high numbers of ESBL E. coli present after enrichment in Bolton broth, also caused overgrowth and masked the few C. jejuni colonies present on the mCCDA plates. The use of Campylobacter spp. specific real-time PCR circumvented these problems and enabled rapid detection of the pathogen after 24h enrichment in both Bolton and Preston broth, for both healthy and freeze stressed cells.

Resistance of Listeria monocytogenes, Escherichia coli O157:H7 and Campylobacter jejuni after exposure to repetitive cycles of mild bactericidal treatments

While maintaining nutritional and sensorial attributes of fresh foods mild processing technologies generally deliver microbiologically perishable food products. Currently little information exists on possible increase in the resistance of pathogens after repetitive exposure to mild (sub-lethal) treatments. Multiple strain-cocktails of Listeria monocytogenes, Escherichia coli O157:H7 and Campylobacter jejuni were exposed to 20 consecutive cycles of sub-lethal inactivation by three different techniques. Used techniques comprised inactivation with lactic acid (LA), chlorine dioxide (ClO(2)) and intense light pulses (ILP). Results showed that the selection of resistant cells was both species and technique dependent. While repetitive cycles of ClO(2) treatment did not result in increased resistance, repetitive inactivation with LA yielded L. monocytogenes culture of higher resistance in comparison to the parental culture. The increased resistance, expressed as decreased level of reduction in bacterial counts in subsequent inactivation cycles, was also observed with ILP for both L. monocytogenes and E. coli O157:H7 strains. Visual trend observations were confirmed through statistical linear regression analysis. No such effects were noted for C. jejuni which became undetectable after first 2-5 cycles. Current findings indicate the ability of foodborne pathogens to adapt to mild bactericidal treatments creating new challenges in risk assessment and more specifically in hazard analysis.

Influence of type of food on the kinetics and overall production of Bacillus cereus emetic toxin

Potato puree and penne pasta were inoculated with cereulide producing B. cereus 5964a and B. cereus NS117. Static incubation at 28 degrees C proved these two foods to be a better substrate for higher cereulide production (4,080 ng/g in puree and 3,200 ng/g in penne were produced by B. cereus 5964a during 48 h of incubation) compared with boiled rice (2,000 ng/g). This difference occurred despite B. cereus counts of more than 10(8) CFU/g in all three products. Aeration of cultures had a negative effect on cereulide production, causing concentrations more than 10-fold lower than in some statically incubated samples. Cereulide production remained undetectable in shaken milk, whereas it reached 1,140 ng/ml in statically incubated milk. At 12 and 22 degrees C, presence of background flora was also a determinative factor. A total B. cereus count of more than 106 CFU/ml did not necessarily lead to uniform cereulide production and was also dependent on the B. cereus strain involved. In this study, we confirm that a number of factors play a crucial role in the determination of the extent to which, if at all, cereulide will be produced. Among those, type of the food, temperature, pH, and whether additional aeration (via incubation on an orbital shaker) is induced had an important role. An important effect was also induced by the cereulide-producing strain involved.

Immunoquantitative Real-Time PCR for Detection and Quantification of Staphylococcus aureus Enterotoxin B in Foods

ABSTRACT A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of immunocapture of SEB and real-time PCR amplification of the DNA probe linked to the detection antibody. iqPCR was compared to an in-house enzyme-linked immunosorbent assay (ELISA) using the same couple of capture-detection antibodies and to commercial kits for detection of S. aureus enterotoxins (SE). The iqPCR was approximately 1,000 times more sensitive (<10 pg ml−1) than the in-house ELISA and had a dynamic range of approximately 10 pg ml−1 to approximately 30,000 pg ml−1. iqPCR was not inhibited by any of the foods tested and was able to detect SEB present in these foods. No cross-reactivity with SE other than SEB was observed. Application of iqPCR for detection of SEB in cultures of S. aureus revealed the onset of SEB production after 4 h of incubation at 22, 37, and 42°C, which was in the first half of the exponential growth phase. The total amounts of SEB produced by the two strains tested were larger at 42°C than at 37°C and were strain dependent.

Multi-method approach indicates no presence of sub-lethally injured Listeria monocytogenes cells after mild heat treatment

Application of mild inactivation treatments follows an increasing trend in the food industry and is often combined with sub-optimal intrinsic product conditions to ensure appropriate level of microbial safety. Listeria monocytogenes was subjected to mild heat treatment (20 min at 60 degrees C) and subsequently exposed to various mild preservation conditions based on increased NaCl concentration and decreased pH. Recovery and resuscitation of L. monocytogenes cells were studied using various methods. Using 12-fold Most Probable Number (MPN) method no difference in the amount of recovered cells under adverse conditions was noted between heat-treated and non-treated L. monocytogenes cells. Time-to-detection method using on-line OD measurements showed that heat-treated L. monocytogenes cells reached detection limit faster in acidified media and NaCl supplemented media in comparison with non-heated control cells. Flow cytometry (FCM) analysis using 5-6-carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) staining showed presence of low numbers of viable cells. Overall, there was no indication of sub-lethal injury in L. monocytogenes cells after mild heat treatment.

Enterotoxin Production by Bacillus cereus Under Gastrointestinal Conditions and Their Immunological Detection by Commercially Available Kits

Currently, three commercial kits for Bacillus cereus enterotoxins Nhe and/or Hbl detection are available, namely, the Bacillus diarrheal enterotoxin visual immunoassay (BDE VIA™) kit (3M Tecra), B. cereus enterotoxin reversed passive latex agglutination (BCET-RPLA) kit (Oxoid), and the Duopath(®) Cereus Enterotoxins (Merck). The performance of the kits and their applicability to gastrointestinal simulation samples were evaluated. Then, the stability and production of enterotoxins Hbl and Nhe under gastrointestinal conditions were investigated. Enterotoxin production was absent or impaired at acidic pH, i.e., in gastric medium with pH 5.0 and lasagne verde with pH 5.5. B. cereus did produce enterotoxins Nhe and Hbl during anaerobic growth in intestinal medium at pH 7.0, but the toxins were instantly degraded by the enzymes in the hosts digestive secretions. Preformed enterotoxins did not withstand gastrointestinal passage under the simulated conditions, which suggests that preformed enterotoxins in food do not contribute to the diarrheal food poisoning syndrome. In conclusion, diarrhea is probably caused by de novo enterotoxin production by B. cereus cells located closely to the hosts intestinal epithelium.

Effects of CO2 on the resuscitation of Listeria monocytogenes injured by various bactericidal treatments

To assure the microbiological safety and quality of a food product, a combination of preservation hurdles is often used. Therefore, the effects of carbon dioxide at concentrations of 0, 20, 40 and 60% in modified atmospheres on the resuscitation of Listeria monocytogenes cells injured by mild bactericidal treatments during storage at 7 degrees C were examined. The bactericidal treatments were intense light pulses (ILP), chlorine dioxide (ClO(2)), lactic acid (LA) and heat. The results indicated additional bactericidal effects of CO(2) on cultures treated with LA, ClO(2) and ILP, with additional reductions in viable L. monocytogenes of 0.5-1.0 log cfu/ml. Lag phase duration was significantly different between the different treatments, with non-treated cells having the shortest lag phase, followed by that of heat, intense light pulses, lactic acid and finally ClO(2) treated cells. Maximum growth rate was also estimated and results showed a negative correlation with increasing CO(2) concentrations. A relationship was found between the amount of sub-lethally damaged cells after a mild inactivation treatment and the lag phase duration in the CO(2) environment. Current findings demonstrate the possibility that combining mild decontamination treatments and packaging in a CO(2) enriched environment could reduce the risk of L. monocytogenes infections in food due to an extension of the lag phase.