Polymerase chain reaction for detection of Clostridium botulinum types A, B and E in food, soil and infant faeces

The application of the polymerase chain reaction (PCR) for detection of Clostridium botulinum types A, B and E in foods, environmental and clinical samples was evaluated and compared to the mouse bioassay. Samples inoculated with 10, 100 and 1000 spores of Cl. botulinum types A and B included pasteurized milk, UHT milk, infant formula, infant faeces, meat juice, canned tuna, mushrooms, blood sausage and soil. Clostridium botulinum type E spores were inoculated into fish eggs, canned tuna, picked herring, raw fish and soil at similar levels. Spores were added to 2.5 g of each sample with the exception of soil which was inoculated in 10 g samples. The presence of Cl. botulinum in sample enrichments was determined by both PCR and the bioassay. An overall correlation of 95.6% was observed between PCR results and the mouse bioassay. Of the total of 114 samples tested there was disparity between the mouse bioassay and the PCR in three samples of soil inoculated with 100 type A or E spores and 10 type B spores per 10 g, respectively, and two samples of infant faeces inoculated with 10 type A or B spores per 2.5 g. All of these samples gave negative animal results and positive PCR results.     

Application of real-time PCR for quantitative detection of Clostridium botulinum type A toxin gene in food

The TaqMan real-time PCR method for the quantitative detection of C. botulinum type A was developed based on sequence-specific hybridization probes. The validity of this assay was verified by using 10 genera of 20 strains, including reference strains of C. botulinum types A, B, C, D, E and F. The detection limit of this assay was evaluated on C. botulinum type A, using a 10-fold dilution series of DNA and spores . The DNA and spores were detected up to level of 0.1 ng/ml and 10(2)spores/ml, respectively. Spore spiked food sample preparation prior to the real-time PCR was performed by two methods, heat treatment and GuSCN. The detection limits after heat treatment showed 10(2) spores/ml for spiked sausage slurry, and 10(3) spores/ml for spiked canned corn slurry, while detection limits after GuSCN precipitation showed 10(2) spores/ml in both sausage and canned corn. Therefore the real-time PCR assay after GuSCN precipitation is useful for the quantification of C. botulinum type A because it showed identical CT values in both pure spore solutions and food slurries. We suggest that quantitative analysis of C. botulinum type A by TaqMan real-time PCR can be a rapid and accurate assessment method for botulinal risk in food samples.     

Use of the Anaerobic Pouch in Isolating Clostridium botulinum Spores from Fresh Meats

The anaerobic film pouch was demonstrated to be an effective device for the primary isolation of Clostridium botulinum types A and B spores from raw pork, beef, and chicken. Optimal pasteurization of these meats (for reduction of nonspore microflora without affecting indigenous putrefactive anaerobic spore levels) was 50 min at 60 C. C. botulinum spores were recovered with good precision from meat samples inoculated with mixtures of C. botulinum and Putrefactive Anaerobe 3679 at 1:1 and at 1:99 ratios. Verification of C. botulinum isolates was accomplished by protection testing of subcultures in mice.     

Development of a combined selection and enrichment PCR procedure for Clostridium botulinum Types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70 degrees C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30 degrees C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 x 10(3) spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.     

Factors influencing Clostridium botulinum spore germination, outgrowth, and toxin formation in acidified media

Clostridium botulinum type A spores were inoculated at a level of 10(7) spores per ml into sterile beef media with protein concentrations of 1, 2, 3, 4, or 6% and acidified to pH values of 2.01 to 4.75 with hydrochloric acid or 4.19 to 4.60 with citric acid. All experimental manipulations, including blending, acidification, inoculation, incubation (30 degrees C), and analyses, were conducted in an anaerobic chamber-incubator in which atmospheric oxygen levels were maintained below 2 ppm (2 microliters/liter). Under these strict anaerobic conditions (oxidation-reduction values in media ranging from -370 to -391 mV), C. botulinum spores were consistently found to germinate, grow, and produce toxin below pH 4.6. The boundary between toxic and atoxic samples in HC1-acidified beef media was mediated by titratable acidity, pH, and protein concentration. A limiting acidity was not established for the citrate-acidified samples; all blends tested (1, 2, 3, and 4% protein and titratable acidities of 0.091 to 0.453%) became toxic within 5 weeks. At the same pH and protein concentration, citric acid was less effective than HC1 in preventing the germination of C. botulinum spores. Higher levels of cell proliferation in the beef protein, as well as enhanced gas production and putrefactive degradation, indicated that beef was a better substrate than soy for C. botulinum spores under these conditions. Reducing the inoculum to 10(4) delayed but did not prevent spore outgrowth and toxin release at pH levels below 4.6.     

Factors influencing Clostridium botulinum spore germination, outgrowth, and toxin formation in acidified media.

Clostridium botulinum type A spores were inoculated at a level of 10(7) spores per ml into sterile beef media with protein concentrations of 1, 2, 3, 4, or 6% and acidified to pH values of 2.01 to 4.75 with hydrochloric acid or 4.19 to 4.60 with citric acid. All experimental manipulations, including blending, acidification, inoculation, incubation (30 degrees C), and analyses, were conducted in an anaerobic chamber-incubator in which atmospheric oxygen levels were maintained below 2 ppm (2 microliters/liter). Under these strict anaerobic conditions (oxidation-reduction values in media ranging from -370 to -391 mV), C. botulinum spores were consistently found to germinate, grow, and produce toxin below pH 4.6. The boundary between toxic and atoxic samples in HC1-acidified beef media was mediated by titratable acidity, pH, and protein concentration. A limiting acidity was not established for the citrate-acidified samples; all blends tested (1, 2, 3, and 4% protein and titratable acidities of 0.091 to 0.453%) became toxic within 5 weeks. At the same pH and protein concentration, citric acid was less effective than HC1 in preventing the germination of C. botulinum spores. Higher levels of cell proliferation in the beef protein, as well as enhanced gas production and putrefactive degradation, indicated that beef was a better substrate than soy for C. botulinum spores under these conditions. Reducing the inoculum to 10(4) delayed but did not prevent spore outgrowth and toxin release at pH levels below 4.6.     

Survival Studies with Spores of Clostridium botulinum Type E in Pasteurized Meat of the Blue Crab Callinectes sapidus

Clostridium botulinum type E studies reported in this paper include the incidence of the organism in selected Chesapeake Bay areas, growth and toxin production in crabmeat homogenates, and the effect of pasteurization upon varying levels of spores in crabmeat. Type E spores were detected in 21 of 24 bottom mud samples taken at locations from which blue crabs were being harvested. Sterilized crabmeat homogenates inoculated with as little as five spores per 10 g became toxic after 8 days at 50 F, 2 days at 75 F, and 1 day at 85 F. Growth at 50 F and above was accompanied by gas production and a slightly sour odor. Growth and toxin production at 40 F required 55 days or longer and inocula of 10(3) spores or higher per 10 g of homogenate. At 40 F gas production was usually not apparent and no off odors could be detected. A recommended minimum pasteurization of 1 min at 185 F internal meat temperature reduced type E spore levels in inoculated packs of crabmeat from 10(8) spores per 100 g to 6 or less spores per 100 g, and the pasteurized meat remained nontoxic during 6 months of storage at 40 F.     

Multiplex PCR assay for detection and identification of Clostridium botulinum types A, B, E, and F in food and fecal material.

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10(2) cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10(-2) spore/g for types A, B, and F to 10(-1) spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.     

The effect of recovery medium on the estimated heat-inactivation of spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic strains of Clostridium botulinum at 85°C, followed by enumeration of survivors on a highly nutrient medium indicated a 5 decimal kill in less than 2 min. The inclusion of lysozyme or egg yolk emulsion in the recovery medium substantially increased apparent spore heat-resistance, with as little as 0.1 μg lysozyme/ml sufficient to give an increase in the number of survivors. After heating at 85°C for 2 min between 0.1% and 1% of the spores of 11 strains (5 type B, 4 type E, 2 type F) formed colonies on medium containing 10 μg lysozyme/ml. Enumeration of survivors on a medium containing lysozyme showed that heating at 85°C for 5 min resulted in an estimated 2.6 decimal kill of spores of strain 17B (type B). These findings are important in the assessment of heat-treatments required to ensure the safety with respect to non-proteolytic Clostridium botulinum of processed (pasteurized) refrigerated foods for extended storage such as sous-vide foods.     

Common Mesophilic Anaerobes, Including Clostridium botulinum and Clostridium tetani, in 21 Soil Specimens

A relatively rich medium was markedly superior to a dilute medium for the isolation of anaerobic bacteria from soil. The obligate anaerobes isolated from 21 soil samples were all clostridia and the counts ranged from 2.7 X 10-2 to 3.3 X 10-6 per g. The organisms most frequently isolated were Clostridium subterminate, C. sordelii, C. sporogenes, C. indolis, C. bifermentans, C. mangenoti, and C. perfringens. Seventeen other species were also recognized but almost one-third of the isolates could not be identified with any known species of Clostridium. C. botulinum type A was demonstrated in six soil samples, and type B in one. These soils were neutral to alkaline in reaction (average pH 7.9) and low in organic matter content (1.4%). The association of C. botulinum types A and B with neutral to alkaline soils was statistically significant (P = 0.001) as was their association with soils low in organic matter (P = 0.005). C. botulinum types E and F were found in one soil sample, pH 4.5, with organic matter 13.7%. C. tetani was isolated from two soil samples, both of intermediate pH value and higher than average organic matter content.     

Honey consumption in the state of São Paulo: a risk to human health?

Infantile botulism was recognized in 1976 as a paralyzing disease caused by the ingestion of viable spores that would germinate and colonize the intestinal tract of infants, with local production and absorption of Clostridium botulinum toxin. The possible origins of botulinic spores are dust and honey, which has been identified as a dietary risk factor for infantile botulism. The objectives of the present study were to investigate 100 honey samples obtained in the state of S?o Paulo (Brazil) in terms of incidence of botulinic spores and of microbiologic quality, in agreement with Decree 367/9. All 100 samples analysed were negative for the presence of Salmonella, Shigella, total coliforms. C. botulinum spores were present in 3 samples (3%) and molds and yeasts, in 64 samples (64%), but only 25 (25%) exceeded established criteria, with counts ranging from zero to 1.5 x 10(5)CFU/g. The presence of small sporogenic Gram-positive rods was observed in 42 (42%) of the 100 samples tested but these bacteria were not identified.     

Effect of Nitrite and Nitrate on Toxin Production by Clostridium botulinum and on Nitrosamine Formation in Perishable Canned Comminuted Cured Meat

Comminuted ham was formulated with different levels of sodium nitrite and nitrate, inoculated with Clostridium botulinum, and pasteurized to an internal temperature of 68.5 C. When added to the meat, nitrite concentrations decreased, and cooking had little effect on them. Nitrite concentrations decreased more rapidly during storage at 27 than at 7 C; however they remained rather constant at formulated levels throughout the experiment at both incubation temperatures. The level of nitrite added to the meat greatly influenced growth and toxin production of C. botulinum. The concentration of nitrite necessary to effect complete inhibition was dependent on the inoculum level. With 90 C. botulinum spores/g of meat, botulinum toxin developed in samples formulated with 150 but not with 200 mug of nitrite per g of meat. At a spore level of 5,000/g, toxin was detected in samples with 400 but not with 500 mug of nitrite per g of the product incubated at 27 C. At lower concentrations of nitrite, growth was retarded at both spore levels. No toxin developed in samples incubated at 7 C. Nitrate showed a statistically significant inhibitory effect at a given nitrite level; however, the effect was insufficient to be of practical value. Analyses for 14 volatile nitrosamines from samples made with varying levels of nitrite and nitrate were negative at a detection level of 0.01 mug of nitrite or nitrate per g of meat.     

Fate of Salmonella enterica serovar Typhimurium on carrots and radishes grown in fields treated with contaminated manure composts or irrigation water

Three different types of compost, PM-5 (poultry manure compost), 338 (dairy cattle manure compost), and NVIRO-4 (alkaline-pH-stabilized dairy cattle manure compost), and irrigation water were inoculated with an avirulent strain of Salmonella enterica serovar Typhimurium at 10(7) CFU g(-1) and 10(5) CFU ml(-1), respectively, to determine the persistence of salmonellae in soils containing these composts, in irrigation water, and also on carrots and radishes grown in these contaminated soils. A split-plot block design plan was used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but with contaminated water applied) and five replicates for a total of 25 plots for each crop, with each plot measuring 1.8 x 4.6 m. Salmonellae persisted for an extended period of time, with the bacteria surviving in soil samples for 203 to 231 days, and were detected after seeds were sown for 84 and 203 days on radishes and carrots, respectively. Salmonella survival was greatest in soil amended with poultry compost and least in soil containing alkaline-pH-stabilized dairy cattle manure compost. Survival profiles of Salmonella on vegetables and soil samples contaminated by irrigation water were similar to those observed when contamination occurred through compost. Hence, both contaminated manure compost and irrigation water can play an important role in contaminating soil and root vegetables with salmonellae for several months.     

Morphology of the spore of some strains of Clostridium botulinum type E

Hodgkiss, W. (Torry Research Station, Aberdeen, Scotland), and Z. John Ordal. Morphology of the spore of some strains of Clostridium botulinum type E. J. Bacteriol. 91:2031-2036. 1966.-The spores of four strains of C. botulinum type E show an unusual and elaborate morphology. Numerous tubular appendages radiate from the surface of the spore. The spore and its appendages are enclosed in a delicate exosporium. The electron microscopic morphology of the spores, as seen in metal-shadowed and negatively stained preparations, and by the carbon-replica technique, is described.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5.2 with HCl and incubated at 30 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10-20 weeks. Citric acid concentrations of 10-50 mmol/l at pH 5.2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10(6). The citric acid also reduced the concentration of free Ca2+ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5.2 could be prevented by the addition of Ca2+ or Mg2+ and greatly reduced by Fe2+ and Mn2+. The addition of Ca2+, but not of the remaining divalent metal ions, restored the concentration of free Ca2+ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca2+. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5.2. The effect of citric acid and Ca2+ at pH 5.2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

Influence of transition metals added during sporulation on heat resistance of Clostridium botulinum 113B spores.

Sporulation of Clostridium botulinum 113B in a complex medium supplemented with certain transition metals (Fe, Mn, Cu, or Zn) at 0.01 to 1.0 mM gave spores that were increased two to sevenfold in their contents of the added metals. The contents of calcium, magnesium, and other metals in the purified spores were relatively unchanged. Inclusion of sodium citrate (3 g/liter) in the medium enhanced metal accumulation and gave consistency in the transition metal contents of independent spore crops. In citrate-supplemented media, C. botulinum formed spores with very high contents of Zn (approximately 1% of the dry weight). Spores containing an increased content of Fe (0.1 to 0.2%) were more susceptible to thermal killing than were native spores or spores containing increased Zn or Mn. The spores formed with added Fe or Cu also appeared less able to repair heat-induced injuries than the spores with added Mn or Zn. Fe-increased spores appeared to germinate and outgrow at a higher frequency than did native and Mn-increased spores. This study shows that C. botulinum spores can be sensitized to increased thermal destruction by incorporation of Fe in the spores.     

A high prevalence of Clostridium botulinum type E in Finnish freshwater and Baltic Sea sediment samples

The distribution of Clostridium botulinum serotypes A, B, E and F in aquatic environments of the Baltic Sea and Finnish mainland was examined. A total of 110 samples were tested with a neurotoxin-specific PCR assay. Clostridium botulinum type E was found in 81% of sea and 61% of freshwater samples. No other toxinotypes were found. Spore counts were quantified by the most probable number method, Cl. botulinum type E kg(-1) averaging 940 in sea and 370 in freshwater samples. The overall prevalence and spore counts of Cl. botulinum type E in aquatic sediments correlated significantly with offshore bottom oxygen content, depth, and bioturbation activity, whereas there was no correlation with bottom water temperature. These findings indicate the possibility of Cl. botulinum type E multiplication or at least, suitable conditions for spore survival, in anoxic sediments.     

Influence of transition metals added during sporulation on heat resistance of Clostridium botulinum 113B spores

Sporulation of Clostridium botulinum 113B in a complex medium supplemented with certain transition metals (Fe, Mn, Cu, or Zn) at 0.01 to 1.0 mM gave spores that were increased two to sevenfold in their contents of the added metals. The contents of calcium, magnesium, and other metals in the purified spores were relatively unchanged. Inclusion of sodium citrate (3 g/liter) in the medium enhanced metal accumulation and gave consistency in the transition metal contents of independent spore crops. In citrate-supplemented media, C. botulinum formed spores with very high contents of Zn (approximately 1% of the dry weight). Spores containing an increased content of Fe (0.1 to 0.2%) were more susceptible to thermal killing than were native spores or spores containing increased Zn or Mn. The spores formed with added Fe or Cu also appeared less able to repair heat-induced injuries than the spores with added Mn or Zn. Fe-increased spores appeared to germinate and outgrow at a higher frequency than did native and Mn-increased spores. This study shows that C. botulinum spores can be sensitized to increased thermal destruction by incorporation of Fe in the spores.     

Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/ microliters and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between C(T) value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.     

Sodium nitrite and sorbic acid effects on Clostridium botulinum spore germination and total microbial growth in chicken frankfurter emulsions during temperature abuse.

Samples of (i) a control or of (ii) sodium nitrite-containing or (iii) sorbic acid-containing, mechanically deboned chicken meat frankfurter-type emulsions inoculated with Clostridium botulinum spores, or a combination of ii and iii, were temperature abuse at 27 degrees C. Spore germination and total microbial growth were followed and examined at specified times and until toxic samples were detected. The spores germinated within 3 days in both control and nitrite (20, 40 and 156 micrograms/g) treatments. Sorbic acid (0.2%) alone or in combination with nitrite (20, 40, and 156 micrograms/g) significantly (P less than 0.05) inhibited spore germinations. No significant germination was recorded until toxic samples were detected. A much longer incubation period was necessary for toxin to be formed in nitrite-sorbic acid combination treatments as contrasted with controls or nitrite and sorbic acid used individually. Total growth was not affected by the presence of nitrite, whereas sorbic acid appeared to depress it. Possible mechanisms explaining the effects of nitrite and sorbic acid on spore germination and growth are postulated.