INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1988, p. 375-381 Vol. 38, No. 4 0020-7713/88/040375-07$02.00/0 Clostridiurn argentinense sp. nov. : a Genetically Homogeneous Group Composed of All Strains of Clostridium botulinum Toxin Type G and Some Nontoxigenic Strains Previously Identified as Clostridium subterminale or Clostridium hastiforme JANE C. SUEN, CHARLES L. HATHEWAY," ARNOLD G. STEIGERWALT, AND DON J. BRENNER Division of Bacterial Diseases, Center for Infectious Diseuses, Centers for Disease Control, Atlanta, Georgia 30333 We determined deoxyribonucleic acid (DNA) relatedness among toxigenic strains of Clostridium botulinum toxin type G and nontoxigenic strains labeled Clostridium subterminale and Clostridium hastiforme by using the hydroxyapatite method at 50 and 65°C. Seven DNA hybridization groups were detected. All of the strains were anaerobic, gram-positive, rod shaped, asaccharolytic, and proteolytic; produced acetic, butyric, isobutyric, and isovaleric acids; and were separable by additional phenotypic characteristics into the seven hybridization groups. One hybridization group was composed of all nine C. botulinum type G strains, two strains received as C. subterminale, and one strain received as C. hastiforme. These strains showed an average level of intragroup relatedness of 94% and were less than 25% related to the type strains of C. botulinum (BL 4847), C. subterminale (BL 4856), and C. hastiforme (BL 4858). The guanine-plus-cytosinecontents of four strains in this group were 28 to 30 mol%. A new species, Clostridium argentinense, is proposed for the genotypically homogeneous group that is unrelated to the strains of C. botulinum and other clostridial species which we studied. The type strain of C. argentinense sp. nov. is strain ATCC 27322. Two of the six other DNA hybridization groups are represented by the type strains of C. subterminale and C. hastiforme. The four remaining DNA hybridization groups represent undescribed species that contain one or two strains each. -___ As currently classified, Clostridium botulinum is distin- The nine strains of C. botulinum type G include two (BL guished from other clostridia by the production of a neuro- 4853 and BL 1353) derived from the original Argentinian soil toxin that causes botulism paralysis. Seven immunologically isolate (strain 89) (9), as well as a second Argentinian soil distinct botulinal toxins (types A through G) are produced by isolate (BL 714). strains of C. botulinum (22). Toxin types A through F have Phenotypic characterization. The procedures and media been implicated in human botulism or animal botulism or used for culturing and characterizing the organisms have both. Toxin type G has been isolated from soil samples (9) been described by Dowell et al. (6-8) and Lombard and and autopsy specimens (23) but has not been clearly impli- Dowell (15, 16). Each strain was inoculated into chopped cated as the cause of paralytic illness or death. C. botulinum meat-glucose-starch broth (8) and incubated for at least 48 h is divided into four metabolically distinct groups; groups I at 35°C in an anaerobic glove box containing 85% N,, 10% through 111 contain multiple toxin types, and group IV H,, and 5% CO,. Subsequent streaking onto anaerobic blood contains only toxin type G (22). Each group has a phenotyp- agar (8) and Centers for Disease Control-modified McClung- ically similar, nontoxigenic counterpart, which for groups I Toabe egg yolk agar plates (8) yielded isolated colonies, and 111 are Clostridium sporogenes and Clostridium novyi which were observed for their colonial and cellular morphol- type A, respectively (22). A species name has not been ogy, Gram stain reaction, production of spores, motility, and proposed for the nontoxigenic strains of group 11. ability to grow in the presence or absence of oxygen. They Clostridium subterminale is the nontoxigenic phenotypic were tested for their ability to ferment 15 carbohydrates; for counterpart of C. botulinum toxin type G (22). Clostridium production of indole or indole derivatives (7, 16), urease, hastiforme is another nontoxigenic species that is phenotyp- catalase, lecithinase, lipase, deoxyribonuclease, and hydro- subterminale, ically similar to C. except that it may be less gen sulfide; for hydrolysis of esculin, starch, and gelatin; and proteolytic, does not produce hydrogen, and produces ter- for digestion of milk. They were also tested for their ability minal rather than subterminal spores (4). Genetic relatedness to degrade 21 substrates in the AN-IDENT (Analytab Prod- among toxigenic strains of C. botulinum groups I through 111 ucts, Plainview, N.Y.) and 18 substrates in the RapID ANA and their nontoxigenic counterparts has been studied previ- (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) micro- ously (12, 13, 20, 21, 25), but similar studies to compare C. test systems. Strains were analyzed for their metabolic acid botulinum toxin type G, C. subterminale, and C. hastiforme end products in peptone-yeast extract-glucose medium (8) have not been done. by gas-liquid chromatography (15). Indole and indole deriv- The purpose of this study was to examine the taxonomic ative(s) were tested by adding 2 drops of 1%p-dimethylami- relationships among C. botzilinum type G, nontoxigenic C. nocinnamaldehyde reagent to a paper disk on Lombard- subterminale, and nontoxigenic hastiforme. C. Dowell agar containing a 48-h growth of the test organism. The presence of indole and derivative compounds was MATERIALS AND METHODS detected by color changes after the addition of the p- dimethylaminocinnamaldehyde reagent; a positive indole Bacteria. The strains which we used are listed in Table 1. reaction was indicated by development of a blue or bluish green color within 30 s, while a lavender to violet color * Corresponding author. denoted a positive reaction for an indole derivative. 375 376 SUEN ET AL. INT. J. SYST.BACTERIOL. TABLE 1. List of Clostridium strains and their sources Strain" Initial identification Received from? Source No. of sender BL 714 C. botulinum type G Gimenez Soil' 117 BL 1353 C. botulinum type G Ciccarelli Soild 89 BL 2738 C. botulinum type G Sonnabend Autopsy' SN 143 BL 2739 C. botulinum type G Sonnabend Autopsy' GM 140 BL 2740 C. botulinum type G Sonnabend Autopsy'' GM 73 BL 2741 C. botulinum type G Sonnabend Autopsy' GM 77 BL 2742 C. botulinum type G Sonnabend Autopsy' GM 56 BL 3659 C. botulinum type G Sonnabend Amniotic fluid HO 4170 BL 4853 C. botulinum type G ATCC Smith, Gimenez, soif ATCC 27322 AL 750 C. subterminule Missouri Health Department Brain abscess AL 1453T C. subterminale ATCC Smith, VPI 2023 ATCC 25774T AL 4422 C. subterminale New Jersey Health Department Blood AL 14431 C. subterminale New Jersey Health Department Wound AL 14764 C. subterminate Health and Welfare Canada Eye infection AL 16525 C. subterminale Mississippi Health Department Wound BL 4856T C. subterminale ATCC Smith, VPI 2023 ATCC 25774T NH 134 C. subterminale Hodinka Hand abscess 134 NH 137 C. subterminale Hodinka 137 NH 138 C. subterminale Hodinka Clinical isolate 138 NH 239 C. subterminale Hodinka 239 AL 1449 C. hastiforme ATCC Smith, VPI 2461; Beerens 736L ATCC 25772 AL 12677 C. hastiforme Texas Health Department Wound BL 485gT C. hastiforme ATCC Moore, VPI 12193; California State Health ATCC 3326gT Department BL 4847* C. botulinum type A ATCC Smith, VPl 1550; CDC KA38 ATCC 25763* BL 6185 C. botulinum type B ATCC National Canners Association 213B ATCC 7949 (January 1941) BL 4851 C. botulinum type F ATCC Smith, VPI 4404; Walls 8G; crab ATCC 25764 BL 4859T C. sporogenes ATCC Hall 338, Peterson IF015950; McClung ATCC 3584T 2004 AL 1443= C. butyricum ATCC NCTC 8237 (J. Howie strain Rowett), pig ATCC 1939gT AL 1442T C. beijerin c kii ATCC Smith, VPI 5481; McClung 1671 ATCC 25752T AL 1778gT C. perfringens ATCC NCTC 8237, Wellcome Lab CN 1491 ATCC 13124T AL 1440 C. baratii ATCC Seo, VPI 670714 (Nakamura strain G) ATCC 27639 BL 4857T C. baratii ATCC Nakamura 2227 (Institut Pasteur 2227) ATCC 2763gT BL 5262 Clostridium sp. CDC isolate Infant fecesR CDC 5262 BL 2990 Clostridium sp. CDC isolate Infant fecesh CDC 2990 " BL, Botulism Laboratory, Centers for Disease Control, Atlanta, Ga.; AL, Anaerobe Laboratory, Centers for Disease Control, Atlanta, Ga.; NH, N. Hodinka, Innovative Diagnostic Systems, Inc., Atlanta, Ga. A superscript "T" indicates a type strain. Gimenez, D. F. Gimenez, Universidad Nacional de San Luis, San Luis, Argentina; Ciccarelli, A. S. Ciccarelli, Universidad Nacional de Cuyo, Mendoza, Argentina; Sonnabend, W. Sonnabend, Kantonsspital, St. Gallen, Switzerland; ATCC, American Type Culture Collection, Rockville, Md.; Hodinka, N. Hodinka, Innovative Diagnostic Systems, Inc., Atlanta, Ga. A. S. Ciccarelli, A. M. Giulietti, and D. F. Gimenez, Primer Congreso y IV Jornadas Argentinas de Microbiologia, abstr. no. 21, 1976. Reference S. ' Reference 23. Reference 9. Reference 19; available as ATCC 43181. Reference 11. Toxicity and neutralization tests. All strains of C. botuli- cells were lysed by a modification of methods described by num and the two strains designated as Clostridium sp. were Lee and Riemann (13) and Nakamura et al. (21). Lysozyme tested for their ability to produce toxin by mouse toxicity (Worthington Diagnostics, Freehold, N.J.) was added to a tests, and the toxins were identified by neutralization with final concentration of 1 mg/ml, and the mixture was incu- specific antitoxins (6). All strains received as C. subtermi- bated for 30 min in a 37°C water bath. Following this, 0.1 nale and C. hastiforme were previously determined to be volume of 1% sodium dodecyl sulfate-O.l M tris(hydroxy- nontoxigenic in the Botulism Laboratory, Centers for Dis- methy1)aminomethane-0.1 M NaCl (pH 9.0) was added, and ease Control. pronase (grade B; Behring Diagnostics, La Jolla, Calif.) was DNA studies. For the deoxyribonucleic acid (DNA) stud- added to a final concentration of 1.0 mglml. This mixture was ies, each strain was inoculated into chopped meat-glucose- incubated for 30 min at 37°C.