Lucia Fenicia

Intestinal Toxemia Botulism in Two Young People, Caused by Clostridium butyricum Type E

Two unconnected cases of type E botulism involving a 19-year-old woman and a 9-year-old child are described. The hospital courses of their illness were similar and included initial acute abdominal pain accompanied by progressive neurological impairment. Both patients were suspected of having appendicitis and underwent laparotomy, during which voluminous Meckels diverticula were resected. Unusual neurotoxigenic Clostridium butyricum strains that produced botulinum-like toxin type E were isolated from the feces of the patients. These isolates were genotypically and phenotypically identical to other neurotoxigenic C. butyricum strains discovered in Italy in 1985-1986. No cytotoxic activity of the strains that might explain the associated gastrointestinal symptoms was demonstrated. The clinical picture of the illness and the persistence of neurotoxigenic clostridia in the feces of these patients suggested a colonization of the large intestine, with in vivo toxin production. The possibility that Meckels diverticulum may predispose to intestinal toxemia botulism may warrant further investigation.

Neurotoxin Gene Profiling of Clostridium botulinum Types C and D Native to Different Countries within Europe

ABSTRACT Clostridium botulinum types C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of the C. botulinum subtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, including C. botulinum types C (n = 12), C-D (n = 29), D (n = 5), and D-C (n = 10), other botulinum neurotoxin (BoNT)-producing Clostridium strains (n = 20), non-BoNT-producing clostridia (n = 20), and other bacterial species (n = 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection of C. botulinum in 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n = 108), poultry (n = 60), and bovines (n = 56). Among the 292 samples, 144 were positive for either the bont/C-D or the bont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.

Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical, Food, and Environmental Samples

ABSTRACT Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.

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

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%).

Clostridium botulinum spores and toxin in mascarpone cheese and other milk products.

A total of 1,017 mascarpone cheese samples, collected at retail, were analyzed for Clostridium botulinum spores and toxin, aerobic mesophilic spore counts, as well as pH, a(w) (water activity), and Eh (oxidation-reduction potential). In addition 260 samples from other dairy products were also analyzed for spores and botulinum toxin. Experiments were carried out on naturally and artificially contaminated mascarpone to investigate the influence of different temperature conditions on toxin production by C. botulinum. Three hundred and thirty-one samples (32.5%) of mascarpone were positive for botulinal spores, and 7 (0.8%) of the 878 samples produced at the plant involved in an outbreak of foodborne botulism also contained toxin type A. The chemical-physical parameters (pH, a(w), Eh) of all samples were compatible with C. botulinum growth and toxinogenesis. Of the other milk products, 2.7% were positive for C. botulinum spores. Growth and toxin formation occurred in naturally and experimentally contaminated mascarpone samples after 3 and 4 days of incubation at 28 degrees C, respectively.

A CASE OF INFANT BOTULISM ASSOCIATED WITH HONEY FEEDING IN ITALY

A case of infant botulism in a 9 week-old female is described. A strain of C. botulinum type B was isolated from the feces of the baby. The epidemiologic study detected in a sample of home canned honey Clostridium botulinum spores of the same serotype that was isolated from the patient. The honey had been used only to sweeten the pacifier of the baby. This is the first case of infant botulism in Europe linked conclusively to honey.

Botulism and Preserved Green Olives

To the Editor: In March 2004, a total of 16 suspected cases of botulism were reported to the Italian National Institute of Health by hospitals in 3 adjoining regions in central and southern Italy (Molise, Campania, and Puglia). Initial investigation showed that all patients had eaten at the same restaurant in Molise on February 22 or 24, 2004. The restaurant provided reservation lists for those dates (the restaurant was closed on February 23). It also provided a list of foods that had been served each evening. Persons on the reservation lists were contacted and asked to provide the names of others who had been at their tables to ensure that as many diners as possible were traced. Of 73 persons who had been identified as having eaten at the restaurant on either evening, 66 were successfully contacted and interviewed in person or by telephone about symptoms and food consumed at the restaurant. For purposes of the investigation, a probable case-patient was defined as a person who had dined at the restaurant on February 22 or 24 and had experienced diplopia or blurred vision and at least 1 of the following symptoms: dysphagia, dry mouth, dysarthria, upper/lower extremity weakness, dyspnea, and severe constipation. Those who met the probable case definition and had laboratory-confirmed botulism were considered definite case-patients. We tested for botulinum neurotoxin in serum and spores in stool samples as described (1). Serum or stool specimens from 24 patients with ≥2 symptoms were sent to the Italian National Institute of Health for testing. Twenty-eight persons reported ≥2 symptoms (42% attack rate); 25 (89%) were considered probable cases and 3 (11%) were considered confirmed cases. Two members of the restaurant owners family and 1 employee were among the probable case-patients. Onset of symptoms occurred 4 hours to 6.5 days after eating at the restaurant (median 36 hours). Twenty persons (71%) had been seen in emergency rooms, 15 (53%) were admitted to a hospital, and 18% were admitted to intensive care. None required ventilatory support, and no deaths occurred. The main symptoms reported by 28 probable and confirmed patients included dry mouth in 25 (89%), dysphagia in 25 (89%), severe constipation in 22 (79%), and blurred vision in 27 (96%). Three weeks after onset of symptoms, 15 (68%) reporting severe constipation, 11 (41%) reporting blurred vision, 10 (40%) reporting dry mouth, and 11 (44%) reporting dysphagia still had these symptoms. Of the 24 patients for whom rectal swabs were available, 3 were culture-positive for Clostridium botulinum type B. None of 5 serum samples tested positive. Food-specific attack rates, relative risks (RRs), and 95% confidence intervals (CIs) were calculated. A Poisson model with robust error variance was used to estimate RR with adjustments for possible confounding and effect modification (2). Foods associated with illness with p values <0.20 were considered in the model. In a univariate analysis in which all 28 patients were considered, the RR of illness was higher among diners who ate home-preserved green olives in salt water (RR 5.2, 95% CI 1.4–19.8), ate cream pastries (RR 2.5, 95% CI 1.8–3.4), and drank homemade lemon liqueur (RR 2.1, 95% CI 1.3–3.4). After multivariate analysis, only the risk associated with eating green olives remained significant (RR 5.2, 95% CI 1.4–19.8). None of the food items served on February 22 or 24 was available for sampling, and none of the other 13 food samples obtained from the restaurant was positive for C. botulinum. However, the pH of a jar of olives that had been prepared at the same time as those eaten on February 22 and 24 was 6.2, far above the level of 4.6 required to prevent growth of C. botulinum. No salinity testing was performed by the local laboratory, and inadequate storage during transit made it impossible to conduct salinity and water activity tests at the national reference laboratory. Interviews with the restaurant proprietors indicated that the olives were prepared on site during the fall of 2003 from local olives. After soaking in salt water for ≈35 days, the olives had been decanted into jars, and salt water had been replaced with fresh water. Neither the amount of salt used in the salt water mixture nor the pH at any stage was standardized during preparation. Both epidemiologic evidence and information obtained regarding preparation of the olives strongly suggest that they were the likely source of the outbreak. This outbreak highlights the previously documented risk associated with improperly prepared olives (3–5). In Italy and elsewhere in Europe, an increasing trend favors traditional foods and preparation methods over large-scale industrial products. This outbreak underlines the importance of providing training and periodic monitoring of those involved in small-scale preparation to ensure that disease risks from improperly prepared or stored foods are minimized.

Intestinal toxemia botulism in Italy, 1984–2005

Botulism in humans is caused by botulinum neurotoxins, produced in most cases by Clostridium botulinum, although other Clostridia species are implicated as well. Of the five forms of botulism in humans, three are referred to as “infective”: wound botulism, infant botulism, and adult intestinal botulism; the latter two forms are also referred to as “intestinal toxemia botulism” because the organism colonizes the lumen of the intestinal tract and produces botulinum neurotoxin in vivo. Twenty-three cases of infant botulism and three cases of adult intestinal botulism occurred in Italy between 1984 and 2005. Microbiological analyses of clinical, environmental, and food samples and analysis of clinical and epidemiological data revealed two main characteristics of intestinal toxemia botulism in Italy that are not common in cases in other countries: the isolation of a strain of C. butyricum that produced botulinum neurotoxin type E in 6 of 26 cases, including two cases of adult intestinal toxemia botulism, and the onset of botulism in these cases with concomitant severe gastrointestinal symptomatology. This report summarizes the microbiological, clinical, and epidemiological data of all cases of intestinal toxemia botulism that have occurred in Italy in the period 1984–2005.

Neurophysiological assessment in the diagnosis of botulism: usefulness of single-fiber EMG.

We report the clinical, serological, and neurophysiological findings in seven patients with foodborne botulism caused by ingestion of black olives in water. The clinical picture was characterized by mild symptoms with a long latency of onset and by involvement of cranial and upper limb muscles; only one patient, a child, developed respiratory failure. Spores of Clostridium botulinum were found in stools in some but not all cases. Conventional neurophysiological tests had low sensitivity; abnormal findings were present only in the patient with severe clinical involvement, in whom compound muscle action potentials (CMAPs) appeared reduced. Repetitive nerve stimulation at a high rate showed pseudofacilitation and not true posttetanic facilitation, but single‐fiber electromyography (SFEMG) showed abnormalities of neuromuscular transmission in every case. Neurophysiological evaluation, particularly SFEMG, is important because it allows rapid identification of abnormal neuromuscular transmission while bioassay studies are in progress.

Bioinformatic tools for using whole genome sequencing as a rapid high resolution diagnostic typing tool when tracing bioterror organisms in the food and feed chain

The rapid technological development in the field of parallel sequencing offers new opportunities when tracing and tracking microorganisms in the food and feed chain. If a bioterror organism is deliberately spread it is of crucial importance to get as much information as possible regarding the strain as fast as possible to aid the decision process and select suitable controls, tracing and tracking tools. A lot of efforts have been made to sequence multiple strains of potential bioterror organisms so there is a relatively large set of reference genomes available. This study is focused on how to use parallel sequencing for rapid phylogenomic analysis and screen for genetic modifications. A bioinformatic methodology has been developed to rapidly analyze sequence data with minimal post-processing. Instead of assembling the genome, defining genes, defining orthologous relations and calculating distances, the present method can achieve a similar high resolution directly from the raw sequence data. The method defines orthologous sequence reads instead of orthologous genes and the average similarity of the core genome (ASC) is calculated. The sequence reads from the core and from the non-conserved genomic regions can also be separated for further analysis. Finally, the comparison algorithm is used to visualize the phylogenomic diversity of the bacterial bioterror organisms Bacillus anthracis and Clostridium botulinum using heat plot diagrams.