scholarly journals Clostridium botulinum Spores Found in Honey from Small Apiaries in Poland

2016 ◽  
Vol 60 (2) ◽  
pp. 89-100 ◽  
Author(s):  
Joanna Wojtacka ◽  
Beata Wysok ◽  
Zbigniew Lipiński ◽  
Małgorzata Gomółka-Pawlicka ◽  
Helena Rybak-Chmielewska ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Multiplex Pcr ◽  
Clostridium Botulinum ◽  
Type A ◽  
Type B ◽  
Average Quantity ◽  
Pcr Method ◽  
Centrifugation Method ◽  
Cooked Meat

Abstract A total of 102 honey samples collected from small apiaries (≤ 20 hives) in Poland were analysed for the presence of Clostridium botulinum spores. The samples were prepared using the dilution centrifugation method and cultured in parallel in cooked meat medium (CMM) and tripticase peptone glucose yeast (TPGY) enrichment broths. Identification of toxin types A, B, and E of Clostridium botulinum strains was performed with the use of the multiplex PCR method. Positive samples were also subjected to quantitative analysis with the use of Clostridium botulinum Isolation Agar Base (CBAB). The prevalence analysis showed 22 (21.6%) samples contaminated with C. botulinum spores. The major serotype detected was botulin neurotoxin type A – 16 (72.7%) whereas type B was found in 3 (13.6%) honey samples and type E also only in 3 (13.6%) honey samples. Dual-toxin-producing strains were noted. The average quantity of spores in PCR - C. botulinum positive samples was 190 in 1 gram of honey.

scholarly journals Honey sold directly by producers in the Silesian region of Poland as a source of Clostridium botulinum types A, B, E and F

2017 ◽  
Vol 35 (No. 3) ◽  
pp. 194-199 ◽  
Author(s):  
Fatma Hayıt ◽  
Hülya Gül
Keyword(s):  
Botulinum Toxin ◽  
Multiplex Pcr ◽  
Clostridium Botulinum ◽  
Type A ◽  
Honey Sample ◽  
Type B ◽  
Subsequent Processing ◽  
Pcr Method ◽  
Centrifugation Method ◽  
Cooked Meat

The level of contamination of honey with Clostridium botulinum spores is considered as an indicator of the adequacy of hygienic practices during collection, extraction, and subsequent processing. A total of 39 honey samples purchased directly from beekeepers at outdoor markets and from small amateur apiaries in Silesia were analysed for Clostridium botulinum spores. The samples were prepared using a dilution centrifugation method and cultured in parallel in cooked meat medium (CMM) and tripticase peptone glucose yeast (TPGY) enrichment broths. Identification of C. botulinum toxin types A, B, E, and F was performed with the use of a multiplex PCR method. The analysis showed six (15.4%) samples to be contaminated with C. botulinum spores. The major serotypes detected were type A – in two (5.1%) and type B – in two (5.1%) honey samples, respectively. Types E and F were found in 1 (2.6%) and 1 (2.6%) positive honey sample analysed, respectively.

scholarly journals Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material

2001 ◽  
Vol 67 (12) ◽  
pp. 5694-5699 ◽  
Author(s):  
Miia Lindström ◽  
Riikka Keto ◽  
Annukka Markkula ◽  
Mari Nevas ◽  
Sebastian Hielm ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Bacterial Species ◽  
Type A ◽  
Food Samples ◽  
Type B ◽  
Fecal Material ◽  
Pcr Assay ◽  
Multiplex Pcr Assay

ABSTRACT Botulism is diagnosed by detecting botulinum neurotoxin andClostridium 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 102 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.

Incidence and Origin of Clostridium botulinum Spores in Honey

1981 ◽  
Vol 44 (11) ◽  
pp. 812-814 ◽  
Author(s):  
C. N. HUBTANEN ◽  
D. KNOX ◽  
H. SBIMANUKI
Keyword(s):  
New Jersey ◽  
Apis Mellifera ◽  
Clostridium Botulinum ◽  
Water Solution ◽  
Type A ◽  
Type B ◽  
Sugar Water ◽  
Quantitative Estimates ◽  
Foreign Countries ◽  
Cooked Meat

Eighty honey samples, including some from foreign countries, were obtained from a local processor or from apiaries in Pennsylvania, Illinois and New Jersey. They were analyzed for Clostridium botulinum spores by a dilution-centrifugation (DC) procedure and by direct addition (DA) of honey to two different enrichment media. All were negative by the DC method; five were positive by DA in fluid thioglycollate media and six by DA in cooked meat media. Some samples positive in fluid thioglycollate media were negative in cooked meat media and vice versa. Bees (Apis mellifera, 25,000 per hive) were experimentally inoculated with spores of C. botulinum by feeding a 50% sugar-water solution containing 1.6 × 105 spores of 20 strains (equal numbers of 11 type A and 9 type B). Honey collected from the hive 2 weeks later contained 1100 spores per g; that collected after 5 weeks contained 50 spores per g. Quantitative estimates of honey yield and spore contents indicated that all the spores originally ingested by the bees had been incorporated into the honey. No botulinal spores were found in the intestinal or rectal contents of the bees 2 weeks or more after spore ingestion.

Outgrowth of Clostridium botulinum in Shredded Cabbage at Room Temperature Under a Modified Atmosphere

1990 ◽  
Vol 53 (10) ◽  
pp. 831-833 ◽  
Author(s):  
HAIM M. SOLOMON ◽  
DONALD A. KAUTTER ◽  
TIMOTHY LILLY ◽  
E. JEFFERY RHODEHAMEL
Keyword(s):  
Clostridium Botulinum ◽  
Room Temperature ◽  
Type A ◽  
Modified Atmosphere ◽  
Type B

The ability of Clostridium botulinum types A and B spores to grow and produce toxin in shredded cabbage at room temperature under a modified atmosphere was investigated. Seven type A and seven type B strains of C. botulinum, mostly of vegetable origin, were used as inocula. Shredded cabbage in high barrier bags, 250 g/bag, was inoculated with various numbers of spores, sealed under a modified atmosphere of 70% CO2 and 30% N2 and incubated at room temperature. Duplicate bags were examined for organoleptic acceptability and the presence of toxin from day 3 by blending the entire contents of each bag and injecting mice with dilutions of the extracts. Toxic extracts were typed with appropriate antitoxins. Only type A spores grew and produced toxin in the cabbage. An inoculum of approximately 100–200 type A spores/g of cabbage, whether in single strains or in various combinations, produced toxin on days 4, 5, and 6, while the cabbage was still organoleptically acceptable, as determined by appearance, odor, and texture.

Failure of Clostridium botulinum to Grow in Fresh Mushrooms Packaged in Plastic Film Overwraps with Holes

1978 ◽  
Vol 41 (5) ◽  
pp. 348-350 ◽  
Author(s):  
H. SUGIYAMA ◽  
KANDEE S. RUTLEDGE
Keyword(s):  
Botulinum Toxin ◽  
Clostridium Botulinum ◽  
Type A ◽  
Plastic Film ◽  
Type B ◽  
Chloride Film

Fresh, commercially grown mushrooms were inoculated in the stem with Clostridium botulinum spores (2.5 × 104 of each of four type A with l × 105 of each of four type B strains). One lb (454 g) of mush-rooms. including two spore-inoculated ones. was packaged in paper-borad trays and overwrapped with a ploybinyl chloride film. The packages were incubated 6 days at 24–26 C after making one or two holes of 1/8 inch (3.175 mm) diameter in the overwrap of test packages. Botulinum toxin. either type A only or mixed with type B, was found in the spore-inoculated mushrooms of all 28 control packages (no hole), but was not detected in any from the 123 packages with one hole and the 4 7 with two holes. The O2% inside the packages after incubation averaged 1.5 for the controls. 4.0 for packages with one hole and 6.2 for the 2-hole package group.

Combined High Pressure and Thermal Processing on Inactivation of Type A and Proteolytic Type B Spores of Clostridium botulinum

2013 ◽  
Vol 76 (8) ◽  
pp. 1384-1392 ◽  
Author(s):  
N. RUKMA REDDY ◽  
KRISTIN M. MARSHALL ◽  
TRAVIS R. MORRISSEY ◽  
VIVIANA LOEZA ◽  
EDUARDO PATAZCA ◽  
...  
Keyword(s):  
High Pressure ◽  
Thermal Processing ◽  
Clostridium Botulinum ◽  
High Pressures ◽  
Type A ◽  
Test System ◽  
High Pressure Processing ◽  
Type B ◽  
Botulinum Type ◽  
D Values

The aim of this study was to determine the resistance of multiple strains of Clostridium botulinum type A and proteolytic type B spores exposed to combined high pressure and thermal processing and compare their resistance with Clostridium sporogenes PA3679 and Bacillus amyloliquefaciens TMW-2.479-Fad-82 spores. The resistance of spores suspended in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.0) was determined at a process temperature of 105°C, with high pressures of 600, 700, and 750 MPa by using a laboratory-scale pressure test system. No surviving spores of the proteolytic B strains were detected after processing at 105°C and 700 MPa for 6 min. A >7-log reduction of B. amyloliquefaciens spores was observed when processed for 4 min at 105°C and 700 MPa. D-values at 105°C and 700 MPa for type A strains ranged from 0.57 to 2.28 min. C. sporogenes PA3679 had a D-value of 1.48 min at 105°C and 700 MPa. Spores of the six type A strains with high D-values along with C. sporogenes PA3679 and B. amyloliquefaciens were further evaluated for their pressure resistance at pressures 600 and 750 MPa at 105°C. As the process pressure increased from 600 to 750 MPa at 105°C, D-values of some C. botulinum strains and C. sporogenes PA3679 spores decreased (i.e., 69-A, 1.91 to 1.33 min and PA3679, 2.35 to 1.29 min). Some C. botulinum type A strains were more resistant than C. sporogenes PA3679 and B. amyloliquefaciens to combined high pressure and heat, based on D-values determined at 105°C. Pulsed-field gel electrophoresis (PFGE) was also performed to establish whether strains with a similar restriction banding pattern also exhibited similar D-values. However, no correlation between the genomic background of a strain and its resistance to high pressure processing was observed, based on PFGE analysis. Spores of proteolytic type B strains of C. botulinum were less resistant to combined high pressure and heat (700 MPa and 105°C) treatment when compared with spores of type A strains.

Screening for Clostridium botulinum Type A, B, and E in Cooked Chilled Foods Containing Vegetables and Raw Material Using Polymerase Chain Reaction and Molecular Probes

2001 ◽  
Vol 64 (2) ◽  
pp. 201-207 ◽  
Author(s):  
AGNÈS BRACONNIER ◽  
VÉRONIQUE BROUSSOLLE ◽  
SYLVIE PERELLE ◽  
PATRICK FACH ◽  
CHRISTOPHE NGUYEN-THE ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Raw Materials ◽  
Type A ◽  
Molecular Probes ◽  
Raw Material ◽  
Type B ◽  
Molecular Method ◽  
Chain Reaction ◽  
Botulinum Type ◽  
Polymerase Chain

A molecular method was used for the detection of Clostridium botulinum spores of type A, B, and E in commercial cooked and pasteurized vegetable purées and in the raw materials (vegetables and other ingredients). The method allowed the detection of less than 8 spores/g of product for C. botulinum type A, less than 1 spore/g for proteolytic type B, less than 21 spores/g for nonproteolytic type B, and less than 0.1 spore/g for type E. Thirty-seven samples of raw vegetables and ingredients were tested for the presence of C. botulinum type A, B, and E; 88 and 90 samples of vegetable purées were tested, respectively, for the presence of C. botulinum type A and B and for the presence of C. botulinum type E. All samples were negative, suggesting that the prevalence of C. botulinum in these vegetable purées and the raw ingredients is probably low.

Growth and Toxin Production by Clostridium botulinum in Sliced Raw Potatoes Under Vacuum With and Without Sulfite

1994 ◽  
Vol 57 (10) ◽  
pp. 878-881 ◽  
Author(s):  
HAIM M. SOLOMON ◽  
E. JEFFERY RHODEHAMEL ◽  
DONALD A. KAUTTER
Keyword(s):  
Sensory Evaluation ◽  
Clostridium Botulinum ◽  
Room Temperature ◽  
Type A ◽  
Toxin Production ◽  
Inoculum Size ◽  
Consumer Acceptability ◽  
Human Consumption ◽  
Type B ◽  
Botulinum Type

The ability of Clostridium botulinum type A or B spores to grow and produce toxin in fresh raw potatoes under vacuum with or without sulfite at 22°C was investigated. Fresh, peeled, sliced potatoes, untreated or dipped for 2 min in sulfite (NaHSO3) and drained, were surface-inoculated at several levels with a mixture of C. botulinum spores, either type A or B, and placed in oxygen-impermeable bags (200 g/bag) that were then vacuum-sealed and incubated at room temperature (22°C). Toxicity was tested on days 0, 3, 4, 5 and 6. After incubation, the potatoes were blended and centrifuged, and the millipore-filtered supernatant fluid was injected intraperitoneally into mice. Sensory evaluation, except taste, was also performed. Potatoes inoculated with C. botulinum type A spores, but untreated with NaHSO3 became toxic in 3 days, which coincided with the sensory evaluation, “Unfit for human consumption.” However, despite inoculum size or residual SO2 levels, potatoes treated with NaHSO3 appeared acceptable for human consumption through day 6, even though they were toxic after 4 days of incubation. Toxicity from type B spores occurred later and in fewer test samples than type A. Again, the potatoes appeared acceptable but were toxic. Thus, although NaHSO3 markedly extended the consumer acceptability of peeled, sliced, raw potatoes at the abuse temperature, it did not inhibit outgrowth and toxin production by C. botulinum under these same conditions.

scholarly journals SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A

2007 ◽  
Vol 73 (9) ◽  
pp. 2891-2896 ◽  
Author(s):  
Lucia Fenicia ◽  
Fabrizio Anniballi ◽  
Dario De Medici ◽  
Elisabetta Delibato ◽  
Paolo Aureli
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clostridium Botulinum ◽  
False Negative ◽  
Type A ◽  
Sybr Green ◽  
Mouse Bioassay ◽  
Chelex 100 ◽  
Microbiological Methods ◽  
Pcr Method

ABSTRACT Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 101 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).

Outgrowth and Toxin Production by Clostridium botulinum in Bottled Chopped Garlic

1988 ◽  
Vol 51 (11) ◽  
pp. 862-865 ◽  
Author(s):  
HAIM M. SOLOMON ◽  
DONALD A. KAUTTER
Keyword(s):  
Soybean Oil ◽  
Clostridium Botulinum ◽  
Room Temperature ◽  
Type A ◽  
Toxin Production ◽  
Type B ◽  
Botulinum Type

The ability of Clostridium botulinum types A and B spores to grow and produce toxin in commercially bottled chopped garlic in soybean oil was investigated. Eight type A and seven type B strains of C. botulinum, mostly of vegetable origin, were used as inocula. Various numbers of spores were inoculated directly into the jars containing garlic, incubated at 35°C and sampled for organoleptic acceptance and presence of toxin every 5th d. In parallel studies conducted at room temperature, jars were sampled at 15-d intervals. At 35°C, when 1 spore/g of garlic was used as inoculum, toxin was produced in 15 d by type A and in 20 d by type B strains. At room temperature, five spores of type A or B per g of garlic produced toxin throughout 75 d. Even when highly toxic, garlic looked and smelled acceptable. Five strains of C. botulinum type A were isolated from 115 bulbs of fresh garlic.

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