Nachweis von Clostridium botulinum Typ A, B, E und F mittels real-time-PCR

Clostridium botulinum is a Gram-positive, anaerobic, spore-performing bacterium with the ability to produce under certain conditions a protein with a characteristical neurotoxicity. Intoxications with C. botulinum toxin belong to the rare occuring food-poisonings; the mortality, however, is very high. C. botulinum produce seven different toxin types (type A to G), human intoxications are currently described to caused by toxin type A, B, E and F. C. botulinum is a strictly anaerobic growing bacterium, so the risk for the consumer’s health is mainly due to non-commercially produced food cans. A special form of botulism is the „infant botulism“. In contrast to the botulism of adults, where the disease is caused through toxin-containing food, spores of C. botulinum can sporulate and produce toxin in the intestines of an infant. The source of infant botulism can be honey, because it contains as a natural product C. botulinum spores. Because of the difficult and time-consuming cultural detection of C. botulinum, PCR methods to screen for the toxin genes A, B, E and F, which are relevant in the human medicine, have been used increasingly during the last years. In this presentation two real-time-PCR assays for C. botulinum, which can be applied in the routine laboratory, will be shown.     

Incidence and Heat Resistance of Clostridium botulinum Type E Spores in Menhaden Surimi

Raw menhaden surimi (Brevoortia tyrannus) was examined to determine the incidence of Clostridium botulinum spores. Seven of 565 test portions (1.2%) were positive for type E spores. The thermal death time (TDT) tube method was used to determine the heat resistance of C. botulinum type E spores inoculated into the remaining 558 negative portions. Calculated mean D-values in mm were 8.66 at 73.9°C, 3.49 at 76.7°C, 2.15 at 79.4°C, and 1.22 at 82.2°C. The z-value of the phantom TDT curve was 9.78C^o. Our data indicate previously reported minimal time/temperature thermal processes used to set surimi gel provide an adequate margin of safety with regard to C. botulinum type E.     

Inhibition of Clostridium botulinum by Antioxidants and Related Phenolic Compounds in Comminuted Pork

Antioxidants, esters of p-hydroxybenzoic acid and gallic acid, and related phenolic compounds were evaluated for their activity against growth and toxin production of C. botulinum types A and B in comminuted pork. With an inoculum level of 8,000 spores/g of meat most of the chemicals at a concentration of 1,000 ppm delayed first swell formation for less than 3 days beyond the control. For most compounds, the time to first swell formation beyond that of the control increased as the spore level per g of meat decreased. 8-Hydroxyquinoline at a concentration of 200 ppm or in combination with sodium nitrite (40 ppm) inhibited the growth and toxin production of C. botulinum in comminuted pork for 60 days at 27°C. 8-Hydroxyquinoline was more active in inhibition of growth and toxin production of C. botulinum in comminuted pork than in prereduced Thiotone-yeast extract-glucose broth.     

Growth and Toxin Production by Clostridium botulinum in Model Acldified Systems

TPGY medium was acidified to pH 4.20, 4.60, 5.00 and 5.40 with acetic or citric acid. The media were inoculated with spores from three strains of C. botulinum type A, B or E. Growth, pH, and toxin production under anaerobic conditions were monitored for 8 wk. Spores of C. botulinum types A and B were incapable of outgrowth and toxin production at pH 4.60 or below when incubated at 35°C. Spores of C. botulinum type E were capable of growth and toxin production at 26°C in citric acid acidified systems at pH 4.20. Growth and toxin production were not detected below pH 5.00 when acetic acid was used.     

Inhibition of Clostridium botulinurn Type E in Model Acidified Food Systems

The potential for growth and toxigenesis of a mixture of three strains of C. botulinum type E in model food systems at pH levels of 4.600 and 4.200 was investigated. Whole shrimp, shrimp puree, tomato puree, and tomato and shrimp puree were acidified to pH levels of 4.200 and 4.600 with acetic or citric acid. Inoculated and uninoculated control samples were tested during 8 wk incubation at 26°C. No significant C. botulinum growth or toxigenesis was detected in the food systems over the 8 wk test period and the pH of the various foods remained generally constant. This investigation affirms the safety of the generally accepted inhibitory pH level of 4.6 in the food systems studied.     

Antibacterial action on pathogenic bacterial spore by green tea catechins

Antibacterial effects of catechins, the major green tea polyphenols, were studied using Clostridium and Bacillus spores. Incubation with crude catechins decreased the number of C botulinum and C butyricum spores but not B cereus spores. Furthermore, the effects of six catechin derivatives on spores were investigated. (−)‐Epicatechin gallate (ECg), (−)‐epigallocatechin (EGC), (−)‐epigallocatechin gallate (EGCg) and (+)‐gallocatechin gallate (GCg) were more effective in decreasing C botulinum and C butyricum spore numbers than (+)‐catechin (C) and (−)‐epicatechin (EC). The vegetative growth of C botulinum and B cereus was inhibited by crude extracts of the catechins. Specifically, purified GCg and EGCg inhibited the vegetative growth of C botulinum and B cereus. The inhibitory effect of ECg on B cereus was similar to that of GCg. However, toxin‐production by B cereus was not inhibited by catechin. Damage to the membrane of C butyricum spores by catechin derivatives was shown using fluorescent microscopy. This study shows that low concentrations of catechins, although requiring a long exposure time, inhibited the growth of bacterial spores. However, the effects of the purified derivatives of the catechins were not the same and GCg and EGCg were found to be the most potent. Spores that are generally resistant to many disinfectants were sensitive to catechins. Copyright © 2005 Society of Chemical Industry     

Detection of C. Botulinum Types in Honey by mPCR

The aim of this study was to determine the prevalence of Clostridium botulinum in honey samples using conventional methods and multiplex PCR (mPCR). A total number of 150 honey samples were randomly collected from apiaries, retail shops, weekly open bazaars, and supermarkets in Samsun, Turkey. Of 150 honey samples, 4 (2.6%) were positive for the botulinum neurotoxin gene by mPCR analysis. A total of 4 C. botulinum isolates were obtained from the mPCR positive samples, of which 3 were type A and 1 was type B. No samples were positive regarding the type E and type F neurotoxin genes. This is the first report of type A and type B spores of C. botulinum being detected and isolated in Turkey. This study revealed that some honey samples may present a potential hazard for food borne and infant botulism.     

Nachweis von Clostridium botulinum Typ A, B, E und F mittels real-time-PCR

Clostridium botulinum is a Gram-positive, anaerobic, spore-performing bacterium with the ability to produce under certain conditions a protein with a characteristical neurotoxicity. Intoxications with C. botulinum toxin belong to the rare occuring food-poisonings; the mortality, however, is very high. C. botulinum produce seven different toxin types (type A to G), human intoxications are currently described to caused by toxin type A, B, E and F. C. botulinum is a strictly anaerobic growing bacterium, so the risk for the consumer’s health is mainly due to non-commercially produced food cans. A special form of botulism is the „infant botulism“. In contrast to the botulism of adults, where the disease is caused through toxin-containing food, spores of C. botulinum can sporulate and produce toxin in the intestines of an infant. The source of infant botulism can be honey, because it contains as a natural product C. botulinum spores. Because of the difficult and time-consuming cultural detection of C. botulinum, PCR methods to screen for the toxin genes A, B, E and F, which are relevant in the human medicine, have been used increasingly during the last years. In this presentation two real-time-PCR assays for C. botulinum, which can be applied in the routine laboratory, will be shown.

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Zusammenfassung: Clostridium botulinum z?hlt zu den anaeroben sporenbildenden Bakterien, die unter bestimmten Bedingungen in der Lage sind, sich in Lebensmitteln zu vermehren und ein Protein mit charakteristischer Neurotoxizit?t zu bilden. Intoxikationen mit Clostridium botulinum-Toxin geh?ren zu den seltenen lebensmittelassoziierten Intoxikationen; die Mortalit?t bei einer Erkrankung ist allerdings sehr hoch. C. botulinum produziert sieben unterschiedliche Toxintypen (Typ A-G), wobei für menschliche Erkrankungsf?lle bisher die Toxintypen A, B, E und F beschrieben sind. Da es sich bei den genannten Keimen um strikt anaerob wachsende Bakterien handelt, stellen vor allem nicht kommerziell hergestellte Konserven, wie z. B. Kesselkonserven, ein Risiko für den Verbraucher dar. Als besondere Form des Botulismus wird der so genannte „S?uglingsbotulismus“ beschrieben. Im Gegensatz zur der Erkrankung, die bei Erwachsenen auftritt und die durch die Aufnahme des bereits toxinhaltigen Lebensmittels verursacht wird, k?nnen Sporen von C. botulinum im Darm von S?uglingen auskeimen und dort Toxine bilden. Ursache für den S?uglingsbotulismus ist h?ufig Honig, der als Naturprodukt C. botulinum-Sporen enthalten kann. Da der kulturelle Nachweis von C. botulinum aufwendig und eine endgültige Differenzierung schwierig ist, wird im Bereich der Routinediagnostik seit einigen Jahren verst?rkt mit PCR-Nachweisverfahren gearbeitet, die ein schnelles Screening auf das Vorhandensein der vier in der Humanmedizin relevanten Toxingene A, B, E und F erm?glichen. In dieser Arbeit werden zwei real-time-PCR-Systeme vorgestellt, die im Bereich der Routinediagnostik einsetzbar sind.

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Eingegangen: 19. Januar 2007     

Temperature, pH, and Spore Load Effects on the Ability of Nisin to Prevent the Outgrowth of Clostridium botulinum Spores

The effectiveness of nisin in preventing the outgrowth of spores of Clostridium botulinum types A, B, and E in TPYG broth was profoundly affected by pH, temperature of heat-shocking, length of the heat-shocking period, and spore load. Nisin was considerably more effective at pH 6 than at either pH 7 or pH 8 in limiting the outgrowth of all six tested strains. Heat-damaged spores were also more sensitive to nisin. Both higher heat-shocking temperatures in the range 20-30°C higher than the optimal heat-shocking temperatures for the particular strain and longer heat-shocking periods served to lower the levels of nisin required to inhibit spore outgrowth. Nisin was more effective against spore loads of 10²spores/ml. than higher spore loads of 10³ or 10⁴ spores/ml with all of these variables taken into consideration, the order of sensitivity of the spores of the various strains of C. botulinum was strain 56A < strain 69A < strain 113 B = strain 213 B < strain Beluga E < strain Minnesota E     

Thermal resistance ofAlicyclobacillus acidoterrestrisspores in different buffers and pH

The influence of buffers and pH on the thermal resistance of a novel flat sour-type of spoilage bacterium,Alicyclobacillus acidoterrestris, was investigated. At a low concentration (20 mM), decimal reduction times (D-values) in citrate and phosphate buffers were 13.6 and 12.9 min, respectively, showing little variation. At a high concentration (100 mM), however, theD-value in citrate buffer, 14.4 min, was significantly higher than that in the phosphate buffer, 12.3 min.D-values in McIlvaine buffers did not vary widely within the pH range of 3.0 to 8.0. At 88, 90, 92 and 95°C in McIlvaine buffers,D-values were 24.1–29.1, 14.8–16.8, 5.7–7.1 and 2.2–2.8 min, respectively. The thermal resistance ofA. acidoterrestrisspores was affected very little by varying the pH of the heating menstruum. TheZ-values also did not vary widely as the pH varied among the different buffers, ranging from 6.4 to 7.1°C. It was evident thatA. acidoterrestrisspores showed a special relationship, unknown among other bacterial spores, between their thermal resistance and the pH of the heating menstruum. Therefore,A. acidoterrestrisshould be targeted when the quality of acidic food products is being tested.     

Antagonistic effect of fat on the antibotulinal activity of food preservatives and fatty acids

The effect of fat on the antibotulinal activity of 11 food preservatives, 12 free fatty acids, and nine lots of enzyme-modified cheese (EMC) was evaluated in a media system. Anhydrous milkfat or soybean oil was added to tubes of Trypticase–peptone–glucose–yeast extract medium (TPGY) supplemented with the additives (final pH adjusted to 5.9). Treatments were inoculated with 3-log₁₀ proteolytic Clostridium botulinum spores/ml (10-strain mixture of serotypes A and B) and incubated anaerobically at 30°C for up to 14 days. For the preservative and fatty acids studies, growth of C. botulinum was determined by measuring optical changes at OD_{640 nm}. Botulinal toxin production was determined in EMC-treatments using the mouse bioassay. Data revealed that the antibotulinal effects of nisin, and free fatty acids caprylic, capric, lauric, myristic, oleic, and linoleic acids were each significantly reduced in treatments supplemented with 20% fat (P<0.05). Similar trends were observed in TPGY supplemented with 20% fat and potassium sorbate, sorbic acid, monolaurin, polyphosphate emulsifier, or EDTA–lysozyme, but the differences were reduced. Fat was also antagonistic to the antibotulinal activity of five EMC-treatments. This study suggests that fat may reduce the efficacy of some antimicrobials added to or found naturally in foods.     

Effect of the Parabens With and Without Nitrite on Clostridium botulinum and Toxin Production in Canned Pork Slurry

Antimiciobials were evaluated in thioglycollate broth at pH 6.5 for the ability to inhibit growth and toxin production by C. botulinum 12885A and ATCC 7949 (Type B). Methyl, ethyl, propyl, and butyl parabens (0.1%) and sorbic acid (0.2%) were effective in inhibiting growth of C. botulinum 12885A and ATCC 7949 in broth. Ethyl, propyl, and butyl parabens (0.1%) and sorbic acid (0.2%) inhibited toxin production by both strains in culture medium. Ethyl, propyl, butyl parabens (0.1%) and sorbic acid (0.2%) were individually added to a comminuted pork slurry having salt and sugar, with or without 40 ppm sodium nitrite. Cans were inoculated with a mixture of C. botulinum 12885A and ATCC 7949 spores. The canned product was abused by holding at 27°C and was observed over a 3-month period for swollen cans. Swollen cans were examined for botulinal toxin by the mouse bioassay. Propyl and butyl paraben did not inhibit or delay toxin production. Ethyl paraben with or without nitrite delayed toxin production for 4 wk. Sorbic acid inhibited toxin for 3 wk; when 40 ppm nitrite was added to the sorbic acid treatment, toxin production was delayed for 4 wk.     

Effect of input data variability on estimations of the equivalent constant temperature time for microbial inactivation by HTST and retort thermal processing

Abstract: Consumer demand for food safety and quality improvements, combined with new regulations, requires determining the processor's confidence level that processes lowering safety risks while retaining quality will meet consumer expectations and regulatory requirements. Monte Carlo calculation procedures incorporate input data variability to obtain the statistical distribution of the output of prediction models. This advantage was used to analyze the survival risk of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) and Clostridium botulinum spores in high‐temperature short‐time (HTST) milk and canned mushrooms, respectively. The results showed an estimated 68.4% probability that the 15 sec HTST process would not achieve at least 5 decimal reductions in M. paratuberculosis counts. Although estimates of the raw milk load of this pathogen are not available to estimate the probability of finding it in pasteurized milk, the wide range of the estimated decimal reductions, reflecting the variability of the experimental data available, should be a concern to dairy processors. Knowledge of the C. botulinum initial load and decimal thermal time variability was used to estimate an 8.5 min thermal process time at 110 °C for canned mushrooms reducing the risk to 10^?9 spores/container with a 95% confidence. This value was substantially higher than the one estimated using average values (6.0 min) with an unacceptable 68.6% probability of missing the desired processing objective. Finally, the benefit of reducing the variability in initial load and decimal thermal time was confirmed, achieving a 26.3% reduction in processing time when standard deviation values were lowered by 90%. Practical Application: In spite of novel technologies, commercialized or under development, thermal processing continues to be the most reliable and cost‐effective alternative to deliver safe foods. However, the severity of the process should be assessed to avoid under‐ and over‐processing and determine opportunities for improvement. This should include a systematic approach to consider variability in the parameters for the models used by food process engineers when designing a thermal process. The Monte Carlo procedure here presented is a tool to facilitate this task for the determination of process time at a constant lethal temperature.     

Production and Gelatin Entrapment of Laccase from Trametes versicolor and its Application to Quantitative Determination of Phenolic Contents of Commercial Fruit Juices

Laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) is a particularly promising enzyme for several industrial fields, including food industries, since this enzyme catalyzes the oxidation of ortho and para-diphenols, amino-phenols, polyphenols, polyamines, lignins, and aryl diamines as well as some inorganic ions coupled to the reduction of molecular dioxygen to water. In this study, laccase was produced from one of the best laccase-producing organisms, Trametes versicolor. For this purpose, several phenolic acids were tested as laccase inducers. Caffeic acid and ferulic acid were determined to be the best inducers among the tested phenolic acids. Also, it was shown that laccase activity could be determined by using caffeic acid and ferulic acid as phenolic substrates by measuring the rates of oxygen consumption. Laccase was immobilized in gelatin under optimized conditions. Kinetic constants K _m and V _max for immobilized enzyme were estimated to be 74.758 μM and 0.744 μmol.ΔO₂/ml.min for caffeic acid and 0.999 μM and 57.80μ mol.ΔO₂/ml.min for ferulic acid, respectively. The immobilized enzyme exhibited the maximal activity at pH 4.5, and at 35°C. Immobilized enzymes were used for the determination phenolic contents of commercially prepared fruit juices. Caffeic acid contents of black cherry, apricot, and peach juice were determined to be 1640±33, 679±24 and 408±29 mg/L, and their ferulic acid contents were determined to be 1786±28, 800±30, and 444±28 mg/L, respectively.     

Inhibition of Clostridium botulinum Growth from Spore Inocula in Media Containing Sodium Acid Pyrophosphate and Potassium Sorbate with or without added Sodium Chloride

The effects of sodium acid pyrophosphate (SAPP), sodium chloride (NaCl) and/or potassium sorbate (PS) on the growth from heat-activated spores of three individual strains or a mixture of ten strains of Clostridium botulinum in peptone-yeast extract-glucose broth at pH 5.55 or 5.85 were measured spectrophotometricalty at A_630nm. Growth ratios (GR = treatment/control) based on time to reach A₆₃₀= 0.35 or 0.04 were calculated and used to compare effects of additives on strains. SAPP, NaCl, PS, and pH exhibited independent significant main effects (p≦0.01) on delaying growth in most C. botulinum strains tested. Combinations of additives without NaCl consistently caused an increase in the GR and an increase in organism sensitivity to additives in the medium. Treatments containing SAPP (0.2 or 0.4%) and PS (0.13 or 0.26%) were more effective for delaying growth than other formulations tested.     

Proteolytic Clostridium botulinum growth at 12–48°C simulating the cooling of cooked meat: development of a predictive model

The objective of this study was to develop a model to predict the germination, outgrowth and lag (GOL), and exponential growth rates of Clostridium botulinum from spores at temperatures (12–48°C) applicable to the cooling of cooked meat products. The growth medium, Reinforced Clostridial medium (RCM) supplemented with oxyrase enzyme to create suitable anaerobic conditions, was inoculated with approximately 4 log₁₀spores ml⁻¹. Clostridium botulinum populations were determined at appropriate intervals by plating onto RCM. Clostridium botulinum growth from spores was not observed at temperatures <12°C or >48°C for up to 3 weeks. Growth curves were determined by fitting Gompertz functions to the data. From the parameters of the Gompertz function the growth characteristics, GOL times and exponential growth rates were calculated. These growth characteristics were subsequently described by Ratkowsky functions using temperature as the independent variable. Closed form equations were developed that allow for predicting relative growth for a general cooling scenario. By applying multivariate statistical procedures, the standard errors and confidence intervals were computed on the predictions of the amount of relative growth for a cooling scenario. The predictive model is capable of predicting spore outgrowth and multiplication for general cooling scenarios, for suitable but un-verified mathematical assumptions, and should aid in evaluating the safety of cooked products after cooling.     

Thermal Inactivation of Clostridium botulinum Type E Spores in Oyster Homogenates at Minimal Processing Temperatures

D-values of Clostridium botulinum type E in oyster homogenates were calculated from the slopes of survivor curves for each variable tested (control, 1.0% NaCl, 0.13% potassium sorbate (KS), and a combination of NaCl and KS) at five temperatures. Mean D-values ranged from 0.78 min at 80°C to 7943 min at 50°C. The addition of salt and/ or KS affected heat resistance of the spores, but differences were not consistent among the temperatures tested, z-Values calculated for the curve between 70°C and 80°C ranged from 6.9 C° to 7.6 C°, while z-values calculated for the curve between 55°C and 70°C were 12.1–14.70°. These data indicate that C. botulinum type E would survive in oysters that are minimally processed. A suitable process could be determined using the data presented; however, oysters would have a cooked appearance.     

Effect of Irradiation on Antibotulinal Efficacy of Nitrite

Ground pork meat was cured with the addition of 0, 50, 100, 156, and 200 mg/kg NaNO₂, pasteurized and irradiated with 0, 3, 6, 9, 10, 20, 30, 40, and 50 kGy. After irradiation the samples were inoculated with a mixture of Clostridium botulinum type A, B and E spores, incubated at 30°C for 28 days and tested for the presence of botulinal toxin. Doses up to 9 kGy caused a decrease of NaNO₂ content with a corresponding decrease in antibotulinal activity. High irradiation doses resulted in a dose-dependent decrease in nitrite content with complete loss of antibotulinal activity.     

Effect of Nisin on the Outgrowth of Clostridium botulinum Spores

Nisin, an antibiotic produced by certain strains of Streptococcus lactis, is effective in preventing the outgrowth of Clostridium botulinum spores. Type A C. botulinum spores were the most resistant to the inhibitory action of nisin requiring 1000-2000 I.U. of nisin/ml for a 50% inhibition of outgrowth on TPYG agar plates. Type E spores were more sensitive requiring only 50-100 I.U./ml for 50% inhibition of outgrowth on TPYG agar plates. Type B spores displayed an intermediate level of sensitivity requiring 500-1000 I.U. of nisin/ml for 50% inhibition of outgrowth on TPYG agar plates. Similar levels of nisin were necessary to prevent spore outgrowth in TPYG broth and BHI broth over a 7-day incubation period. With prolonged incubation periods of up to 65 days in TPYG broth, spore outgrowth was observed sporadically at higher nisin levels with the type A and B spores which may indicate some decomposition of nisin with storage. Nisin levels of 5000 I.U./ml for the type A spores and 2000 I.U./ml for the type B spores and the Minnesota E spores were insufficient to prevent spore outgrowth by C. botulinurn in cooked meat medium. For the Beluga E spores, a nisin level of 2000 I.U./ml was necessary to prevent spore outgrowth in cooked meat medium. The need for higher levels of nisin in cooked medium to prevent spore outgrowth may be due to the binding of the nisin by meat particles.     

The Investigation of the Presence of Clostridium Botulinum Spores in Honey in Serbia

The presence of Clostridium botulinum spores in 59 honey samples originating from different regions of the Republic of Serbia was studied. In addition to microbiological methods, after enrichment, centrifugation and membrane filtration, molecular methods (PCR methods) were utilized. The number of spores in PCR positive samples was estimated by the most probable number (MPN) method. PCR confirmed C. botulinum spores in 5 (8.47%) honey samples. MPN of spores varied from 20/kg to 204/kg honey. PCR was more sensitive than cultural methods. Natural honey contamination with C. botulinum spores is low-level and not homogeneous, and therefore, PCR methods require multiple sub-sampling.