JOURNAL OF CLINICAL MICROBIOLOGY, June 1991, p. 1114-1124 Vol. 29, No. 60095-1137/91/061114-11$02.00/0Copyright © 1991, American Society for Microbiology

Identification of Clostridium botulinum, Clostridium argentinense,and Related Organisms by Cellular Fatty Acid AnalysisFATMA M. GHANEM,W ANN C. RIDPATH,2 W. E. C. MOORE,2 AND L. V. H. MOORE2*

Department of Veterinary Medicine, Suez Canal University, Ismailia, Egypt,' and Department ofAnaerobicMicrobiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061_03052

Received 14 December 1990/Accepted 7 March 1991

On the basis of 686 analyses of 285 strains of Clostridium botulinum, Clostridium argentinense (formerly C.botulinum type G), and phenotypically related organisms, 14 cellular fatty acid (CFA) groups of toxicorganisms and 6 CFA groups of nontoxic organisms were delineated. The CFA groups of toxic strains includedtwo of type A, three of proteolytic strains of type B, two of proteolytic strains of type F, one each ofnonproteolytic strains of types B, E, and F, and one each of types Ca, CoI, and D and C. argentinense. Thegroups of phenotypically similar nontoxic strains included Clostridium sporogenes, Clostridium putrificum,nontoxic strains with phenotypic characteristics similar to those of nonproteolytic strains of C. botulinum typesB, E, and F (BEF-like), two groups of nontoxigenic organisms with phenotypic characteristics similar to thoseof C. botulinum types C and D and Clostridium novyi (CDN-like), and Clostridium subterminale, which hasphenotypic characteristics similar to those of C. argentinense. Within the toxin types, 89 to 100% of the strainswere correctly identified by CFA analysis, and 74 to 100% of the analyses were correct. Of 36 strains of C.sporogenes, 30 (83%) were correctly identified; 17% of the strains of C. sporogenes were incorrectly identifiedas C. botulinum type A or B. All analyses of C. putrificum and C. subterminale were correctly identified. Therewas no significant level of similarity between strains of C. botulinum and phenotypically similar organisms and85 other species of clostridia or 407 other taxa of gram-positive and gram-negative bacteria. Additionally, theone strain each of Clostridium baratii and Clostridium butyricum previously reported to produce C. botulinumtoxin could be differentiated from C. botulinum types as well as from strains of C. baratii and C. butyricum thatdid not produce a neurotoxin.

Clostridium botulinum produces a potent exotoxin that isthe cause of botulism. This neurotoxin causes a severeneuroparalytic disease characterized by sudden onset andswift course, terminating in paralysis and pulmonary arrest.Although the disease caused by botulinum toxin is rare inhumans, it is relatively common in birds and animals (17).Holdeman and Brooks (8) divided C. botulinum types A

through F into three major groups according to their culturalcharacteristics and metabolic products. Other clostridia thathave cultural characteristics similar to those of C. botulinumalso are included in these groups. The phenotypic groupingof Holdeman and Brooks (8) has been recognized by others(12, 17), but these other authors rearranged the groups asfollows. Group I contains C. botulinum type A, proteolyticstrains of C. botulinum types B and F, and Clostridiumsporogenes. Group II contains C. botulinum type E, non-proteolytic strains of types B and F, and related nontoxicorganisms. Group III contains C. botulinum types C and Dand Clostridium novyi type A. Smith (17) proposed a fourthgroup for Clostridium argentinense (C. botulinum type G)which was expanded to include Clostridium subterminale(18).

Following the metabolic grouping by Holdeman andBrooks (8), high levels of DNA relatedness were reported(12) among proteolytic strains of types A, B, and F and C.sporogenes (group I). Lee and Reimann (13) also showedhigh levels ofDNA relatedness among nonproteolytic strainsof types B and F, strains of type E, and some metabolicallysimilar nontoxic strains that made up group II. Repre-sentative strains of C. botulinum types C and D (group III)

* Corresponding author.

have no significant DNA relatedness to reference DNA ofeither group I or group II organisms. Suen et al. (18) reportedthat C. argentinense (type G) strains are unrelated by DNAhybridization to C. sporogenes or strains of C. botulinum ingroup I and proposed a new species, C. argentinense, for thetype G strains. Also included in C. argentinense are somenontoxic strains that they previously had identified as C.subterminale or Clostridium hastiforme.The most reliable method for differentiation of strains of

C. botulinum from their nontoxic counterparts is by specificantitoxin neutralization of toxic activity in a mouse assay.Inasmuch as many laboratories do not have the capability todo such neutralization assays, it would be highly desirable tohave an in vitro assay available that could differentiate C.botulinum from its nontoxic counterparts.The use of cellular fatty acid (CFA) composition for

taxonomy and identification of clostridia has been investi-gated previously. Moss and Lewis (16) examined 41 strainsrepresenting 13 species of clostridia and found that both thepresence and relative amounts of fatty acids with a chainlength of 10 to 20 carbon atoms (as determined by gas-liquidchromatography) could distinguish among Clostridium per-fringens, C. sporogenes, and Clostridium bifermentans aswell as separate these 3 species from the other 10 speciestested. In an expanded study, Elsden et al. (6) mapped thefatty acid compositions of the lipids of 23 species of proteo-lytic clostridia using capillary gas-liquid chromatographyand gas-liquid chromatography-mass spectrometry. Theyidentified the relative quantities of 55 fatty acids in the C12 toC,8 range and correlated these with amino acid metabolism.Elsden et al. (6) did not, however, attempt to use the resultsfor purposes of identification.Hardware and software (Microbial Identification System,

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CELLULAR FATTY ACIDS OF C. BOTULINUM 1115

or MIS system), distributed by Microbial ID, Inc. (Newark,Del.), now is available to identify bacteria by comparison ofCFA profiles with those of standardized reference patterns.The MIS system includes a gas chromatograph with a flameionization detector with an integrator and a computer that,by retention time, can differentiate more than 140 com-pounds including fatty acid methyl esters (FAMEs), di-methyl acetyls (DMAs), aldehydes, and unknown com-pounds that are distinctive for individual species. FAMEsextracted from strains to be tested are compared withidentification libraries that contain profiles of CFAs of eachspecies for which there is a library entry.Each library entry is developed by testing FAME extracts

of multiple properly identified strains of the taxon or multipleanalyses of only a few strains if only a few are available. Thestrains are grown under standardized conditions (especiallyof medium composition and incubation temperature). Eachanalysis contributes to a library entry; multiple libraryentries make up a library. Each library entry is composed ofthe means and standard deviations of the peak areas as apercentage of the total chromatographic area of all peaksmost commonly detected in the tested reference strains.Microbial ID, Inc., periodically provides updated and im-proved libraries to its subscribers. The MOORE AnaerobeBroth Library is one of several libraries distributed byMicrobial ID, Inc. The MOORE library has been developedby using broth cultures, because we found that results weremore reproducible from broth than from solid medium and itoften was easier to get sufficient growth of slowly growingstrains in broth than on plates (unpublished data).

Library entries should include reasonable variation thatwill be seen among properly identified strains of a speciesand slight variations that will occur under even "standard-ized" conditions such as different lots of medium, slightlydifferent incubation times (16 versus 24 or 36 h), replicatecultures tested on the same days, replicate cultures tested ondifferent days, and cultures prepared and extracted bydifferent technologists.

Software for developing library entries also is availablefrom Microbial ID, Inc. This software includes programs tocompare strains thought to represent the same species byprincipal component analysis of their FAMEs. The relation-ships of individual strains can be seen by cluster analysesthat compare the variable components, e.g., 1 versus 2, 2versus 3, and 1 versus 3. These comparisons portray spatialrelationships among the analytical results of the individualstrains and can indicate analyses (or strains) that appear tobe outliers. Each library entry should be "broad" enough toinclude real variation within species, yet "narrow" enoughto differentiate each species from all other species.The user in a clinical laboratory would not be expected to

generate library entries. Rather, the identification librariescan be obtained from Microbial ID, Inc. CFA extracts of testorganisms grown under standardized conditions are chro-matographed, and a chromatogram of the fatty acids and alisting of the names of the fatty acid derivatives, retentiontimes, amounts, and suggested identifications with similarityvalues are produced. If there is an insufficient amount ofCFA for adequate comparison, the report indicates that theanalysis is questionable and a repeat analysis is requested.The derivation of "similarity unit" is proprietary informa-tion. By general usage, a value of about 0.500 or greater isconsidered an acceptable identification and a value of<0.500 generally is interpreted as a questionable identifica-tion. For any given analysis, more than one species mayhave a similarity value of >0.500, in which case the higher

value would be the preferred suggested species. Becausethere are different species with similar fatty acid composi-tions, e.g., both giving similarity values of >0.500, it iscritical that a library include as many species as possible sothat an unknown is not misidentified as library entry "A"with a similarity value of 0.600 when it really is species "Z"that is not yet included in the library but that would have asimilarity value with the unknown strain of 0.9 if it were.The MOORE Anaerobe Broth Library is continuously

being modified and expanded. Here we report the results ofa study to evaluate a CFA system for the identification of C.botulinum, C. argentinense, and phenotypically related or-ganisms.

MATERIALS AND METHODS

Bacterial strains used in the generation of library entries.When this study was initiated, we were using a modificationof the 2.2 version of the VPI (Virginia Polytechnic Instituteand State University) Anaerobe Library that was distributedby Microbial ID, Inc. The C. botulinum libraries in the 2.2version were based on 202 analyses of 58 strains of types Athrough F plus 30 analyses of 7 strains of C. argentinense. Inthis study, 95 additional strains of C. botulinum types A, Bproteolytic (BP), C, and E were tested. No new strains oftypes D, F, or G were available for study.Type A. The 2.2 library version contained results of

analyses of VPI 1550 (parent strain of ATCC 25763T [Amer-ican Type Culture Collection [ATCC], Rockville, Md.]) (2analyses) and ATCC 17862 (2 analyses), CDC KA-95B(Centers for Disease Control [CDC], Atlanta, Ga.) (1 analy-sis) and CDC 297 (2 analyses), Prevot (Pasteur Institute,Paris, France) strains 146 (2 analyses) and 729 (2 analyses),L. S. McClung (Indiana University, Bloomington, Ind.)strains 465 (labeled type B) (1 analysis) and 2058 (Loch-Maree strain) (3 analyses), and Gimenez (Argentina) strain84-2(4 analyses) and 15 analyses of five other strains. In thisstudy, results of analyses of Food and Drug Administration(FDA) type A strains (received from Warren Landry, FDA,Houston, Tex.) 106, 107, 108, 109, and 110; Prevot strains729, PP, 910, 969, 62NCA, P179, 878, 621, 865, F18, F16,F57, dewping, F60, 892, P64, 697B, and F5G; and Ivan C.Hall type A strains 174, 3685A, 10468d, 10637, 11569, 17544,6581Aa, 5675, and 10174A were added to the library entry.

Proteolytic strains of type B. The 2.2 library versionincluded results of analyses of ATCC 7949 (2 analyses),ATCC 8083 (1 analysis), CDC KA-40 (4 analyses), andPrevot strain 25NCASE (3 analyses) and 22 analyses ofseven other strains. Results of analyses of 37 proteolyticstrains of type B added in this study were of ATCC 844; Hallstrains 80, 179, 6517B, 6660, 6706, and 10007; Landry (FDA,Houston, Tex.) strains 111, 112, 113, 114, 115, and 120;McClung strains 450, 457, and 2239; Prevot strains B, Bli,B12 (8 analyses), CM, Fll, PPois, 594, 1490, 1504, 1542,1552, 1662, 1687, 1740, 1837, 1962, and 2345; VPI 1750A; and5 other strains.

Nonproteolytic strains of type B. Results of analyses ofEklund strain 17B (12 analyses) and Prevot strain 59 (5analyses) constituted the type B nonproteolytic (BNP) li-brary entry.Type C. Results of analyses of Jensen strain X220B1-5

type Ca (2 analyses), Jensen strain X220B2-4 type Ca (1analysis), Jensen strain 468(6)d type C, (1 analysis), Prevotstrain 468 type C,B (2 analyses), and Smith strain type C, (2analyses) and 45 analyses of 11 other strains constituted the

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1116 GHANEM ET AL.

2.2 library version. Results of analyses of Prevot type Cstrains 571T, 2233, and 2266 were added in this study.Type D. Results of analyses of Smith strains D#1 (seven

analyses), Onderstepoort received from Jensen and theparent strain of ATCC 27517 (five analyses), and M'Bour(six analyses) and four analyses of one other strain wereincluded in the library entry.Type E. Results of analyses of ATCC 9564 (four analyses),

Hobbs 4248 Beluga strain (four analyses), Hobbs 4249 TennoE strain (two analyses), Hobbs strain FT18 (four analyses),Kautter (FDA, Washington, D.C.) strain 066B TOX (fouranalyses), and CDC KA-95B (three analyses) and threeanalyses of one other strain were included in the libraryentry.Type F (proteolytic). Results of analyses of CDC KA-1 (=

Langeland strain) (seven analyses), Wall strain 8-G (sixanalyses), and Gimdnez strain 160 (three analyses) wereincluded in the library entry.Type F nonproteolytic. Results of analyses of Eklund strain

F202 (five analyses), Hobbs strain FIO (one analysis), andSmith strain 5436 (six analyses) were included in the libraryentry.Type G. Results of analyses of Gimenez strain 89 (four

analyses); Microbial Diseases Laboratory of the CaliforniaDepartment of Health Services strain 0547 (five analyses);and Fort Detrick strains 2738 (five analyses), 2739 (fouranalyses), 27440 (six analyses), 2741 (two analyses), and2742 (four analyses) constituted the library entry.

Also included in this study were analyses of two strainsreceived from C. L. Hatheway, CDC, that produce C.botulinum toxin but that have phenotypic characteristics ofother species: VPI 14304, which is CDC strain 5262 andwhich resembles Clostridium butyricum (14), and VPI 14484,which is CDC strain 3112 and which resembles Clostridiumbaratii (7).The C. sporogenes library entry (Table 1) is based on the

results of 44 analyses of 36 strains, including ATCC 3584Tand 35 other strains, mostly from human or animal clinicalspecimens. The Clostridium putrificum library entry (Table1) is based on the results of 4 analyses of ATCC 25784Tand 18 analyses of 13 other strains. The nontoxic and non-proteolytic BEF-like entry (Table 2) is based on the resultsof 12 analyses of three strains, including Kautter (FDA,Washington, D.C.) strain 28-2 isolated from Lake Huronmud. The nontoxic CDN-like group 1 and group 2 libraryentries (Table 2) are based on the results of analyses ofstrains labeled types Ccx, Cp, D, and C. novyi (all previouslytoxic) and of clinical isolates, some of which lost their toxi-cities before they could be typed and some in which toxinwas never demonstrated. The C. subterminale libraryentry (Table 3) contains the results of 28 analyses of 21strains, including ATCC 25774T. The C. butyricum libraryentry (Table 3) contains the results of 27 analyses of 18strains, which included three cultures of the type strain(ATCC 19398, NCTC 7423, and McClung 2391), 13 DNAhomology strains (3), and 4 clinical isolates. The C. baratiilibrary entry is based on the results of 21 analyses of 15strains, including ATCC 27638, the type strain of C. baratii,and the type strains of Clostridium perenne and Clostridiumparaperfringens (ATCC 25782T and ATCC 27639T, respec-tively), which have been shown to be subjective synonymsof C. baratii (2).

Bacterial taxa used to test the specificities of library entriesand identifications. Extracts of all strains automatically werecompared with 517 library entries on the basis of more than20,000 analyses. These entries included analyses of 85 spe-

cies of clostridia (all named species of clostridia that areassociated with human and animal infections and mostnamed species of Clostridium and clostridial toxin types,except for a few species of thermophiles). Library entries ofanalyses of nonclostridial species with which the results ofthe C. botulinum strains automatically were compared con-tain 9 named species (13 serotypes) and 2 unnamed speciesof Actinomyces, Bacterionema matruchotii, 25 named and 3unnamed species of Bifidobacterium, Coprococcus catus, 21named and 16 unnamed species of Eubacterium, 56 namedand 5 unnamed species of Lactobacillus, 7 named and 7unnamed species of Peptostreptococcus, 5 species of Propi-onibacterium, Rothia denticariosa, 11 species of Staphylo-coccus, 15 named and 15 unnamed species of Streptococcus,Acidaminococcus fermentans, 21 named and 28 unnamedspecies of Bacteroides, Biophila wadsworthia, Campylobac-ter concisus, 5 named and 2 unnamed species of Capnocy-tophaga, Centipeda periodontii, Eikenella corrodens, 15named and 2 unnamed species of Fusobacterium, 1 namedand 2 unnamed species of Leptotrichia, Mitsuokella multi-acida, 18 species of Prevotella, 3 species of Porphyromonas,6 named and 1 unnamed species of Selenomonas, 5 speciesof Veillonella, and 2 named and 1 unnamed species ofWolinella. Almost all library entries contained at least 20analyses.

Characterization. All reference strains were cultured andcharacterized phenotypically as described previously byfermentation and antimicrobial susceptibility tests in prere-duced media (9), by toxin-antitoxin neutralization assays (9),and by polyacrylamide gel electrophoreses (15). Antitoxinsfor types A, B, C, D, E, and F were prepared by L. DeSpainSmith in burros; and those for type C1 and C. argentinensewere prepared in rabbits. These antitoxins were used at adilution that contained about 10 IU/ml. The small amountof type CoL antitoxin available was received from L. S.McClung; the original source and the potency were un-known. After the supply of Smith type A antitoxin wasexhausted, type A antitoxin prepared in goats was obtainedfrom TechLab (Blacksburg, Va.).CFA analyses. Cells for CFA analysis were grown in

PYG-Tween (PYG-T) broth medium that was slightly mod-ified from that of Holdeman et al. (9), and this change madea significant difference in the analytical results. The PYG-Tbroth contained the following, per 100 ml of medium: 0.5 g ofBacto-Peptone (Difco Laboratories, Detroit, Mich.), 0.5 g ofpepticase (Sheffield, Norwich, N.Y.), 1 g of yeast extract(Difco), 1 g of glucose, 2.0 ml of salts solution (0.5 g ofCaCl2 2H20 and 0.4 g of MgSO4 dissolved in 1,000 ml ofdeionized water, to which was added a solution containing2.0 g of K2HPO4, 2.0 g of KH2PO4, 20.0 g of NaHCO3, and100 g of NaCl dissolved in 1,000 ml of deionized water), 0.4ml of resazurin (25 mg of resazurin [Difco] dissolved in 100ml of deionized water), 1 ml of hemin solution (50 mg ofbovine crystalline hemin [Aldrich Chemical Co., Milwaukee,Wis.] mixed with 1.0 ml of 1 N NaOH and 99 ml of deionizedwater, boiled, cooled under oxygen-free carbon dioxide,tubed anaerobically under nitrogen, and sterilized at 121°Cfor 15 min), 0.02 ml of vitamin K1 solution (0.15 ml ofvitamin K1 [Sigma Chemical Co., St. Louis, Mo.] added to30 ml of95% ethanol), 0.25 ml of Tween 80 solution (10 ml ofTween 80 in 90 ml of deionized water), and 0.05 g ofL-(+)-cysteine hydrochloride. Unless otherwise specified,chemicals were from Fisher Scientific Co., Pittsburgh, Pa.,and were certified as American Chemical Society grade. ThepH was adjusted to 7.2 to 7.3 with 8 N NaOH, and themedium was dispensed in 3- or 10-ml amounts and auto-

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CELLULAR FATTY ACIDS OF C. BOTULINUM 1117

TABLE 1. Methylated cellular components of washed cells of proteolytic strains of C. botulinum and related organisms

% (mean + SD) of total chromatographic area inb:

Componenta C. botulinum toxin type and CFA group: C. C.A A2 BP BP2 BP3 FP FP type sporogenes putrificum

(56/103) (2/20) (33/75) (4/21) (12/55) (2/18) (1/20) (36/44) (14/22)UN 9.740/nC13 ?10:0 FAME12:0 FAME11:0 DMA14:0 iso FAME14:1 cis 9 FAME14:0 FAME13:0 iso 30H FAME14:1 cis 7 DMA14:0 DMA15:0 FAME16:0 iso FAME16:1 cis 9 FAME16:1 cis 11 FAME16:0 FAME16:1 cis 9 DMA16:0 DMA17:cyc FAME16:0 30H FAME18:0 ALDE18:1 cis 9 FAME18:1 cis 13 FAME18:0 FAME18:1 cis 9 DMA18:1 cis 11 DMA22:0 NHC19 cyc 9,10/:1 FAME19:0 cyc 9,10 DMA20:1 cis 11 FAME13:1 cis 12 FAME or

14:0 alde or 11:1 20HFAME

UN 14.762 15:2 ? or 15:2or 15:1 cis 7 FAME

15:0 DMA or 14:0 30HFAME

15:0 anteiso 30H FAMEor 16.1 cis 7 DMA

17:2 FAME at 16.76ECL or 17:1 cis 8FAME

18:1 cis 11 FAME orUN-17.834 ECL

2.2 ± 2.4 0.9 ± 1.1 2.3 ± 2.3

1.6 ± 0.4 0.6 ± 0.4

1.8 ± 0.45.6 ± 2.3 7.4 ± 1.5 6.0 ± 2.4

5.2 ± 1.5 4.4 ± 2.3 5.7 ± 1.2

11.6 ± 3.0 18.0 ± 4.0 10.3 ± 3.21.9 ± 0.7 2.0 ± 0.7

43.1 ± 5.7 34.4 ± 11.8 44.0 ± 6.4

0.8 ± 0.8 0.9 ± 0.7 0.8 ± 0.78.3 ± 2.2 8.0 ± 2.1

1.8 ± 1.3 1.0 ± 1.7 2.0 ± 1.0

0.6 ± 0.5

1.6 ± 1.9

1.2 ± 0.6

19.3 ± 4.60.7 ± 0.4

3.4 ± 1.60.6 ± 0.31.9 ± 0.92.8 ± 0.60.7 ± 0.2

33.5 ± 7.62.4 ± 0.81.7 ± 0.8

12.8 ± 2.9

2.6 ± 0.83.1 ± 0.5

2.9 ± 2.4

1.3 ± 0.8

1.8 ± 3.8

0.4 ± 0.3

1.2 ± 1.2

21.6 ± 3.31.2 ± 0.91.2 ± 0.56.1 ± 2.31.7 ± 0.52.1 ± 2.03.1 ± 0.90.9 + 0.515.0 + 3.84.1 ± 0.91.1 ± 0.5

0.5 ± 0.4

15.0 ± 6.0

1.3 ± 0.95.3 ± 2.30.7 ± 0.6

2.1 ± 1.2

2.7 ± 1.2

0.8 + 0.5

0.5 ± 0.6

8.5 ± 5.8

1.4 ± 1.6

4.5 ± 0.6

10.1 ± 3.62.4 ± 0.5

43.3 ± 8.5

0.9 ± 0.811.0 ± 1.9

2.0 ± 0.3

0.6 ± 0.7

1.5 ± 2.30.02 ± 0.10.1 ± 0.40.1 ± 0.20.3 ± 0.5

11.6 ± 3.60.3 ± 0.50.1 ± 0.22.6 ± 0.70.1 ± 0.30.7 ± 0.33.0 + 0.41.0 ± 0.2

28.7 ± 2.82.7 ± 0.40.8 ± 0.30.7 ± 0.3

0.2 ± 0.815.2 ± 3.30.1 ± 0.33.2 ± 1.52.3 ± 0.80.1 ± 0.20.1 ± 0.4

10.5 ± 2.00.5 ± 0.30.5 ± 0.41.0 ± 0.4

3.6 ± 4.0

0.5 ± 0.6

1.9 ± 1.8

22.8 ± 3.61.8 ± 1.31.2 ± 0.66.3 ± 2.61.9 ± 0.82.9 ± 2.23.1 ± 0.60.6 ± 0.6

13.8 ± 3.83.4 ± 1.01.0 ± 0.5

0.7 ± 0.7

14.0 ± 5.0

4.1 ± 1.4

2.6 ± 2.0

3.2 ± 2.0

2.7± 1.7

0.8 ± 0.9

1.6 ± 1.3

22.4 ± 3.00.9 ± 0.81.2 ± 0.65.7 ± 2.52.2 ± 0.82.0 ± 2.14.0 ± 0.90.8 ± 0.6

15.5 ± 4.63.4 ± 1.20.9 ± 0.7

0.6 ± 0.9

16.8 ± 6.7

1.0 ± 1.23.4 ± 1.1

0.6 ± 0.6

2.8 ± 1.4

0.6 ± 0.5 0.8 ± 0.6 1.8 ± 0.6 1.0 ± 0.5 1.0 ± 0.4 1.5 ± 0.7 0.9 ± 0.8

1.0 + 0.6 - 1.1 ± 1.3 1.0 ± 0.6

-- - - 0.5 ± 0.3

2.8 ± 1.2 2.4 ± 1.0 0.7 ± 0.5 1.6 ± 0.6 3.9 ± 1.5 0.1 ± 0.2 1.3 ± 0.9 0.9 ± 0.8

13.6 ± 7.0 27.9 ± 15.0 12.3 ± 5.4 4.3 + 0.7 4.3 + 1.3 7.6 + 2.6 10.7 ± 2.5 3.8 ± 1.6 4.4 ± 1.3

a FAME, fatty acid methyl ester; DMA, dimethyl acetyl; ECL, equivalent chain length; UN, unknown compound; NHC, normal hydrocarbon.b Values are the percentage of the total chromatographic area of products; -, minor peaks that were highly variable or undetected and not used in the MIS

system's differential analysis. Values in parentheses are number of strains/number of analyses.

claved as described previously (9). The same medium isavailable tubed in 10-ml amounts from Carr-ScarboroughMicrobiologicals, Inc. (Stone Mountain, Ga.), and this com-mercial medium was used as an alternative for many of theanalyses in our current library entries.Ten milliliters of PYG-T medium in a screw-cap tube (16

by 100 mm) closed with a rubber stopper was inoculatedunder a stream of oxygen-free CO2 with 0.2 to 0.3 ml of aculture in PYG-T medium that had been incubated for 16 to24 h. The inoculated tubes were incubated overnight at 37°C.Cultures were centrifuged, and the supernatant was dis-carded. The cell pellets were processed immediately orfrozen at -60°C for later CFA analysis.

Following the MIS system instructions for anaerobe iden-tification with broth cultures (Microbial ID, Inc.), the cells

were lysed and saponified with 1.0 ml of a sodium hydroxide-methanol solution (45 g ofNaOH, 150 ml of methanol, 150 mlof deionized water) at 100°C for 5 min and then mixed on avortex mixer for 5 to 10 s and heated at 100°C for anadditional 25 min. Tubes were cooled, and fatty acids weremethylated with 1 ml of a methanol-HCl solution (275 ml ofmethanol carefully mixed with 325 ml of 6 N HCI) and 1 mlof H2SO4-methanol (162.5 ml of H2SO4 gradually added to162.5 ml of water, mixed, and then mixed with 275 ml ofmethanol), vortexed for 5 to 10 s, and heated at 80 + 1°C for10 + 1 min. Tubes were cooled rapidly in an ice bath.Methylated compounds were extracted with 1.25 ml ofhexane ether (200 ml of high-pressure liquid chromatograph-ic-grade hexane and 200 ml of high-pressure liquid chromato-graphic-grade methyl tert-butyl ether) by rotating the tubes

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TABLE 2. Methylated cellular components of washed cells of nonproteolytic strains of C. botulinum types B, and E, and F andC. botulinum types C and D and related organisms

% (mean ± SD) of total chromatographic area inb:

Componenta C. botulinum type: NT-NPc C. botulinum type: Nontoxic CDN-like:

B E F BEF-like Cat and Co Ca C,B D Group 1 Group 2(2/17) (13/37) (3/25) (3/12) (21/49) (9/17) (10/19) (4/22) (7/16) (7/12)

10:0 FAME12:0 FAME13:0 FAME14:0 FAME14:0 DMA16:0 ALDE15:1 cis 9/t8 FAME15:0 FAME16:1 cis 7 FAME16:1 cis 9 FAME16:0 FAME16:1 cis 9 DMA16:0 DMA17:1 cis 11 FAME17:0 FAME18:1 at 17.25 ECLDMA

18:1 cis 9 FAME18:0 FAME18:1 cis 9 DMA18:1 cis 11 DMA18:0 DMA19 cyc 9,10/:1FAME

13:1 cis 12 FAMEor 14:0 ALDE or11:1 20H FAME

12:0 30H FAMEor 13:0 DMA

UN 14.762 15:2 ?or 15:2 or 15:1cis 7 FAME

15:0 DMA or 14:030H FAME

15:0 anteiso 30HFAME or 16:1cis 7 DMA

17:2 FAME at16.76 ECL or17:1 cis 8 FAME

17:1 cis 9 FAMEor 17:2 FAME at16.801 ECL

18:1 cis 11 FAMEor UN 17.834ECL

2.5 ± 1.0 1.3 ± 0.8 1.6 ± 0.8

22.5 ± 4.4 18.2 ± 4.2 15.3 ± 2.4

1.1 ± 0.3 0.9 ± 0.5 0.8 ± 0.7

2.32.4

21.42.14.7

2.2 ± 2.0 1.8 ± 1.11.6 ± 1.0

22.4 ± 5.0 18.2 ± 5.81.4 ± 0.8

1.3 ± 0.5 -

0.6 ± 0.71.3 ± 0.8 2.3 ± 1.3 1.1 ± 0.52.1 ± 0.8 0.9 ± 0.8 0.9 ± 0.7

20.1 ± 3.1 13.6 ± 5.8 17.2 ± 4.61.7 ± 0.6 0.8 ± 0.8 2.3 ± 1.1

- -- - - 0.6 ± 0.68.7 ± 6.2 15.4± 3.5

± 0.7 2.1 ± 0.9 1.5 ± 0.5 3.0 ± 0.9 1.3 ± 0.8 2.1 ± 0.5± 0.4 2.4 ± 0.6 2.6 ± 0.9 3.0 ± 0.6 3.8 ± 0.9 3.9 ± 1.0± 3.9 26.9 ± 3.8 18.7 ± 5.7 22.0 ± 3.6 9.5 ± 3.2 11.7 ± 3.3± 0.7 1.8 ± 1.0 1.3 ± 0.5 2.3 ± 0.5 1.5 ± 0.6 1.4 ± 0.6± 1.1 4.0 ± 1.7 3.3 ± 2.3 4.6 ± 1.3 0.9 ± 0.9 1.1 ± 0.7-_ - - - - 0.7 ± 0.6

0.6 ± 0.7 1.2 ± 0.7- -- - - 0.7 ± 0.4

13.1 ± 3.51.5 ± 1.0

11.3 ± 2.82.6 ± 0.40.6 ± 0.6

13.0 ± 4.0 21.6 ± 10.53.0 ± 1.7 2.2 ± 1.39.4 ± 3.4 14.9 ± 2.13.6 ± 1.3 2.8 ± 0.91.4 ± 0.8 1.0 ± 1.0

13.1 + 2.7 26.6 ± 10.51.2 ± 0.78.0 ± 2.03.5 ± 1.2

0.6 ± 0.77.8 ± 4.60.5 ± 0.5

-_- - 0.7 t 0.6

2.5 ± 1.6 10.2 ± 5.10.6 ± 0.5 1.5 ± 0.84.6 ± 0.8 3.2 + 1.06.9 ± 1.3 14.5 ± 5.41.7 ± 0.4 1.9 ± 0.5

1.4 ± 1.0

1.1 ± 1.00.5 ± 0.5

18.0 ± 5.3 35.5 ± 9.00.9 ± 0.9 -3.5 ± 1.9 12.8 ± 1.90.5 ± 0.4 0.6 ± 0.6

0.6 ± 0.8

0.7 ± 0.4

21.7 ± 12.11.6 ± 1.26.8 ± 3.80.6 ± 0.5

1.2 ± 0.6 2.5 ± 1.61.3 ± 0.8 2.0 ± 1.9

18.0 ± 4.5 13.5 ± 3.81.8 ± 0.7 0.7 ± 0.8

12.1 ± 6.21.3 ± 0.43.0 ± 0.6

14.6 ± 4.21.3 ± 0.61.4 ± 0.5

1.2 ± 0.8

1.3 ± 1.1

4.7 + 0.96.8 ± 1.71.4 ± 0.3

22.8 ± 8.0 43.7 + 5.51.4 ± 1.0 0.6 ± 0.65.8 ± 2.40.6 ± 0.6

- 1.6 ± 1.3

1.1 + 0.9 0.7 + 0.5

0.4 + 0.4

1.3 ± 0.7 1.2 ± 0.9 0.9 ± 0.9 2.0 ± 0.4 1.6 ± 1.1 2.3 ± 0.9 1.1 ± 1.0 1.9 ± 1.5

---- 1.2 ± 0.8 1.3 + 0.4 1.2 ± 1.2 1.0 ± 0.6

2.4 ± 0.7 1.8 ± 1.1 1.5 ± 0.7

2.7 ± 1.3 2.3 ± 1.1 3.6 ± 1.0

2.4 ± 0.8 0.6 ± 0.5

2.3 ± 1.0 3.0 ± 2.1

0.8 ± 0.5 0.5 ± 0.5 1.0 ± 0.6

1.6 + 1.1 4.7 ± 2.1 2.2 ± 1.4

1.2 ± 0.6 -

1.1 ± 0.5 1.0 ± 1.3

0.6 ± 0.4

1.5 ± 0.8 2.6 ± 1.1

-- - - 1.6 ± 1.0 0.6 ± 0.9

4.4 ± 1.0 5.8 ± 1.5 5.6 ± 1.4 5.4 ± 1.2 4.2 ± 1.5 3.3 + 1.0 5.2 ± 1.6 3.7 ± 1.9 3.8 ± 1.1 6.0 ± 0.9

a FAME, fatty acid methyl ester; DMA, dimethyl acetyl; ECL, equivalent chain length; UN, unknown compound; ALDE, aldehyde.b Values are the percentage of the total chromatographic area of products; -, minor peaks that were highly variable or undetected and not used in the MIS

system's differential analysis. Values in parentheses are number of strains/number of analyses.c NT, nontoxigenic; NP, nonproteolytic (neither milk or meat digested).

end over end for 10 min. The bottom aqueous phase wasremoved, and the top phase was washed with 3 ml of diluteNaOH (5.4 g of NaOH certified by the American ChemicalSociety [Fisher], 450 ml of deionized distilled water satu-rated with approximately 125 g of NaCl) by rotating thetubes end over end for 5 min. The aqueous phase was frozen,and a portion of the organic phase was transferred to a gaschromatography sample vial which was capped or coveredwith thin aluminum foil disks and sealed with Elmer's schoolglue (Borden, Inc., Columbus, Ohio) and placed on thesample changer.

Two microliters of the organic phase was chromato-graphed on a fused-silica capillary column (0.2 mm by 25 m)with a liquid phase consisting of5% phenylmethyl silicone inan HP-5890A gas chromatograph (Hewlett-Packard, Avon-dale, Pa.) equipped with a flame ionization detector and anHP-3392A integrator. Gas flow rates were ca. 400 ml/min forair, 30 ml/min for hydrogen, and 30 ml/min for nitrogen.Temperatures were 250°C for the injection port and 300°C forthe detector. After injection, the oven temperature wasincreased from 170 to 270°C at 5°C/min and then from 270 to310°C at 30°C/min, held at 310°C for 2 min, and then returned

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CELLULAR FATTY ACIDS OF C. BOTULINUM 1119

TABLE 3. Methylated cellular components of washed cells of C. argentinense, C. subterminale, toxic strains phenotypically resemblingC. butyricum and C. baratii, and C. butyricum and C. baratii

% (mean ± SD) of total chromatographic area inb:

Component' C. C. Toxic C. Toxic C. baratiiargentinense subterminale C. butyricum" butyricum C. baratii' 15/21

(7/30) (21/28) (1/6) (18/27) (1/3) (

10:0 FAME 0.8 ±0.8 0.7 ±0.412:0 FAME 2.5 ± 0.7 2.1 ± 0.9 0.6 ± 0.4 0.6 ± 0.2 1.6 ± 0.4 1.4 ± 0.411:0 DMA 1.5 ±0.5 0.9 ± 0.814:1 cis 9 FAME 1.0 ± 0.214:0 FAME 4.2 ± 1.0 7.0 ± 3.1 6.7 ± 1.5 8.0 ± 1.8 5.2 ± 1.3 6.5 ± 1.814:0 DMA 5.4 1.1 4.2 ± 3.2 -16:0 ALDE 1.4 ±0.8 0.7 ± 0.7 -16:1 cis 7 FAME 0.6 ± 1.0 3.9 ± 0.8 3.6 ± 0.8 1.4 ± 0.516:1 cis 9 FAME 3.0 ± 0.7 4.5 ± 2.4 2.2 ± 0.4 2.1 ± 0.3 6.6 ± 1.1 3.4 ± 0.516:0 FAME 9.0 ± 2.2 12.8 ± 5.2 40.0 ± 3.3 40.4 ± 2.9 9.2 ± 3.1 14.3 ± 5.016:1 cis 9 DMA 3.8 ±0.9 3.0 ± 1.2 0.7 ± 0.4 0.6 ± 0.4 1.1 ± 0.416:0 DMA 5.3 ±2.2 3.4 ± 1.5 1.3 ± 0.3 1.9 ± 0.8 1.0 ± 0.518:1 cis 9 FAME 35.1 ± 4.3 31.6 ± 11.8 13.7 ± 2.4 11.5 ± 1.4 28.4 ± 3.5 17.9 ± 5.818:0 FAME 1.1 ± 0.8 1.5 ± 0.8 8.6 ± 1.7 7.3 ± 2.9 2.5 ± 0.2 2.5 ± 0.618:1 cis 9 DMA 8.3 ± 2.7 8.2 ± 2.8 3.2 ± 1.2 3.8 ± 1.3 20.2 ± 3.0 18.5 ± 4.818:1 cis 11 DMA 2.0 ± 2.4 2.0 ± 0.3 1.9 0.5 3.5 ± 1.018:0 DMA 1.7 ± 0.922:0 NHC 0.6 0.8 -19 cyc 9,10/:1 FAME 0.7 ± 0.6 2.0 ±0.6 4.2 ± 0.6 2.7 ± 1.619 cyc 11, 12/:1 FAME 2.0 ± 1.3 3.7 ± 0.919:0 cyc 9,10 DMA - 0.7 0.4 4.3 ± 0.5 4.6 ± 1.919:0 cyc 11,12 DMA 0.7 0.520:1 cis 11 FAME - 0.5 ± 0.313:1 cis 12 FAME or 14:0 ALDE or 11:1 20H 2.9 ± 0.8 2.0 ± 1.7FAME

UN 14.762 15:2 ? or 15:2 or 15:1 cis 7 FAME 2.3 ± 0.9 1.6 ± 1.2 1.0 ± 0.3 0.7 ± 0.5 1.2 ± 2.015:0 anteiso 30H FAME or 16:1 cis 7 DMA 0.8 ± 1.1 1.9 ± 0.3 2.0 ± 0.4 1.0 ± 0.617:2 FAME 16.76 ECL or 17:1 cis 8 FAME 3.0 ± 1.6 2.1 ± 1.7 0.5 ± 0.3 0.6 ± 0.4 8.0 ± 1.4 5.3 ± 1.818:1 cis 11 FAME or UN 17.834 ECL 6.8 ± 1.4 8.6 ± 2.3 9.3 ± 1.0 6.5 ± 1.4 7.3 ± 1.1 8.6 ± 1.2UN 18.62 ECL or 19:0 iso FAME 0.8 ± 0.5

a FAME, fatty acid methyl ester; DMA, dimethyl acetyl; ECL, equivalent chain length, UN, unknown compound; ALDE, aldehyde; NHC, normalhydrocarbon.

b Values are the percentage of the total chromatographic area of products; -, minor peaks that were highly variable or undetected and not used in the MISsystem's differential analysis. Values in parentheses are number of strains/number of analyses.

c Produces neurotoxin.

to 170°C before the next sample was injected. A standardfrom Microbial ID, Inc., containing known fatty acids (Cgthrough C20 straight-chain acids and C10:0 20H, C10:0 30H,C14:0 20H, C14:0 30H, and C16:0 20H) was chromatographedat the beginning of each day on which samples were run andafter each set of 10 samples was chromatographed. Identifi-cation of peaks (by retention time), area, area/height ratio,equivalent chain length, total area, and total area of thenamed compounds were determined by the MIS systemsoftware package, which also calculated the percent area foreach named compound compared with the total area of thenamed compounds.

Identification. The MIS system (Microbial ID, Inc.) wasused for computer identification, which was based on com-parisons of the profile of extracts of the unknown strain withthose of library entries to find the reference that most closelyresembled the bacterium being tested.

RESULTS

The major CFA derivatives of C. botulinum and relatedorganisms grown under the described conditions were 16:0FAME, 14:0 FAME, and 18:1 cis 9 FAME, followed by 18:1cis 9 DMA and 18:1 cis 11 FAME or an unknown compoundwith an equivalent chain length of 17.834 (Tables 1 through

3). Differences in the relative proportions of the various fattyacid derivatives that we detected permitted correct strainidentification for 74 to 100% (depending on the toxin type orgroup) of the analyses (Table 4), 89 to 100% of the toxicstrains tested, and 60 to 100% of the nontoxic strains tested(Table 5). Table 5 shows all results that were not correct(first choice) for all toxic and nontoxic strains tested. InTable 4, the seven analyses of C. botulinum type C3 thatwere identified as C. botulinum type C were considered to becorrect.

Metabolic group I (proteolytic strains). Fifty-three (95%) ofthe type A strains were correctly identified by at least one

analysis (Table 5). For 104 analyses of the 56 type A strains,the first identification choice was either type A or type Bproteolytic and the second choice was type A, type Bproteolytic, or type F proteolytic (1 strain) (Tables 4 and 5).For the 79 analyses giving the correct identification of C.botulinum type A, the mean difference between the first andsecond choices was 0.101 similarity unit. The three strainsthat were not correctly identified by any analysis were

Landry strain 107 (two analyses), McClung strain 465 (oneanalysis), and Prdvot strain 729 (one analysis).Two strains (Hall strains 5675 and 10174A) of C. botuli-

num that produced toxin neutralized by type A antitoxin andthat had characteristics typical of proteolytic strains of C.

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TABLE 4. Agreement of CFA identification with toxigenic and phenotypic identification

No. (%) of analysesSpecies or No fcorrect Ma E

C. botulinum toxin No. ofstMean t SEM Most common second choice when first choice correct (no.)type or CFA groupa First Second

choice choice

A 56 79 (76) 103 (99) 0.101 ± 0.009 C. botulinum type BP (76)A2 2 20 (100) 0.445 ± 0.038 C. botulinum type A (8), Bifidobacterium D02 (7), C. botulinum

BP (3), other (2)BP 33 61 (81) 75 (100) 0.072 ± 0.008 C. botulinum type A (61)BP2 4 21 (100) 0.600 ± 0.370 C. botulinum type FP (6), C. sporogenes (4), Eubacterium

saburreum (4), other (7)BP3 12 48 (89) 55 (100) 0.189 ± 0.180 C. sporogenes (24), C. putrificum (19), C. novyi type A (5)FP, type 1 20 (100) 0.732 ± 0.060 C. botulinum type BP2 (9), Bifidobacterium animalis (9), other

(2)FP 2 17 (74) 18 (78) 0.243 + 0.077 C. botulinum type D (8), type BP (7); C. botulinum type Cp (2)C. sporogenes 30 42 (78) 44 (81) 0.215 ± 0.018 C. botulinum type BP3 (30), C. putrificum (11), C. botulinum

type BP2 (1)C. putrificum 14 22 (100) 0.221 ± 0.026 C. sporogenes (11), C. botulinum type BP3 (11)BNP 2 13 (76) 17 (100) 0.182 ± 0.028 C. botulinum type E (6), type FNP (2), BEF/NT (5)E 13 37 (100) 0.293 + 0.017 C. botulinum type BNP (25), type FNP (3), BEF/NT (9)FNP 3 22 (88) 25 (100) 0.213 ± 0.034 C. botulinum type E (7), type BNP (6), type C (2), C. tetani (7)NT-NP BEF-like 5 9 (75) 12 (100) 0.220 ± 0.017 C. botulinum type E (5), type BNP (4)Ca (or C) 9 15 (88) 17 (100) 0.172 ± 0.040 C. botulinum type C (10), CDN1/NT (5)Co (or C) 10 17 (89) 19 (100) 0.105 ± 0.026 C. botulinum type C (7), CDN2/NT (2), C. limosum (1); 7

strains identified as C. botulinum type C (first choice) with C.botulinum type C, as the second choice

D 4 17 (77) 22 (100) 0.126 ± 0.017 C. botulinum CDN1/NT (10), type C (5), type Ca (1), C. novyitype B (1)

CDN1-NT 7 12 (75) 16 (100) 0.117 ± 0.029 C. botulinum type D (4), type Ca (4), type C (2), C.haemolyticum (2)

CDN2-NT 7 10 (83) 12 (100) 0.305 ± 0.614 C. novyi type A (4), C. botulinum type C (4), type C, (2)C. argentinense 7 28 (93) 30 (100) 0.223 ± 0.023 C. subterminale (28)C. subterminale 21 28 (100) 0.309 ± 0.023 C. argentinense (18), other (10)C. butyricum 1 6 (100) 0.302 ± 0.044 C. butyricum (6)

(type E toxin)C. butyricum 20 27 (100) 0.443 ± 0.031 C. butyricum (toxic E) (23), C. fallax (4)C. baratii (type E 1 3 (100) 0.592 ± 0.030 C. sardiniense (2), C. baratii (1)

toxin)C. baratii 15 21 (100) 0.430 + 0.028 C. sardiniense (16), C. baratii, toxic type E (2), other (3)

a A, B, Ca, C,B, D, E, and F, toxin types of C. botulinum; A2, second cellular fatty acid group of C. botulintum type A; BP2 and BP3, additional CFA groupsof proteolytic strains of C. botulinum type B; FP, type F proteolytic; BNP, type B nonproteolytic; FNP, type F nonproteolytic; NT-NP BEF-like, nontoxigenicand nonproteolytic strains phenotypically similar to nonproteolytic strains of C. botulinum type B, E, or F; CDN1-NT, nontoxigenic strains phenotypically similarto C. botulinum type C or D or C. novyi; CDN2-NT, second CFA group of CDN1-NT strains.

b Mean difference between first and second choice similarity values and standard error of the mean.

botulinum type A could not be identified as any describedclostridial species by CFA analysis and produced FAMEresults that did not cluster with those of other type A strains.Results of replicate analyses of these strains were used toform a new library entry, C. botulinum type A2 (Tables 1 and4). There was good separation between the similarity valuesfor the first and second choices (mean, 0.445).

Results of analyses of proteolytic strains of type B clus-tered into three groups that we designated BP (which in-cludes ATCC 7949 and ATCC 8083), BP2, and BP3.Of the 33 strains in the BP CFA group, 29 (88%) were

correctly identified by at least one of the analyses (Table 5).Type B Prevot strain PPois was incorrectly identified as C.botulinum type F (two analyses). The three strains that wereincorrectly identified as type A were ATCC 844 (one analy-sis), Hall strain 6577B (one analysis), and Prevot strain 1552(two analyses). For 75 analyses of the 33 strains in the BPgroup, the first identification choice was either type A or BPand the second choice, likewise, was either type A or BP(Table 4). For the 61 analyses giving the correct identifica-tion of C. botulinum type B, the mean difference between thefirst and second choices was 0.072 similarity unit.

The current BP2 library consists of 21 analyses of fourstrains; all were correctly identified. Three strains receivedfrom Ivan C. Hall (Hall strains 6660 and 6707 isolated from"duck sickness" and strain 10007 isolated from kipperedsalmon) that produced toxin that was neutralized by type Bantitoxin and that were phenotypically like proteolyticstrains of C. botulinum type B initially identified by CFAwith a low similarity value (ca. 0.012) as C. botulinum typeE or type B nonproteolytic. Cluster analysis showed thattheir CFA profiles were unlike those for any other entries forC. botulinum. These three strains and McClung strain 2239were considered to be a new CFA group (BP2), and thelibrary entry developed for them was distinct from those forall other clostridia tested (the mean difference between thefirst and second choices was 0.600).There was only one strain (VPI 1750A) in CFA group BP3

when this study was initiated. As a result of this study, thelibrary entry for BP3 now contains 55 analyses of 12 strains(Table 4). A single replicate of each of three BP3 librarystrains had higher similarity values with C. sporogenes, C.putrificum (one replicate of each of two strains), or C. novyitype A (one analysis of one strain) than it did with C.

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TABLE 5. Accuracy of strain identification'

Species andTotal no. No. (%) of

CFA identification'type or CFA Toftralino. stano. (%)cofrrect__________________________________groupb of strains strains Incorrect No. of strains First choice Second choice

C. botulinumType A 56 3 (4) 2 Type BP (2) Type A (2)

1 Type BP (2) Type FP (2)Type BP 33 4 (11) 3 Type A (4) Type BP (4)

1 Type FP (2) Type A (1), C. limosum (1)

NT-NP BEF-like 5 2 (40) 2 Type E (3) NT-NP BEF-like (3)

C. sporogenes 36 6 (17) 4 Type BP (6) Type A (6)1 Type A (2) Type BP (2)1 Type BP3 (1)d, C. putrificum (1) C. putrificum (1)d, C. sporogenes (1)

a All misidentified (first choice) strains for all strains tested are listed here.b BP, type B proteolytic; FP, type F proteolytic; NT-NP BEF-like, phenotypically like nontoxigenic and nonproteolytic types B, E, or F; BP3, CFA group 3

of type B proteolytic.c Values in parentheses are the total number of analyses.d Same strain.

botulinum group BP3. For these six analyses, BP3 was thesecond choice. Other strains in the BP3 group include FDAstrains 112, 116, and 120 and Prdvot strains Fll, 594, 892,1490, 1504, 1662, 1687, and 2345. No information regardingsources was available for any of these strains. On blood agarplates incubated anaerobically for 48 h, 7 of the 12 strains inthe BP3 group produced filamentous to rhizoid coloniessimilar to those characteristic of C. sporogenes. Four strainsproduced irregular umbonate colonies, and one strain pro-duced the irregular, low convex, grey to white colony typicalof most strains of C. botulinum.Of the three proteolytic strains of C. botulinum type F

available for study, the CFA profile of the type strain(Langeland strain, CDC KA-1) was different from those ofthe other two strains (Wall strain 8-G and Gimdnez strain160). The resulting two library entries were designated "C.botulinum type F proteolytic, type" for the type strain and"C. botulinum type F proteolytic" for the other two proteo-lytic type F strains.

C. sporogenes and C. putrificum. Of 36 strains of C.sporogenes tested, 83% were correctly identified by at leastone analysis. The strains that were not correctly identified(Table 5) were four clinical isolates that were identified as C.botulinum type BP as the first choice and C. botulinum typeA as the second choice. The other two strains were Hallstrain 11727 (two analyses that identified it as C. botulinumtype A as the first choice) and Prevot strain 785 (twoanalyses that identified it as C. botulinum type BP3 and C.putrificum). Of the 54 analyses of 36 strains, 78% werecorrect as the first choice, and 81% were correct as the firstor second choice.

All 22 analyses of the 14 strains of C. putrificum werecorrectly identified (Table 1) and were separated by anaverage of 0.22 similarity unit from the second-choice spe-cies C. sporogenes or C. botulinum type B of the BP3 CFAgroup (Table 4).

Metabolic group II (nonproteolytic strains). Although theCFAs of the nonproteolytic strains of types B and F and thetype E strains were similar to each other, they could bedifferentiated with 76 to 100% accuracy (first choice) (Table4). Two analyses of type BNP and two analyses of nontox-igenic and nonproteolytic (NT-NP) BEF-like strains identi-fied them as type E as the first choice. The poorest discrim-ination was obtained with the small group of nontoxigenic

strains resembling nonproteolytic C. botulinum type B, typeE, or type F strains (BEF-NT). Of five strains in this group,two strains were incorrectly identified (Table 5) as toxigenictype E strains. One was an isolate from a clinical specimenobtained from an ear, and the source of the other wasobscure.

Metabolic group III (C. botulinum types C and D and C.novyi and nontoxic strains phenotypically similar to C. botuli-num types C or D or C. novyi [CDN-NT groups 1 and 2]). The58 analyses of C. botulinum type C and D strains (Table 4)were 77 to 89% correct (first choice) after individual libraryentries were made for strains of types Ca and Cr3, althoughseparation between the first and second choices was rela-tively close (mean difference, 0.105 to 0.172 similarity unit).A type C library entry was retained to represent all type Cstrains, including those strains for which the subgroup wasunknown or not determined. Of the five analyses of threestrains of type D that were incorrect, two strains wereidentified as type Ca as the first choice, two strains wereidentified as nontoxic type CDN2, and one strain wasidentified as nontoxic type CDN1 (CDN indicates pheno-typic similarity to C. botulinum type C or D or C. novyi).Toxin type D was the second choice for each.

Results of the 22 analyses of 14 strains in two CFA groups(CDN1-NT and CDN2-NT) of nontoxic strains phenotypi-cally similar to group III organisms (C. botulinum types Cand D and C. novyi) were 75 to 83% accurate (first choice).All strains were correctly identified by at least one analysis.Repeated toxicity tests in mice confirmed that these strainswere nontoxic.

Metabolic group IV (C. argentinense and C. subterminale).The large amount of 18:1 cis 9 FAME detected in theanalyses of C. argentinense and C. subterminale (Table 3)was similar to that present in C. botulinum CFA groups A,A2, BP, and type F proteolytic (FP) (Table 1). Results of thetwo analyses of C. argentinense that were incorrect (Table 4)identified the strains as C. subterminale as the first choiceand as C. argentinense as the second choice. Replicateanalyses of these two strains produced correct identifica-tions of C. argentinense as the first choice. Results of allanalyses of the 21 strains of C. subterminale were correct(first choice). C. argentinense was the second choice for 18of 28 analyses; other second choices for analyses of C.subterminale were Clostridium limosum, Clostridium clos-

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tridioforme, Clostridium glycolicum, C. botulinum type E,Lactobacillus rogosae, and an unnamed Eubacterium spe-cies (VPI Dll) that is found in the human gingival crevice.Other toxin-producing strains. The neurotoxins produced

by CDC 3112 (VPI 14484) and CDC 5262 (VPI 14304), whichare toxic strains of C. baratii and C. butyriclum, respectively,both were neutralized by our C. botulinum type E antitoxin.By CFA analysis, the strain resembling C. baratii initiallyidentified as C. baratii at a similarity level of 0.247, andreplicate analyses of the strain resembling C. butyricumidentified it as "no match." Each was correctly identifiedwhen individual library entries were made for them with thefew replicate analyses of each that we had. There wasreasonable separation between the first and second choicesfor both strains (Table 4).

Specificity. Among 85 other species of clostridia and 417taxa of nonsporeforming rods and cocci, none of the non-sporeforming rods and cocci and few (less than 0.2%) of theclostridia analyzed, other than those presented in Tables 4and 5, were identified as C. botulinum at a similarity greaterthan 0.2.

DISCUSSION

In view of the phenotypic and genetic similarities (at thespecies level) of several toxin types of C. botulinum and oftheir nontoxic counterparts, it is surprising that they can bedifferentiated so well by their CFA compositions.

Metabolic group I (proteolytic strains). There is consider-able phenotypic and genetic relatedness among members ofthe proteolytic strains of C. botulinum (12, 13). Therefore,we did not expect that the toxin types of the organisms inthis group could be separated from each other or from C.sporogenes by evaluation of their CFA compositions, nordid we expect that there would be subgroups within the toxintypes of the type A and proteolytic type B strains.Among the proteolytic strains studied by Lee and Rei-

mann (13), the DNA of one of two type A strains tested andthe DNA of the Langeland type F strain showed 100%relatedness (100% binding and 100% competition) with theDNA of type A strain A62. The DNAs of two proteolytictype B strains tested showed 69 and 85% relatedness bydirect binding to the DNA of type A strain A62. The DNA ofonly one type B strain was tested by both direct binding andcompetition and gave relatedness values to type A strain A62of 83% (direct binding) and 41% (competition). The related-ness of the DNAs of three strains of C. sporogenes (includ-ing DNA from strain PA 3679) to the DNA of strain A62 bydirect binding was 86 to 100%, and by competition therelatedness was 51 to 90%. The relatedness of the DNAs ofthree other strains of C. sporogenes to strain A62 was lower(48 to 60% by direct binding). The results of Lee andReimann (12) indicate that there is moderate to high related-ness among the DNAs of proteolytic strains of C. botulinumand C. sporogenes. Given the 100% DNA relatedness Leeand Reimann (12) found between the Langeland (type) strainof type F and strain A62 of type A, it is surprising that theLangeland strain and the type A strains appeared to bedistinct by CFA analyses.

Strains in CFA groups BP and BP2 could not be differen-tiated from each other phenotypically, although there wasexcellent separation by CFA profiles. It is possible thatgroup BP2 is represented by only two, rather than fourstrains, because the two Hall strains from duck sicknessmight represent two isolates of one strain and because theMcClung strain, whose source is not known, might have

been obtained directly or indirectly from I. C. Hall. Thus,the BP2 group may be rare.

Strain VPI 1750A (BP3 group) has been an anomaly for ussince 1967. It was a contaminant in a culture labeled"Sphaerophorus abscedens" received from the Prevot Col-lection (Pasteur Institute) and was the only strain of C.botulinum that we had seen that produced a C. sporogenestype of colony, i.e., a rhizoid medusa head. We initiallythought that we had a mixed culture that contained only afew cells of C. botulinum that were overgrown by C.sporogenes and tried several times, on multiple occasions,during the ensuing 20 years to purify C. botulinum from thepresumed mixture, but we were unable to do so. Inasmuchas seven of the additional strains in the C. botulinum BP3library entry also had rhizoid to filamentous colonies, we arenow convinced that VPI 1750A represents a pure culture. ByCFA analysis, these strains are more closely related to C.sporogenes and C. putrificum than they are to other toxicstrains.

Metabolic group II (nonproteolytic strains). As would beexpected from genetic studies (11, 13), nonproteolyticstrains of types B and F and strains of type E were moreclosely related to each other by CFA analyses than theywere to other types of C. botulinum or to other clostridia.The overall accuracy of identification among the toxigenicnonproteolytic strains and between them and the NT-NPBEF-like organisms was 93.5%. Included among the NT-NPBEF-like strains (Table 4) was strain 28-2, which Lee andReimann (13) found to have 89% DNA relatedness with thetoxic type E MINN strain. All three analyses of strain 28-2identified it as CFA group NT-NP BEF-like. Two analysesof Landry strain 116 and one analysis of a clinical isolate(VPI 10428) identified them incorrectly as C. botulinum typeE (first choice) and correctly as NT-NP BEF-like (secondchoice).

Metabolic group III (types C and D and C. novyi). C.botulinum types C and D and C. novyi type A are closelyrelated phenotypically (1, 8, 9, 17). Furthermore, some oftheir lethal toxins, which are found in various ratios in thethree toxin types, are phage mediated (4, 5, 10). Lee andReimann (13) found only 34 to 38% binding and competitionbetween the DNAs of type C strain C573 and type C strain8613, which is comparable to the level between type C strainC573 and type D strain 8265, demonstrating genetic hetero-geneity in the type C strains. It is possible that one of theirtype C strains was type Ccx and the other was type C1. Ourdifferentiation of the type C, D, and nontoxic CDN-likestrains was rather poor until we made separate libraryentries for the Cox and Co strains. Because not all of ourstrains were tested with antitoxin that would differentiatetype Cox and type C1 strains and we ran out of antitoxinbefore all strains could be differentiated, we retained thetype C library entry in addition to the type Ca and type C1library entries.

Metabolic group IV (C. argentinense and C. subterminale).Although Suen et al. (18) reported that three nontoxic strainswere included in the C. argentinense homology group, all ofthe strains that we examined that were identified as C.argentinense by CFA analyses were toxic, and all of thestrains that we previously considered to be nontoxic C.subterminale were identified as C. subterminale with goodseparation between the two CFA groups (a mean differenceof 0.2 to 0.3 similarity unit). Thus, although the two speciesare similar phenotypically, they apparently can be separatedreliably by their CFA patterns.

C. butyricum. Initially, CFA analyses of the CDC strain

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that was phenotypically similar to C. butyricum except thatit produced toxin neutralized by C. botulinum type E anti-toxin produced "no match" CFA results, indicating that itsCFAs were not similar to those of C. butyricum. Multipleanalyses of the toxic strain enabled us to produce a separatelibrary entry for the toxic strain. With this entry, the secondchoice for each analysis was C. butyricum, and first andsecond choices were separated by a mean difference of 0.3(of a possible 0.99) similarity unit.

C. baratii. Hall et al. (7) reported the isolation of a strainthat phenotypically resembled C. baratii that produced aneurotoxin neutralized by C. botulinum type F antitoxin.This strain was isolated from feces in a case of infantbotulism. For us, the strain also was neutralized by type Fantitoxin obtained from CDC. However, it repeatedly wasneutralized by our type E but not by our type F antitoxin. Asconfirmation that vials of our antitoxin were not mislabeled,type F antitoxin from the same vial used to test toxin fromthe C. baratii strain successfully neutralized toxin producedby the type F Langeland (proteolytic) and by Hobbs F10(nonproteolytic) strains, and our type E antitoxin did notneutralize toxins produced by the Langeland and Hobbs F10strains. We cannot explain this discrepancy in toxin neutral-ization, except to suggest that some toxic component(s)shared by types E and F and the toxic C. baratii strain waspresent in different proportions in the type E and F toxoidsused to prepare the different lots of antitoxin.When we initially tested the CFA pattern of this strain,

results of two separate analyses identified it as C. baratii atlow levels (0.20 and 0.25 similarity units, respectively). Aseparate library entry for this toxic strain permitted itsseparation from C. baratii. Additional neurotoxic strains ofboth C. butyricum and C. baratii are needed to increase thereliability of these two library entries.

In addition to the creation of the toxic subgroups, whichallowed identification of the strains that could not have beencorrectly identified by the old C. botulinum library, theadditional strains and analyses considerably improved thelibrary entries. For example, for C. botulinum type BP andfor the strains now available, the old library would be 55%correct as the first choice and 71% correct as the first andsecond choices, whereas the new library entry is 81 and100% correct, respectively (with C. botulinum type A as thealternate choice).

Clinical considerations. C. botulinum types A, B, and E arethe types most often isolated from human clinical specimensor from food implicated in human botulism. Only 7 of 107(6%) of the strains and 45 of 275 (16%) of the analyses of the107 strains of C. botulinum types A and B did not givecorrect results (Tables 4 and 5). However, all analyses of allstrains of types A, A2, BP, and BP2 were identified as typeA, A2, BP, or BP2. Inasmuch as therapeutic antitoxinusually contains antitoxin against both types A and B, themisidentification of type A as type B and vice versa shouldnot cause serious clinical consequences, and all patientsfrom whom these strains were isolated should receive ade-quate treatment. Although all of the BP3 strains werecorrectly identified by at least one analysis of each strain, 6of 55 (11%) of the multiple analyses of strains in the BP3group gave an identification of C. sporogenes as the firstchoice and C. botulinum BP3 as the second choice. Thus,within the type A and proteolytic type B groups, the CFAresults of 6 of 275 analyses (2%) did not indicate type A orproteolytic type B, and antitoxin therapy would not havebeen initiated in 2% of the cases on the basis of these testresults.

Six of 36 strains (17%) and 6 of 54 analyses (11%) of thestrains of the nontoxigenic species C. sporogenes wereidentified as C. botulinum type A, BP, or BP3; one analysiswas identified as C. putrificum, which also is nontoxigenic.By these CFA results, administration of polyvalent type ABantitoxin would have been falsely indicated in 11 to 17% ofthe cases from which C. sporogenes was isolated and wouldhave been correctly indicated in all cases from which C.botulinum type A or proteolytic type B strains were isolated.

All 37 analyses of the 13 strains of C. botulinum type Ewere correctly identified as C. botulinum type E. Multipleanalyses of the less frequently isolated nonproteolyticstrains of types B and F were recognized with only 76 and88% accuracy, respectively, although all strains were cor-rectly identified by at least one analysis. Analyses of thenontoxigenic counterparts of the NT-NP BEF-like groupwere only 75% accurate and erred on the side of indicating atoxigenic rather than a nontoxigenic strain. Only 60% of thestrains were accurately identified (Table 5). More strains areneeded to develop adequate libraries for the toxic andnontoxigenic strains of nonproteolytic C. botulinum and C.botulinum-like organisms.

Likewise, testing of additional strains probably wouldimprove the library entries for C. botulinum types C and Dand their nontoxigenic counterparts, which now range frombeing 75 to 89% accurate.

Limitations and advantages. The fatty acid composition ofbacterial cells is extremely sensitive to the composition ofthe medium. We obtained suitable comparable results withmedium that we prepared according to the described formu-las (see Materials and Methods) and with commercial PYG-Tmedium in 10-ml vials from Carr-Scarborough Microbiolog-icals that is prepared to the same specifications. Successfulidentification requires strict adherence to the analyticalprotocol. We believe that the procedure has general appli-cation.Although the age of the culture and the growth tempera-

ture also affect the composition of the CFAs, we usedreplicate cultures of different ages (generally 18 to 72 h old)held in incubators set at 37°C that were opened from few tomany times a day, so that the library entries would includevariations encountered in normal laboratory operations.For use of the equipment described here, which has an

automatic sample changer, the cost is $5.00 to $7.00 peranalysis, including materials and labor but not includingcapital costs for the equipment or overhead. By allowingtime for changing septa, liners, tanks of gas, etc., 30 to 36test samples and 4 standards (every 10th analysis) can be runeach day. Total setup time is 2.5 to 3 h for 32 to 36 samplesand 1.25 h for 5 to 10 samples. During setup there is freetechnologist time during centrifugation, saponification,methylation, and extraction. Each analysis takes 30 min ofchromatograph time. Thus, if 23 samples were loaded ontothe sample changer at 5:00 p.m., by 8:00 a.m. the nextmorning the printed results would be available for the 23samples (3 calibration standards also would have been run).The equipment requires about 6.5 linear ft (about 200 linearcm) of bench space.

Identification by CFA analyses by using the identificationlibraries available from Microbial ID, Inc., requires attentionto detail but is not cumbersome or time-consuming. Once theequipment has been purchased, the operating cost for iden-tification is less expensive or is comparable to that foridentification by kit systems, but the variety of organismsthat can be identified by the CFA libraries surpasses thevariety of organisms that can be identified by any of the

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panels or kit systems. The rapid methods are not designed toand cannot differentiate the different clostridial toxin types(e.g., those of C. botulinum), and their data bases often donot include recently described organisms.

It is reasonable to assume that mouse assays used todetermine the toxicity of supernatants and toxin neutraliza-tion assays to determine the toxin type will continue to bethe most nearly accurate methods for differentiating betweenC. botulinum and its phenotypically similar but nontoxigeniccounterparts and for determining the antigenic nature of thetoxin produced. However, for those laboratories that do nothave facilities or antitoxin for or expertise in mouse neutral-ization tests, CFA analysis offers a reasonable alternative forthe identification of these organisms.

ACKNOWLEDGMENTS

We gratefully acknowledge the assistance of Dianne Wall Bourneand Joy Soriano for performing many of the CFA analyses and AnnP. Donnelly for performing many of the polyacrylamide gel electro-phoresis analyses. We are indebted to C. L. Hatheway, CDC,Atlanta, Ga., for sending strains CDC 3112 and CDC 5262. Weappreciate critical review of the manuscript by J.-S. Chen and R. M.Smibert.

This research was supported by the Egyptian Peace FellowshipProgram, project 131052 from the Commonwealth of Virginia, andgrant 88-34116-3790 from the U.S. Department of Agriculture.

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