Clostridium botulinum is an anaerobic spore-forming bacterium producing BoNT, which is the cause of botulism in humans and animals (1) and is divided into seven serotypes (A to G) (2). Some strains harbor two different serotypes of BoNT genes in their genome (3). BoNT is encoded by an approximately 3.8 kb gene, which is preceded by several nontoxic component genes (4). BoNT together with the nontoxic component genes are defined as the boNT gene cluster (5). Recently, BoNT was subclassified by BoNT gene sequence analysis, BoNT/A being divided into four subtypes (A1, A2, A3 and A4) (3). There are two types of nontoxic components of gene organization (the HA and Orfx clusters), and C. botulinum type A strains were classified according to their harboring of these clusters (4–6). The HA cluster consists of ha17, ha33, ha70, botR and ntnh genes, and the Orfx cluster consists of orfx3, orfx2, orfx1, botR, p47 (unknown function) and ntnh genes (6). Franciosa et al. have reported that type A strains possess boNT/A1 and HA cluster genes to boNT/A gene cluster type 1; boNT/A2 and Orfx cluster genes to boNT/A gene cluster type 2; and boNT/A1 with unexpressed or expressed boNT/B, HA cluster and Orfx cluster genes to boNT/A gene cluster type 3 (5). BoNT/A gene cluster typing has recently been applied for molecular characterization of type A strains (5).
In Japan, 24 cases of infant botulism have been reported: 16 of type A, 3 of type B, 1 of type C and 1 of C. butyricum producing BoNT/E. The types of toxin in the other three cases were not described (7–9). During 1986 to 1989, nine cases occurred; all were type A and gave a history of feeding with honey before the onset of symptoms. Since 1990, seven cases of type A have occurred, but none had a history of feeding with honey and the origin was not identified in five cases.
In this study, we developed the multiplex PCR method to easily detect boNT/A gene cluster types. In order to better understand the background of infant botulism cases in Japan, we then genotyped C. botulinum type A isolates by boNT/A gene cluster and PFGE types.
Twenty-seven C. botulinum type A strains, including 10 isolates associated with infant botulism in Japan, were cultured in 10 ml cooked meat medium (Difco, Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with 0.3% glucose and 0.2% soluble starch under anaerobic conditions for 18 hr at 30°C (Table 1). Bacterial DNA was extracted according to our previous report (10). Multiplex PCR assay was performed using two sets of primers: “boNT/A1, A2” contained three primers to identify boNT/A1 or boNT/A2 genes; and “ha33, p47” contained four primers to detect the ha33 gene, which is specific for the HA cluster, and the p47 gene, which is specific for the Orfx cluster (Table 2). PCR was performed with a 25 μl reaction mixture containing 0.1–1 ng template DNA, 0.25 μM of each primer, 1.25 U Ex Taq (TaKaRa Shuzo, Kyoto, Japan), 2.5 μl Ex Taq buffer and 200 μM deoxynucleotide-triphosphate. Each PCR cycle consisted of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, and was repeated 30 times. Unexpressed boNT/B gene was detected by another PCR assay for boNT/A to boNT/G genes (11). Sma I digested PFGE was carried out as described in our previous report (10). Dendrogram analysis of the band patterns was generated with FPQuest software ver.4.5 (Bio-Rad, Hercules, CA, USA). Another PFGE type was defined where there was more than one fragment difference in the PFGE band pattern.
|Source and strain||Description† (Reference)||boNT gene‡||Multiplex PCR||boNT/A gene cluster type§||PFGE type|
|boNT/A1, A2||ha33, p47|
|Infant botulism in Japan|
|Chiba H||Chiba, 1986, honey feeding (7, 8)||A||A2||p47||2||S1|
|Kyoto F||Kyoto, 1987, honey feeding (8)||A||A2||p47||2||S2|
|KZ1828||Ishikawa, 1987, honey feeding (8)||A||A2||p47||2||S1|
|7I03 H||Osaka, 1987, honey feeding (8)||A||A2||p47||2||S2|
|7I05 F||Ehime, 1987, honey feeding (8)||A||A2||p47||2||S3|
|7I05 H||Ehime, 1987, honey feeding (8)||A||A2||p47||2||S4|
|Y8036||Kanagawa, 1987, honey feeding (8)||A||A2||p47||2||S5|
|Hiroshima1||Hiroshima, 1999, unidentified (8, 16)||A, B||A1||ha33, p47||3||S6|
|Miyagi2006||Miyagi, 2006, well water (9)||A, B||A1||ha33, p47||3||S6|
|Iwate2007||Iwate, 2007, unidentified (9)||A, B||A1||ha33, p47||3||S7|
|Food-borne botulism in Japan|
|Renkon||Kumamoto, 1984, karashi renkon (8)||A, B||A1||ha33, p47||3||S8|
|CB111||Tokyo, 1999, unidentified||A||A1||ha33||1||S9|
|CB121||Chiba, 1999, vacuum-packed hashed beef (17)||A, B||A1||ha33, p47||3||S6|
|Osaka99||Osaka, 1999, unidentified||A, B||A1||ha33, p47||3||S10|
|Infant botulism in the USA|
|89E00064-3||California, 1989||A, B||A1||ha33, p47||3||S13|
|89E00086-1||California, 1989||A, B||A1||ha33, p47||3||S14|
|802-1||Germany, 1988, red pepper||A||A1||ha33||1||S17|
|804-1H||Brazil, 1988, honey||A||A2||p47||2||S18|
|Primer set||Primer||Sequence (5′-3′)||Product size (bp)||Location on gene (coding region)|
The results of multiplex PCRs of 27 type A strains are summarized in Table 1 and representative results are depicted in Figure 1. The boNT/A1 (665 bp) and ha33 amplicons (534 bp) were detected in 11 strains (CB111, 89E00033-1, 89E00035-1, 83E00080, 2137-1-77, 802-1, Denken, 97A, 62A, 33A and 36A), which were accordingly classified as boNT/A gene cluster type 1. The boNT/A2 (440 bp) and p47 amplicons (344 bp) were detected in eight strains (Chiba H, Kyoto F, KZ1828, 7I03 H, 7I05 F, 7I05 H, Y 8036 and 804-1H), which were accordingly classified as boNT/A gene cluster type 2. The boNT/A1, ha33 and p47 amplicons were detected in eight strains (Hiroshima1, Miyagi2006, Iwate2007, Renkon, CB121, Osaka99, 89E00064-3 and 89E00086-1). These also harbored the unexpressed boNT/B gene (Table 1), and were therefore classified into boNT/A gene cluster type 3. No amplicon was detected in the 12 control strains, which were as follows: three type B (Okra, 111 and Osaka05), one type C (CB-19), one type D (1873), one type E (Iwanai), one type F (Langeland), one BoNT/E producing C. butyricum (5262), one C. sporogenes (ATCC19404), one C. bifementas (ATCC638), one C. perfringens (ATCC13124) and one C. difficile (ATCC43593) by “boNT/A1, A2” PCR. The ha33 amplicon was detected in the three type B strains and the p47 amplicon in the type F strain by “ha33, p47” PCR (data not shown).
The PFGE patterns of Sma I digested DNA from 27 type A strains and a dendrogram based on the similarities between normalized PFGE patterns are presented in Figure 2, and PFGE types are listed in Table 1. The 27 strains were divided into 22 PFGE types (S1-S22) and their similarity ranged from 29.6% to 100%. The seven isolates associated with infant botulism in Japan during 1986–1987 were divided into five PFGE types with 78.6–100% similarity, and 81.5% to 89.7% similarity to strain 804-1H, isolated from Brazil honey. The three isolates associated with infant botulism in Japan during 1999–2007 were divided into two PFGE types with 62.1% similarity. Strains Hiroshima1 and Miyagi2006 showed identical PFGE types to strain CB121, which was associated with food-borne botulism in Japan in 1999.
The new multiplex PCR method for boNT/A gene cluster typing established in this study is able to classify reference strains (62A and Kyoto-F) into their previously described cluster types (1 and 2, respectively) (5). The detection limit of “boNT/A1, A2” PCR was from 5.5 × 10 to 2.8 × 102 cells/ml, and of “ha33, p47” PCR from 2.1 × 102 to 2.1 × 103 cells/ml of culture dilutions (data not shown). The PCR-based boNT/A gene cluster typing method reported is a combination of BoNT/A subtyping by PCR-RFLP and ha33 and p47gene detection by separate PCR (5); however, our PCR method has the advantages of simplicity, rapidity, specificity and sensitivity, so it would be applicable not only for molecular typing, but also the diagnosis of botulism.
A correlation between boNT/A gene cluster types and geographical distribution has been reported (5). Cluster type 1 and 3 strains are predominant in the USA, while cluster type 2 strains are predominant in Europe. Isolates associated with infant botulism in Japan were clearly divided into cluster type 2 and 3 by their time periods, and shown to be related to isolates from honey of South American origin and food-borne botulism, respectively. This is the first report of the genetic relationship between isolates associated with infant botulism and food-borne botulism in Japan. C. botulinum type A is rarely found in Japanese soil, while type C and E are widely distributed (12). There is a possibility that imported goods are related to botulism cases. In other countries, in addition to honey, powdered infant formula (13), baby food (14) and house dust (15) have been reported as causes of infant botulism. Further risk assessment of several food and environmental samples to prevent infant botulism are warranted.
While boNT/A gene cluster typing is less discriminating than PFGE genotyping, it is excellent for genetic comparison among different laboratories or countries. The application of multiplex PCR assays will contribute to understanding the local and geographic epidemiology of C. botulinum.