Dual-Toxin ("Bivalent") Infant Botulism in California, 1976-2020: Epidemiologic, Clinical, and Laboratory Aspects.

OBJECTIVE: To assess the consequences of infant botulism that result from Clostridium botulinum strains that produce 2 botulinum toxin serotypes, termed "bivalent." STUDY DESIGN: Epidemiologic investigations used a standard questionnaire. Clostridium botulinum strains were isolated by standard methods. Botulinum neurotoxin (BoNT) serotypes and the relative amounts of toxins produced were identified using the standard mouse bioassay. BoNT subtypes and genomic locations were identified by DNA nucleotide sequencing. RESULTS: Thirty bivalent cases of infant botulism occurred in the 45 years (1976-2020), representing 2.0% of all California infant botulism cases, in the 3 geographic regions of southern California, the southern Central Valley, and mid-northern California. Toxin serotype combinations were Ba (n = 22), Bf (n = 7), and Ab (n = 1). More patients with illness caused by bivalent C botulinum Ba and Bf strains needed endotracheal intubation at hospital admission, 60.0% (18/30), than did patients with illness caused by monovalent BoNT/B strains, 34.3% (152/443). The Cbotulinum Ba and Bf strains produced BoNT/B5 and either BoNT/A4 or /F2. The Ab strain produced BoNT/A2 and /B1. All toxin gene clusters were on plasmids. CONCLUSIONS: Infant botulism caused by bivalent Cbotulinum strains occurs sporadically and in diverse locations in California. Affected patients with bivalent Ba and Bf strains lacked distinguishing epidemiological features but appeared to be more severely paralyzed at hospital presentation than patients with illness caused by only BoNT/B. These bivalent strains produced BoNT subtypes A2, A4, B1, B5, and F2, and all toxin gene clusters were on plasmids.

Closed Genome Sequence of Clostridium botulinum Strain IBCA10-7060 Type Bh.

Clostridium botulinum strain IBCA10-7060 was isolated from a stool specimen from an infant botulism patient and is the only Clostridium botulinum strain known that produces botulinum toxin type H. We present here its 4.09-Mbp closed genome sequence.

Descriptive Epidemiology of Infant Botulism in California: The First 40 Years.

OBJECTIVE: To ascertain the descriptive epidemiology of infant botulism, the flaccid paralysis that results when neurotoxigenic Clostridium species produce botulinum toxin (BoNT) in the infant colon, in its first 40 years following initial recognition in California in 1976. STUDY DESIGN: Cases were defined by laboratory identification of BoNT and/or neurotoxigenic Clostridium species in patients' feces. Parents were interviewed using a structured questionnaire. Descriptive epidemiologic characteristics were compared between 1976-1996 and 1997-2016. RESULTS: From 1976-2016, 1345 cases of infant botulism occurred in 45 of 58 California counties (6.5 cases/100 000 live-births/year) caused by BoNT types A, B, Ba, Bf, and F; 88% of cases were ≤6 months of age and 51% were female. Cases were white (84.2%), Asian (8.9%), other races (3.8%), and African American (2.8%); 29.4% of cases were Hispanic. More than 99% of cases were hospitalized. Case occurrence peaked in summer-fall. Of 8 designated geographic regions, the Central Coast counties had 3 times the statewide incidence in both 20-year time periods. Breast-fed patients (83%) were more than twice as old at onset as formula-fed patients (median, 4.4 vs 1.7 months, respectively; P < .001). BoNT/A cases were older at onset than BoNT/B cases (median, 3.8 vs 2.9 months, respectively; P < .001). CONCLUSIONS: Comprehensive continuous surveillance of infant botulism for 40 years in a large, diversely populated state identified fundamental epidemiologic characteristics of this uncommon illness. Unusual features included greater than 99% case hospitalization, absence of male preponderance, and a distinctive age distribution.

Seven-Year Case-Control Study in California of Risk Factors for Infant Botulism.

OBJECTIVE: To ascertain possible risk factors for infant botulism, the intestinal infectious form of human botulism, in the years immediately following its initial recognition in California in 1976. STUDY DESIGN: Parents of 159 California laboratory-confirmed cases of infant botulism from 1976 to 1983 and 318 healthy controls were interviewed using a comprehensive (>300 factors) questionnaire. "Neighborhood controls" (n = 184) were matched on date of birth, sex, race/ethnicity, and neighborhood of residence. "County controls" (n = 134) were matched only on date of birth, sex, and county of residence. Age-stratified bivariate and multivariate conditional logistic regression analyses were performed using SAS. RESULTS: All cases required hospitalization. Bivariate analyses identified several risk factors that in multivariate analyses were not significant. In multivariate analyses, risk factors differed with stratification by age. For the ≤2 month-old neighborhood controls comparison, birth order >1, cesarean delivery, ≤1 bowel movements (BMs) per day, and windy residence area were associated with illness hospitalization, and for the county controls comparison, only pacifier use was associated. For the <2 month-old neighborhood controls comparison, <1 bowel movements (BMs) per day, cesarean delivery, birth order >1, and windy residence area were associated with illness hospitalization, and for the county controls comparison, pets in the home was an additional risk factor. CONCLUSIONS: With the exception of the ≤2-month-old county controls group, slower intestinal transit time (≤1 BM/d) was associated with illness. Otherwise, our case-control investigation identified few physiologic, environmental, and maternal factors associated with infant botulism hospitalization in California.

The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA.

BACKGROUND: Botulinum neurotoxins (BoNTs) comprise seven agreed-on serotypes, A through G. In 2014, a novel chimeric neurotoxin produced by clostridial strain IBCA10-7060 was reported as BoNT/H, with subsequent names of BoNT/FA or BoNT/HA based on sequence homology of the N-terminus to BoNT/F, the C-terminus to BoNT/A and neutralization studies. The purpose of this study was to define the immunologic identity of the novel BoNT. METHODS: monoclonal antibodies (mAbs) to the novel BoNT/H N-terminus were generated by antibody repertoire cloning and yeast display after immunization with BoNT/H LC-HN or BoNT/F LC-HN. RESULTS: 21 unique BoNT/H LC-HN mAbs were obtained; 15 from the BoNT/H LC-HN immunized library (KD 0.78 nM to 182 nM) and six from the BoNT/F-immunized libraries (KD 20.5 nM to 1490 nM). A total of 15 of 21 mAbs also bound catalytically inactive BoNT/H holotoxin. The mAbs bound nine non-overlapping epitopes on the BoNT/H LC-HN. None of the mAbs showed binding to BoNT serotypes A-G, nor any of the seven subtypes of BoNT/F, except for one mAb that weakly bound BoNT/F5. CONCLUSIONS: The results, combined with the chimeric structure and neutralization by anti-A, but not anti-F antitoxin indicate that immunologically the novel BoNT is BoNT/HA. This determination has significant implications for existing countermeasures and potential vulnerabilities.

Antimicrobial Susceptibility of 260 Clostridium botulinum Type A, B, Ba, and Bf Strains and a Neurotoxigenic Clostridium baratii Type F Strain Isolated from California Infant Botulism Patients.

Infant botulism is an infectious intestinal toxemia that results from colonization of the infant large bowel by Clostridium botulinum (or rarely, by neurotoxigenic Clostridium baratii or Clostridium butyricum), with subsequent intraintestinal production and absorption of botulinum neurotoxin that then produces flaccid paralysis. The disease is often initially misdiagnosed as suspected sepsis or meningitis, diagnoses that require prompt empirical antimicrobial therapy. Antibiotics may also be needed to treat infectious complications of infant botulism, such as pneumonia or urinary tract infection. Clinical evidence suggests (see case report below) that broad-spectrum antibiotics that are eliminated by biliary excretion may cause progression of the patient's paralysis by lysing C. botulinum vegetative cells in the large bowel lumen, thereby increasing the amount of botulinum neurotoxin available for absorption. The purpose of this antimicrobial susceptibility study was to identify an antimicrobial agent with little or no activity against C. botulinum that could be used to treat infant botulism patients initially diagnosed with suspected sepsis or meningitis, or who acquired secondary infections, without lysing C. botulinum Testing of 12 antimicrobial agents indicated that almost all California infant botulism patient isolates are susceptible to most clinically utilized antibiotics and are also susceptible to newer antibiotics not previously tested against large numbers of C. botulinum patient isolates. No antibiotic with little or no activity against C. botulinum was identified. These findings reinforce the importance of promptly treating infant botulism patients with human botulism immune globulin (BIG-IV [BabyBIG]).

Safety and immunogenicity of investigational recombinant botulinum vaccine, rBV A/B, in volunteers with pre-existing botulinum toxoid immunity.

OBJECTIVES: We undertook an open-label, uncontrolled study of investigational recombinant botulinum vaccine for botulinum neurotoxin (BoNT) serotypes A and B (rBV A/B) to assess its safety and immunogenicity in healthy volunteers who had been previously immunized with investigational pentavalent botulinum toxoid. Study participants who wished to do so could donate their hyperimmune plasma for production of Human Botulism Immune Globulin Intravenous (BIG-IV, BabyBIG®). STUDY DESIGN: A single 0.5 ml (mL), 40-microgram intramuscular injection of rBV A/B was administered to study participants. Post-vaccination sera collected at approximately 2-week intervals were evaluated for anti-BoNT/A and anti-BoNT/B neutralizing antibody concentrations (NAC). Local and systemic treatment-emergent adverse events (TEAEs) were identified by clinical and laboratory monitoring for 12 weeks post-vaccination with a final telephone follow-up for additional safety assessment at 6 months. The primary endpoint for immunogenicity was a ≥4-fold rise in NAC in ≥50% of participants by Week 4 post-vaccination. RESULTS: All 45 enrolled participants completed the study. Forty-two of 45 participants (93.3%) experienced at least one TEAE. Overall, 138 of 218 (63.3%) reported TEAEs were treatment-related, the majority of which were mild injection-site reactions. No serious or unexpected adverse events occurred. The study achieved its primary immunogenicity endpoint with 37/45 (82.2%) participants and 39/45 (86.7%) participants having a ≥4-fold rise in NAC to anti-BoNT/A and to anti-BoNT/B, respectively, by Week 4 post-vaccination. CONCLUSION: A single 0.5 mL dose of rBV A/B was safe, well-tolerated and immunogenic in participants previously immunized with pentavalent botulinum toxoid. The tolerability and immunogenicity characteristics of rBV A/B vaccination of individuals with existing BoNT immunity support its potential future use to provide occupational protection to botulism laboratory workers. Almost all study participants donated hyperimmune plasma for production of BIG-IV. ClinicalTrials.gov registration number: NCT01701999.

More Clinical Mimics of Infant Botulism.

OBJECTIVE: To ascertain the actual diagnoses of 76 patients (2005-2015) whose clinical presentations so closely resembled infant botulism that the patients were treated with Human Botulism Immune Globulin Intravenous (BIG-IV; BabyBIG), but whose illnesses subsequently were not laboratory confirmed as infant botulism ("clinical mimics" of infant botulism). STUDY DESIGN: The California Department of Public Health produces BIG-IV and distributes it nationwide as a public service (ie, not-for-profit) orphan drug to treat patients hospitalized with suspected infant botulism. During the study period, admission records and discharge summaries for all patients treated with BIG-IV but who lacked a laboratory-confirmed diagnosis of infant botulism were collected and abstracted. The patients' discharge diagnoses were identified, categorized, and compared with previously reported clinical mimics categories for 32 patients (1992-2005). RESULTS: From 2005 to 2015, 76 clinical mimic illnesses were identified. These illnesses were distributed into the 5 categories previously reported of (1) probable infant botulism lacking confirmatory testing (26.3%); (2) spinal muscular atrophy (19.7%); (3) miscellaneous (15.8%); (4) metabolic disorders (11.8%); and (5) other infectious diseases (10.6%). Of the 76 clinical mimic illnesses, 15.8% had no alternate diagnosis established and were therefore categorized as undetermined. CONCLUSIONS: Over the 23 years 1992-2015, patients presenting with illnesses so clinically similar to infant botulism that they were treated with BIG-IV had actual diagnoses that were distributed into 5 main categories. These categories and their individual components constitute a working bedside differential diagnosis of infant botulism.

Efficacy of Human Botulism Immune Globulin for the Treatment of Infant Botulism: The First 12 Years Post Licensure.

OBJECTIVES: To report the efficacy of Human Botulism Immune Globulin Intravenous (BIG-IV) in the first 12 years following its licensure in 2003 and to characterize its use nationwide in treating patients with infant botulism. STUDY DESIGN: Medical records and billing information were collected for US patients treated with BIG-IV from 2003 to 2015. Length of hospital stay (LOS) and hospital charge information for treated patients were compared with the BIG-IV Pivotal Clinical Trial Placebo Group to quantify decreases in LOS and hospital charges. RESULTS: The use of BIG-IV reduced mean LOS from 5.7 to 2.2 weeks. This shortened hospital stay resulted in a mean decrease in hospital charges of $88 900 per patient. For all US patients 2003-2015, total decreases in LOS and hospital charges were 66.9 years and $86.2 million, respectively. The decrease in mean LOS was time dependent: BIG-IV treatment on hospital days 0-3 reduced mean LOS by 3.7 weeks (P <.001 vs the BIG-IV Pivotal Clinical Trial Placebo Group), on hospital days 4-7 by 2.6 weeks (P <.001 vs the BIG-IV Pivotal Clinical Trial Placebo Group) and on hospital days 8-10 by just 1 week (P = NS). Since licensure, 1192 patients in 48 states and Washington, DC, have been treated with BIG-IV. CONCLUSIONS: The use of BIG-IV since its licensure in 2003 treated approximately 93% of US patients with laboratory-confirmed infant botulism, and prevented >65 years in hospital stay and >$85 million in hospital charges from occurring. The greatest LOS reduction was achieved when BIG-IV was administered soon after hospital admission. Effective and appropriate use of BIG-IV in the US has continued in the postlicensure period.

Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H.

BACKGROUND: Only Clostridium botulinum strain IBCA10-7060 produces the recently described novel botulinum neurotoxin type H (BoNT/H). BoNT/H (N-terminal two-thirds most homologous to BoNT/F and C-terminal one-third most homologous to BoNT/A) requires antitoxin to toxin ratios ≥1190:1 for neutralization by existing antitoxins. Hence, more potent and safer antitoxins against BoNT/H are needed. METHODS: We therefore evaluated our existing monoclonal antibodies (mAbs) to BoNT/A and BoNT/F for BoNT/H binding, created yeast-displayed mutants to select for higher-affinity-binding mAbs by using flow cytometry, and evaluated the mAbs' ability to neutralize BoNT/H in the standard mouse bioassay. RESULTS: Anti-BoNT/A HCC-binding mAbs RAZ1 and CR2 bound BoNT/H with high affinity. However, only 1 of 6 BoNT/F mAbs (4E17.2A) bound BoNT/H but with an affinity >800-fold lower (equilibrium dissociation binding constant [KD] = 7.56 × 10(-8)M) than its BoNT/F affinity (KD= 9.1 × 10(-11)M), indicating that the N-terminal two-thirds of BoNT/H is immunologically unique. The affinity of 4E17.2A for BoNT/H was increased >500-fold to KD= 1.48 × 10(-10)M (mAb 4E17.2D). A combination of mAbs RAZ1, CR2, and 4E17.2D completely protected mice challenged with 280 mouse median lethal doses of BoNT/H at a mAb dose as low as 5 µg of total antibody. CONCLUSIONS: This 3-mAb combination potently neutralized BoNT/H and represents a potential human antitoxin that could be developed for the prevention and treatment of type H botulism.

Molecular epidemiology of infant botulism in California and elsewhere, 1976-2010.

BACKGROUND: Infant botulism (IB), first identified in California in 1976, results from Clostridium botulinum spores that germinate, multiply, and produce botulinum neurotoxin (BoNT) in the immature intestine. From 1976 to 2010 we created an archive of 1090 BoNT-producing isolates consisting of 1012 IB patient (10 outpatient, 985 hospitalized, 17 sudden death), 25 food, 18 dust/soils, and 35 other strains. METHODS: The mouse neutralization assay determined isolate toxin type (56% BoNT/A, 32% BoNT/B). Amplified fragment-length polymorphism (AFLP) analysis of the isolates was combined with epidemiologic information. RESULTS: The AFLP dendrogram, the largest to date, contained 154 clades; 52% of isolates clustered in just 2 clades, 1 BoNT/A (n=418) and 1 BoNT/B (n=145). These clades constituted an endemic C. botulinum population that produced the entire clinical spectrum of IB. Isolates from the patient's home environment (dust/soil, honey) usually located to the same AFLP clade as the patient's isolate, thereby identifying the likely source of infective spores. C. botulinum A(B) strains were identified in California for the first time. CONCLUSIONS: Combining molecular methods and epidemiological data created an effective tool that yielded novel insights into the genetic diversity of C. botulinum and the clinical spectrum, occurrence, and distribution of IB in California.

Arrangement of the Clostridium baratii F7 toxin gene cluster with identification of a σ factor that recognizes the botulinum toxin gene cluster promoters.

Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. We sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. This TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.

Molecular characterization of a novel botulinum neurotoxin type H gene.

We sequenced the 2 botulinum toxin gene clusters of Clostridium botulinum strain IBCA10-7060 type Bh. The sequence of bont/H differed substantially from the sequences of the 7 known bont genes for toxin types A-G. The 5' one-third terminus of bont/H that codes for the botulinum toxin light chain differed markedly from the light chain coding sequences of toxin types A-G. The 3' two-thirds terminus of bont/H that codes for the botulinum toxin heavy chain contained a novel Hn translocation domain coding sequence and a nonneutralizing type A-like Hc binding domain coding sequence. bont/H was part of an orfX toxin gene cluster that was located at a unique chromosomal site distant from those used by other botulinum toxin gene clusters. The bont/B sequence was similar to that of subtype bont/B2 and was located within its ha toxin gene cluster at the oppA/brnQ site. Our findings further establish that C. botulinum IBCA10-7060 produces novel BoNT/H.

A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins.

BACKGROUND: Clostridium botulinum strain IBCA10-7060, isolated from a patient with infant botulism, produced botulinum neurotoxin type B (BoNT/B) and another BoNT that, by use of the standard mouse bioassay, could not be neutralized by any of the Centers for Disease Control and Prevention-provided monovalent polyclonal botulinum antitoxins raised against BoNT types A-G. METHODS AND RESULTS: The combining of antitoxins to neutralize the toxicity of known bivalent C. botulinum strains Ab, Ba, Af, and Bf also failed to neutralize the second BoNT. Analysis of culture filtrate by double immunodiffusion yielded a single line of immunoprecipitate with anti-A, anti-B, and anti-F botulinum antitoxins but not with anti-E antitoxin. A heptavalent F(ab')2 botulinum antitoxin A-G obtained from the US Army also did not neutralize the second BoNT. An antitoxin raised against IBCA10-7060 toxoid protected mice against BoNT/B (Okra) and against the second BoNT but did not protect mice against BoNT/A (Hall) or BoNT/F (Langeland). CONCLUSION: The second BoNT thus fulfilled classic criteria for being designated BoNT/H. IBCA10-7060 is the first C. botulinum type Bh strain to be identified. BoNT/H is the first new botulinum toxin type to be recognized in >40 years, and its recognition could not have been accomplished without the availability of the mouse bioassay.

Clostridium botulinum strain Af84 contains three neurotoxin gene clusters: bont/A2, bont/F4 and bont/F5.

Sanger and shotgun sequencing of Clostridium botulinum strain Af84 type Af and its botulinum neurotoxin gene (bont) clusters identified the presence of three bont gene clusters rather than the expected two. The three toxin gene clusters consisted of bont subtypes A2, F4 and F5. The bont/A2 and bont/F4 gene clusters were located within the chromosome (the latter in a novel location), while the bont/F5 toxin gene cluster was located within a large 246 kb plasmid. These findings are the first identification of a C. botulinum strain that contains three botulinum neurotoxin gene clusters.

Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing.

A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.

Universal and specific quantitative detection of botulinum neurotoxin genes.

BACKGROUND: Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA)-accepted assay to detect and type botulinum neurotoxins (BoNTs) is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR) technology to determine the specific serotype of the neurotoxin. RESULTS: We assayed purified C. botulinum DNA and crude toxin preparations, as well as food and stool from healthy individuals spiked with purified BoNT DNA, and one stool sample from a case of infant botulism for the presence of the NTNH gene, which is part of the BoNT gene cluster, and for the presence of serotype-specific BoNT genes. The PCR surpassed the mouse bioassay both in specificity and sensitivity, detecting positive signals in BoNT preparations containing well below the 1 LD50 required for detection via the mouse bioassay. These results were type-specific and we were reliably able to quantify as few as 10 genomic copies. CONCLUSIONS: While other studies have reported conventional or quantitative PCR-based assays for the detection of C. botulinum genes, our procedure's high-throughput capability and its portability allows most laboratories to quickly assess the possible presence of BoNTs either in food processing samples or in suspected cases of botulism. Thus, this assay provides rapid and specific detection of BoNT and toxin complex genes and would enable the targeting of appropriate therapeutics to infected individuals in a timely manner.

Presence of soil-dwelling clostridia in commercial powdered infant formulas.

OBJECTIVE: Because Clostridium botulinum was isolated from powdered infant formula (PIF) fed to an infant in the United Kingdom who subsequently developed infant botulism and from unopened PIF from the same manufacturer, we tested PIF manufactured in the United States for the presence of clostridial spores. STUDY DESIGN: Thirty PIF ingested by 19 California infants with botulism within 4 weeks of onset of illness (48% of all patients fed PIF during study) in 2006-2007 were cultured anaerobically to isolate clostridia. All isolated clostridia were identified to the species level and enumerated with standard microbiologic and molecular methods. RESULTS: Five of 30 (17%) PIF samples ingested by patients contained clostridial spores. Spores were also found in 7 of 9 (78%) market-purchased PIF samples. Clostridium sporogenes was isolated most frequently, followed by Clostridium butyricum and at least 10 other soil-dwelling clostridial species. No neurotoxigenic clostridia were isolated. The most probable number of clostridial spores in PIF ranged between 1.1 to >23 per 100 g. CONCLUSIONS: With the notable exception of production of botulinum neurotoxin, C sporogenes is physiologically comparable with proteolytic strains of C botulinum, and both share the same natural reservoir (soils and dust worldwide). The isolation of C sporogenes and potentially pathogenic clostridia from U.S.-manufactured PIF suggests that neurotoxigenic clostridial spores have the potential to be present in these products.