JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1981, p. 526-5310095-1 137/81/03-0526/06$02.00/0

Vol. 13, No. 3

Selective Medium for Isolation of Clostridium botulinum fromHuman Feces

M. DEZFULIAN,t L. M. McCROSKEY, C. L. HATHEWAY, AND V. R. DOWELL, JR.*

Enterobacteriology Branch, Bacteriology Division, Bureau of Laboratories, Centers for Disease Control,Atlanta, Georgia 30333

A selective medium, Clostridium botulinum isolation (CBI) agar, was developedfor the isolation of C. botulinum from human feces. This medium containscycloserine (250 ,ug/ml), sulfamethoxazole (76 ytg/ml), and trimethoprim (4 gg/ml) as selective inhibitory agents. Qualitative tests indicated complete recoveryof C. botulinum types A, B, F, and G on CBI medium. It was more difficult torecognize type G colonies on the medium because of their lack of lipase activity.Except for a few species of Clostridium, the growth of other obligate anaerobesand of the facultative anaerobes tested on CBI medium was suppressed. Quanti-tative studies of C. botulinum on the selective medium yielded counts comparableto those obtained on egg yolk agar control plates. Isolation of C. botulinum typesA, B, and F from seeded fecal specimens was easily achieved with CBI medium.The use of CBI agar should aid the rapid isolation of C. botulinum from fecalspecimens associated with foodborne and infant botulism.

In recent years, coproexamination has beenshown to be an invaluable procedure for labo-ratory confirmation of foodborne and infant bot-ulism (6, 10, 14). The procedure is especiallyuseful for confirmation of infant botulism be-cause botulinal toxin is very rarely detected inthe serum of an infant with the illness (1, 13).Coproexamination involves testing an extract ofthe patient's feces for botulinal toxin with mousebioassay and toxin neutralization techniques andculturing the feces for Clostridium botulinum(1, 6, 10).

Usually, a culture procedure employing aspore selection technique (heat or ethanol treat-ment to kill vegetative cells) in combination withnonselective liquid enrichment and agar platingmedia is used for isolating C. botulinum (6, 17).One ofthe major disadvantages of this procedureis that strains of C. botulinum which do notsporulate readily (e.g., C. botulinum type G[18]) may not be isolated.

It has recently been reported (3) that C. bo-tulinum types A and B have a high level ofresistance to cycloserine, sulfamethoxazole, andtrimethoprim. Also, Swenson et al. (20) haveshown that C. botulinum types A, B, and G andproteolytic type F are highly resistant to a com-bination of sulfamethoxazole and trimethoprim.In this paper, we describe the development of anagar medium, C. botulinum isolation (CBI) me-dium, containing cycloserine, sulfamethoxazole,

t Present address: Johns Hopkins University MedicalSchool, Baltimore, MD 21205.

and trimethoprim as selective agents, which al-lows quantitative as well as qualitative primaryisolation of C. botulinum from stool specimens.

MATERIALS AND METHODS

Organisms. One hundred twenty strains of bacte-ria were used in the study. These included 48 strainsof C. botulinum, 35 strains (16 species) of other clos-tridia, 15 strains (11 species) of nonsporeforming an-aerobes, and 22 strains (8 species) of various faculta-tively anaerobic bacteria. The clostridia, including C.botulinum, came from the collection of the Centers forDisease Control (CDC) Anaerobe Section. Ail of thestrains of C. botulinum type B were proteolytic. Fiveof the type F strains were proteolytic, and two (F-5and F-6) were nonproteolytic. Details regarding long-term and short-term storage and techniques for con-firming the identity of clostridia have been reportedpreviously (3, 4). Pure cultures of the facultative an-aerobes, previously identified by various reference lab-oratories at CDC, were obtained from the CDC Anti-microbics Investigations Section.Medium for isolation of C. botulinum On the

basis of previous findings at CDC (3, 20) we decided tocultivate C. botulinum on CDC-modified McClungToabe egg yolk agar (EYA) containing cycloserine(ICN Pharmaceuticals, Cleveland, Ohio), sulfa-methoxazole (Wellcome Laboratories, Research Tri-angle Park, N.C.), and trimethoprim (Wellcome Lab-oratories) in various concentrations. In preliminaryexperiments, the effects of various concentrations ofcycloserine, sulfamethoxazole, and trimethoprim inEYA on the growth of C. botulinum were studied todetermine optimal concentrations of the antimicrobialagents to use (see Table 1).On the basis of data derived from the preliminary

study and information from the literature relative to

526

on January 29, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

ISOLATION OF C. BOTULINUM FROM HUMAN FECES 527

the antimicrobial action of cycloserine, sulfamethoxa-zole and trimethoprim on other bacteria, we formu-lated a medium we call CBI. The medium has thefollowing composition (per liter): Trypticase peptone(BBL Microbiology Systems, Cockeysville, Md.), 40.0g; Na2HPO4, 5.0 g; NaCi, 2.0 g; MgSO4 (5% aqueoussolution), 0.2 mi; D-glucose, 2.0 g; agar, 20.0 g; yeastextract (Difco Laboratories, Detroit, Mich.), 5.0 g;distilled water, 900.0 ml; egg yolk suspension (50% insaline; Difco) 100.0 ml; cycloserine, 250.0 mg; sulfa-methoxazole, 76.0 mg; and trimethoprim, 4.0 mg.

In preparing the medium, all of the ingredientsexcept the egg yolk suspension and the antimicrobialagents were mixed, dissolved by heating, adjusted topH 7.4, and then autoclaved at 1210C for 15 min. Afterautoclaving, the basal medium was cooled to 55°C ina water bath, and the egg yolk suspension (warmed toambient temperature) and sterile filtered stock solu-tions of the antimicrobial agents were added asepti-cally. The cycloserine stock solution was a 1% solutionin distilled water. The stock solution of sulfamethox-azole (1.9%) was prepared by dissolving the drug indistilled water and adding enough 10% NaOH to putit in solution (19). The stock solution of trimethoprim(0.1%) was made in the same manner as that describedfor sulfamethoxazole, except 0.05 N HC1 was usedinstead of 10% NaOH, and the process was carried outin a 55°C water bath. Twenty-five milliliters of thecycloserine, 4 ml of the sulfamethoxazole, and 4 ml ofthe trimethoprim solutions were added in the prepa-ration of one liter of medium. If there is significant lossof volume due to evaporation during autoclaving thevolume must be adjusted by adding sterile distilledwater. The medium was mixed thoroughly and dis-pensed in 20-ml quantities into 15- by 100-mm plasticpetri dishes and allowed to solidify. The plates wereleft at ambient temperature for several hours to avoidexcessive moisture on the surface of the medium andthen stored in sealed plastic bags at 4°C in a refriger-ator. The plates were held in an anaerobic glove boxfor 4 h before use.

Evaluation of CBI medium. The medium wastested qualitatively by comparing growth obtained onit with that on EYA by using pure cultures of C.botulinum, various nonsporeforming anaerobes andClostridium species, and a variety of facultative an-aerobes (see Table 2). Bacterial suspensions equiva-lent to one-half the turbidity of a McFarland number1 nephelometer standard (21) were prepared in pep-tone-yeast extract-glucose broth (5, 11) by suspendingcells from colonies on EYA which had been incubatedanaerobically at 35°C. For the qualitative compari-sons, the bacterial suspensions were further diluted 1:10 in peptone-yeast extract-glucose before inoculatingthe CBI and EYA media. A plate of each medium wasinoculated with 0.01 ml of diluted bacterial suspensionand streaked for isolation by using disposable plasticloops (A/S Nunc, Roskilde, Denmark). Dilutions weremade, and plates were inoculated on the laboratorybench; immediately after inoculation, the plates wereplaced in a jar flushed with nitrogen until they couldbe transferred to the anaerobic glove box. Growth onthe two media was compared after 48 h of incubationin an anaerobic system at 35°C.

Quantitative recovery of C. botulinum strains (typesA, B, F, and G) on CBI and EYA media were compared(see Table 3). The bacterial suspension in peptone-yeast extract-glucose (one-half the turbidity of aMcFarland no. 1 standard) as described above wasdiluted in 10-fold increments in peptone-yeast extract-glucose. The 103, 104, and 105 dilutions of the suspen-sion were used for inoculation of media. Either adisposable 0.01-ml plastic loop or a 0.025-ml pipettedropper (Cooke Laboratory Products, Alexandria, Va.)was used for inoculation of the media with the dilutedcell suspensions. In the latter case, sterile bent-glassrods ("hockey sticks") were used to spread the inocu-lum over the surface of the medium. The plates wereincubated anaerobically at 35°C for 48 h, and thecolonies were counted to obtain the number of orga-nisms recovered on each medium.

Recovery of C. botulinum from seeded fecal speci-mens on CBI medium was compared with that ob-tained by conventional techniques (spore selection byethanol, heat treatments, and growth on EYA me-dium). Seeded fecal samples were prepared by addingsamples of C. botulinum cell suspensions to fecal spec-imens obtained from healthy adults (CDC employees).A sample (10 to 20 g) of feces was weighed in a sterilemortar and thoroughly mixed with gelatin phosphatediluent (4), 1 g of feces to 3 ml of diluent, until auniform suspension was obtained. Then 1 ml of a 102dilution of the C. botulinum cell suspension (one-halfthe turbidity of a McFarland no. 1 standard in pep-tone-yeast extract-glucose) was mixed with 4 ml offecal suspension. The seeded fecal sample was thendivided equally in two screw-capped test tubes. Oneportion was mixed with an equal volume of absoluteethanol to kil vegetative cells of bacteria as describedpreviously (4). The other portion was mixed with anequal volume of the gelatin-phosphate diluent. This10' dilution of fecal sample was further diluted in 10-fold increments to a final dilution of 103. Plates of theCBI and EYA media were inoculated with samples(0.01 or 0.025 ml) of the diluted feces (10', 102, or 103)and incubated anaerobically at 35°C for 48 h, and thecolonies were counted. In some experiments the 10'dilution of seeded fecal suspensions was held in an800C water bath for 10 min to kil vegetative cellsbefore inoculating the CBI and EYA media (see Table4).

Toxicity tests. Random colonies from fecal sam-ples exhibiting characteristic lipase activity on CBIand EYA media were picked to tubes of choppedmeat-glucose-starch medium (5) and were tested fortoxicity. Toxin neutralization tests to identify the toxintype were performed as described previously (3, 4, 10).

RESULTSOptimal concentrations of antimicrobial

agents. Data from a preliminary experiment todetermine the quantitative recovery of C. botu-linum on EYA medium containing various con-centrations of cycloserine, sulfamethoxazole,and trimethoprim are shown in Table 1. On thebasis of these data and data from several similarexperiments it was decided to use 250 mg of

VOL. 13, 1981

on January 29, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

TABLE 1. Quantitative recovery of C. botulinum on CDC modified McClung-Toabe EYA containing variousconcentrations of cycloserine cycle), sulfamethoxazole (SMX), and trimethoprim (TMP)

Concn (lg/ml) of: Viable C. botulinum per ml of cell suspensionCombination

Cyci SMX TMP Type A (A28K) Type G (G3)

A 500 152 8 4.5 x 106 MicrocoloniesB 250 76 4 4.7 x 106 4.0 x 107C 500 76 4 5.0 x 106 4.8 x 107aD 250 152 8 6.0 x 16 6.8 x 107aE (control) 0 0 0 5.4 x 106 4.3 x 107

a Very small colonies.

cycloserine, 76 mg of sulfamethoxazole, and 4mg of trimethoprim per liter of medium (com-bination B in Table 1), and this formulation,designated CBI medium, was used in all subse-quent experiments to be described. As shown inTable 1, C. botulinum type A grew quite well inail of the media with or without the antimicro-bial agents, and type G was inhibited andshowed only tiny colonies on media preparedwith certain combinations of the antimicrobialagents (combinations A, C, and D in Table 1).Qualitative tests. Growth of 48 strains of C.

botulinum, 35 strains of other clostridia, 15strains of gram-positive and gram-negative non-

sporeforming anaerobes, and 22 strains of facul-tatively anaerobic bacteria on CBI medium was

compared with that on EYA medium (Table 2).All 14 strains of C. botulinum type A, all 21strains of type B, all 7 strains of type F (prote-olytic and nonproteolytic), all 3 strains of typeG, and 1 out of 3 strains of type E grew on CBIplates (Table 2).Among 34 species of obligately anaerobic and

facultatively anaerobic bacteria tested, only fivespecies of Clostridium (C. bifermentans, C. ca-

daveris, C. perfringens, C. sordellii, C. sporo-genes) grew on CBI medium. The C. perfringensstrains were partially inhibited by the selectivemedium (Table 2). All the organisms tested grewon EYA medium control plates.Quantitative tests. The recovery rates for

18 isolates of C. botulinum (including 8 of typeA, 4 of type B, 5 of type F, and 1 of type G) were

determined on CBI and EYA media (Table 3).No difference was observed in the recovery ofthe organisms on CBI versus EYA medium,except the size of the colonies. The coloniesappeared slightly smaller in certain culturesgrown on CBI as compared with EYA.

Ceil morphology of the bacteria grown on CBIplates appeared normal when the gram-stainedsmears from random colonies were examinedunder the microscope. As expected, the coloniesof all type A, B, and F strains were lipase posi-tive, and the type G colonies were lipase nega-tive.

TABLE 2. Growthofvariousspeciesofbacteriaon CBImediuma

No. growing on:

Species Noe CBI EYAme- me-dium dium

Clostridium botulinum type A 14 14 14C. botulinum type B 21 21 21C. botulinum type E 3 1 3C. botulinum type F 7 7 7C. botulinum type G 3 3 3C. bifermentans 3 3 3C. cadavaris 3 3 3C. perfringens 2 (2)b 2C. sordellii 3 3 3C. sporogenes 2 2 2

a Strains that grew on the EYA control plate, but not onCBI medium, included: 3 strains each of Clostridium butyri-cum, C. difficile, C. innocuum, C. paraputrificum, C. septicum,C. tertium, Bacteroides fragilis, Escherichia coli, and Proteusvulgaris; 2 strains each of Clostridium limosum, C. sphen-oides, C. subterminale, Eubacterium limosum, Propionibac-terium acnes, Providencia stuartii, and Staphylococcus au-reus; 1 strain each of Clostridium ramosum, Bacteroidesdistasonis, B. eriksonii, B. ovatus, B. thetaiotaomicron, B.uniforms, B. vulgatus, Eubacterium lentum, E. monileforrne,Citrobacter freundii, C. diversus, Bacillus cereus, and B.subtilis; 4 strains of Proteus mirabilis; and 5 strains of Strep-tococcus faecalis.

b Parentheses indicate partial inhibition.

Recovery of C. botulinum from seededfecal samples. The results of quantitative test-ing with nine fecal specimens seeded with 16pure cultures of C. botulinum (types A, B, andF) are summarized in Table 4. As expected, theEYA medium was nonselective and yielded bac-terial colonies of various types. The C. botu-linum strains on EYA appeared as flat coloniesembedded in the agar and usually were coveredwith multiple colonies of the fecal flora. Thediffused lipase reaction of these colonies differedconsiderably from the typical reaction charac-teristic of pure cultures of C. botulinum on EYAplates. The CBI medium was clearly more ad-vantageous than EYA medium for isolation ofC. botulinum. The number of lipase-positive col-onies appearing on the CBI plates was compa-rable to that on EYA medium (Table 4), and thenumber of (lipase-negative) colonies derived

528 DEZFULIAN ET AL. J. CLIN. MICROBIOL.

on January 29, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

ISOLATION OF C. BOTULINUM FROM HUMAN FECES 529

TABLE 3. Recovery of C. botulinum strains on CBIand EYA media

No. of organism recovered/ml of

Isolated Toxin type culture on:CBI medium EYA medium

a-3N A 6.8 x 106 6.0 x 106a-1 A 5.4 x 108 4.8 x 105a-4 A 2.8 x 106 3.1 x 106A-22 A 6.8 x 106 5.0 x 106A-24 A 6.4 x 106 7.0 x 106A-28K A 4.0 x 107 2.8 x 107A-29 A 5.4 x 107 4.1 x 107b-15 B 9x105 2.2x106B-21 B 2.4 x 106 2.0 x 106B-35 B 1.3 x 106 1 x 106B-23 B 1 x 105 9 x 105F-1 F 3.2 x 107 2.4 x 107F-2 F 3.2 x 107 2 x 107F-3 F 1.6 x 106 4.8 x105F-4 F 3.2 x 107 2 x 107G-3 G 7.6 x 107 7 x 107

a Type A and B isolates designated with lower-caseletters were isolated from infant botulism cases; allstrains used in these experiments were proteolytic.

from fecal flora was reduced approximately 50to 80%. These lipase-negative colonies were lim-ited to a few colonial types and were mostlypinpoint to 0.5 mm in diameter. The highestdilution of feces used in these experiments was103, so that the small number of C. botulinumcells seeded in the fecal specimen could be de-tected. Obviously, at the higher dilutions, theselectivity of the medium could be improved,although at the expense of its sensitivity. Incontrast to the flat colonies of C. botulinum onEYA plates, the colonies of this organism onCBI medium were somewhat raised and morediscrete due to less competition and exhibited atypical lipase reaction. All of 10 lipase-positivecolonies selected at random from CBI platesstreaked with seeded fecal specimens wereproved to be toxic and type specific by toxinneutralization tests.The lecithinase-positive colonies, which ap-

peared on most of the EYA plates inoculatedwith feces, were partially or completely inhibitedby CBI medium, and, at least in one case, thelecithinase activity rather than the growth ofthe organism was completely suppressed.As shown in Table 4, heat (80°C for 10 min)

or alcohol treatments resulted in reduced recov-ery or complete elimination of C. botulinumfrom the fecal specimens.Lack of lipase activity makes C. botulinum

type G distinct from other toxigenic types of thisorganism. With a dilution technique we obtainedan indirect evidence that this type of C. botu-

TABLE 4. Recovery of C. botufecal specimens on CB

Fecal Seed Toxn Spore se-speci- orga- lectionmen nismb ype method

1 a-3N A None

2 A-28K A NoneAlcohol

3 a-1 A None

4 a-4 A NoneAlcohol

5 A-22 A NoneAlcohol

6 A-24 A NoneAlcohol

7 A-29 A NoneAlcohol

8 A-28N A NoneAlcohol

9 a-15N A NoneHeat

10 b-2 B None

11 B-41N B NoneHeat

12 F-lA F None

13 F-1 F None

14 F-3 F None

15 F-2 F NoneAlcohol

16 F-4 F None

linum from seededtI mediumNo. of lipase-positiveorganism recovered

from:

CBI EYAcontrol

2.8 x 106 1.6 x 106

1.12 x 107 7.6 x 1063.1 x 105 1.0 x 10î

4.8 x 108 2.4 x 108

8.0 x 105 3.2 x 105o o

1.0 x 106 4.4 x 10lo o

1.0 x 106 8.4 x 1052.4 x 105 8.0 x 104

2.0 x 107 4.0 x 107o o

1.0 x 106 3.0 x 106o o

5.6 x 106o

2.5 x 106

3.6 x 106o

7.7 x 106

1.6 x 107

2.4 x 107

8.8 x 1063.2 x 105

1.2 x 106

1.3 x 107

2.7 x 106

5.6 x 106O

6.2 x 107

1.6 x 107

1.2 x 107

1.0 x 1074.8 x 105

4.0 x 105a Lipase-positive colonies were considered as C. botulinum

since unseeded specimens did not yield any, and all lipase-positive colonies tested produced botulinal toxin.

b Type A and B strains designated with lowercase letterswere isolated from infant botulism cases; all strains used inthese experiments were proteolytic.

linum could also be recovered from a mixedpopulation. A fecal specimen was diluted in a10-fold series (101 to 106) before or after additionof a smail number of C. botulinum type G. Platesof CB were then inoculated with 0.01 ml ofvarious dilutions of the two specimens. No col-onies resembling type G were recovered fromthe three highest dilutions (104, 105, and 106) ofthe unseeded fecal specimen, but 36, 11, and 1colonies, respectively, were recovered from thesame dilutions of the seeded specimen. Thecharacteristics of these colonies were identical

VOL. 13, 1981

on January 29, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

530 DEZFULIAN ET AL.

with those of the C. botulinum type G on EYAmedium.

DISCUSSIONA method commonly used for isolation of C.

botulinum from fecal samples involves cultiva-tion of primary specimen or enriched culture onEYA medium (6, 10). Though differential forlipase-positive organisms (including most toxi-genic types of C. botulinum), EYA medium isnonselective and allows the growth of a widevariety of bacteria from fecal flora. Spore selec-tion techniques (heat or alcohol treatment) arecommonly used for selective isolation of clos-tridia from fecal specimens (6, 17). Our data,however, indicate that when only a small num-ber of spores are present in the fecal sample,heat or alcohol treatment may lead to the elim-ination of the organism and thus to a false-negative result (Table 4). Use of CBI mediumwould allow recovery of C. botulinum by directstreaking with a specimen without treatment orenrichment.The use of cycloserine, sulfamethoxazole, and

trimethoprim in CDC modified EYA yielded amedium (CBI) which is both selective and dif-ferential for C. botulinum types A, B, and F.This selective medium proved superior to thenonselective method of culturing on EYA andthe selection method involving heat and alcoholtreatment. Whereas the majority of other micro-organisms present in feces were inhibited, somedid grow on CBI medium. The colonies of thesebacteria on CBI medium were mostly small orpinpoint and could easily be distinguished fromthe lipase-positive colonies of C. botulinumtypes A, B, and F. Due to the inhibitory effectof CBI on the fecal microflora, the colonies of C.botulinum on this medium were usually readilyapparent and easy to isolate.A combination of cycloserine, sulfamethoxa-

zole, and trimethoprim was incorporated intothe EYA medium with the knowledge that thepresence of thymidine in the complex EYA me-dium might interfere with the bacteriocidal ef-fect of the combination of sulfamethoxazole andtrimethoprim (12, 19) and thus with the selectiv-ity of the medium. It has been demonstratedthat adding thymidine phosphorylase (presentin hemolyzed horse blood) may improve certainmedia (with limited thymidine) by convertingthymidine to thymine (19). Improved selectivityof CBI medium, however, would interfere withthe isolation of less resistant type G (Table 1)and nonproteolytic type F (20) strains of C.botulinum. Cycloserine alone or in combinationwith cefoxitin has previously been used for theisolation of C. difficile (7, 23). Likewise, trimeth-

oprim combined with sulfamethoxazole (9) orother antimicrobial agents (16) has been em-ployed in various selective media.

Cycloserine in a concentration of 500 ,g/mlhas been shown to affect the morphology ofbacterial cells, converting the gram-positive ba-cilli of C. difficile to filamenteous forms (7). Nosuch effect was observed in the case of C. botu-linum cells growing on CBI medium (with cyclo-serine at 250 ,ig/ml). The morphology and spor-ulation of the cells, as judged by microscopicexamination of gram-stained smears of the col-onies on CBI medium remained normal.Although C. botulinum type G can also be

recovered on CBI medium, lack of lipase activitymakes the differentiation of the colonies of thisorganism from those of other bacteria more dif-ficult. In cases where the results of clinical andtoxigenic studies warrant a search for C. botu-linum type G, further examination of the lipase-negative colonies on CBI medium is justified.Presumptive identification can be achieved bymeans of a spot test for "indole-derivatives" andexamination of volatile and nonvolatile acids bygas-liquid chromatography (15; M. Dezfulian,unpublished data), and the identity can be con-firmed by toxin-neutralization tests in mice (2,8). The immunodiffusion technique of Ferreiraet al. (J. L. Ferreira, M. K. Hamdy, F. A. Za-patka, and W. O. Herbert, Abstr. Annu. Meet.Am. Soc. Microbiol. 1980, P8, p. 190), based onthe interaction of toxin and antitoxin to form a"precipitin zone" around the colonies of C. bo-tulinum type A, could possibly be extended tothe presumptive identification of C. botulinumtype G colonies on the selective medium. In thepresent report, we described the use of dilutedfecal suspensions cultured on CBI for the recov-ery of this organism.

Recently, Wilcke et al. (22) described the useof a medium consisting of heart infusion brothsupplemented with agar, egg yolk (3%), and kan-amycin sulfate (12 ,ug/ml) in the quantitationand isolation of C. botulinum from the feces offour infants with botulism. Although the authorsfound that the medium did not appreciably in-hibit fecal bacteria, they stated that it did sup-press the growth of some organisms sufficientlyso that the presence of lipase-positive coloniesof C. botulinum could be easily detected. It iswell known that a variety of intestinal bacteria(sporeformers and nonsporeformers) are resist-ant to high concentrations (e.g., 100 ,ug/ml) ofaminoglycoside antibiotics. Some, such as kan-amycin, paromomycin, and neomycin are com-monly used for selective isolation of bacteroides,fusobacteria, C. perfringens, and a variety ofothers from clinical materials (4).

J. CLIN. MICROIBIOL.

on January 29, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

ISOLATION OF C. BOTULINUM FROM HUMAN FECES 531

Although CBI medium seems to be superiorto nonselective EYA medium, the simultaneoususe of both media for the isolation of C. botu-linum from the primary specimen, and if neces-sary from the enriched culture, is recommended,to avoid missing drug-susceptible strains of C.botulinum such as some type E strains (20).Our data also indicate that CBI medium, with

or without some modification, can be used forthe isolation of certain other clostridia such asC. bifermentans, C. sordellii, and C. cadaveris(Table 2). The colonies of these lipase-negativeorganisms are easily distinguished from C. bo-tulinum colonies. Another organism which grewon CBI medium, as would be anticipated, was C.sporogenes, which can be differentiated from C.botulinum by the mouse toxicity and neutrali-zation test.The CBI medium should be especially useful

for selective isolation and presumptive identifi-cation of C. botulinum associated with infantbotulism. This assumption is based on the factsthat: (i) the fecal bacterial flora of the humaninfant is less complex than that of the adult; (ii)only types A, B, and F (all proteolytic) havebeen associated with infant botulism to date;(iii) these toxin types are resistant to the anti-microbial agents in CBI medium; (iv) all threetoxin types of C. botulinum exhibit lipase activ-ity on the CBI medium.

LITERATURE CITED

1. Center for Disease Control. 1979. Botulism in theUnited States, 1899-1977. Center for Disease Control,Atlanta, Ga.

2. Ciccarelli, A. S., D. N. Whaley, L. M. McCroskey, D.F. Gimenez, V. R. Dowell, Jr., and C. L. Hatheway.1977. Cultural and physiological characteristics of Clos-tridium botulinum type G and the susceptibility ofcertain animals to its toxin. Appl. Environ. Microbiol.34:843-848.

3. Dezfulian, M., and V. R. Dowell, Jr. 1980. Cultural andphysiological characteristics and antimicrobial suscep-tibility of Clostridium botulinum isolates from food-borne and infant botulism cases. J. Clin. Microbiol. 11:604-609.

4. Dowell, V. R., Jr., and T. M. Hawkins. 1974. Labora-tory methods in anaerobic bacteriology: CDC labora-tory manual. Center for Disease Control, Atlanta, Ga.

5. Dowell, V. R., Jr., G. L. Lombard, F. S. Thompson,and A. Y. Armfield. 1977. Media for isolation, char-acterization and identification of obligately anaerobicbacteria. Center for Disease Control, Atlanta, Ga.

6. Dowell, V. R., Jr., L. M. McCroskey, C. L. Hatheway,

G. L. Lombard, J. M. Hughes, and M. H. Merson.1977. Coproexamination for botulinal toxin and Clos-tridium botulinum: a new procedure for laboratory di-agnosi8 of botulism. J. Am. Med. Assoc. 238:1829-1832.

7. George, W. L., V. L. Sutter, D. Citron, and S. M.Finegold. 1979. Selective and differential medium forisolation of Clostridium difficile. J. Clin. Microbiol. 9:214-219.

8. Gimenez, D. F., and A. S. Ciccarelli. 1970. Anothertype of Clostridium botulinum. Zentralbl. Bakteriol.Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe 215:221-224.

9. Gunn, B. A., D. K. Ohashi, C. A. Gaydos, and E. S.Holt. 1977. Selective and enhanced recovery of groupA and B streptococci from throat cultures with sheepblood agar containing sulfamethoxazole and trimetho-prim. J. Clin. Microbiol. 5:650-655.

10. Hatheway, C. L. 1979. Laboratory procedures for casesof suspected infant botulism. Rev. Infect. Dis. 1:647-651.

11. Holdeman, L. V., E. P. Cato, and W. E. C. Moore.1977. Anaerobe laboratory manual, 4th ed. VirginiaPolytechnic Institute and State University, Blacksburg,Va.

12. Koch, A. E., and J. J. Burchall. 1971. Reversal of theantimicrobial activity of trimethoprim by thymidine incommercially prepared media. Appl. Microbiol. 22:812.

13. Midura, T. F. 1980. Infant botulism. Clin. Microbiol.News Letter 2:1-3.

14. Midura, T. F., and S. S. Arnon. 1976. Infant botulism:identification of Clostridium botulinum and its toxin infeces. Lancet ii:934-936.

15. Moss, C. W., C. L. Hatheway, M. A. Lambert, and L.M. McCroskey. 1980. Production of phenylacetic andhydrophenylacetic acids by Clostridium botulinumtype G. J. Clin. Microbiol. 11:743-745.

16. Skirrow, M. B. 1977. Campylobacter enteritis: a "new"disease. Br. Med. J. 2:9-11.

17. Smith, L. 1977. Botulism: the organism, its toxins, thedisease. Charles C Thomas, Publisher, Springfield, Ill.

18. Solomon, H. M., and D. A. Kautter. 1979. Sporulationand toxin production by Clostridium botulinum type G.J. Food Protect. 42:965-967.

19. Swenson, J. M., and C. Thornsberry. 1978. Suscepti-bility tests for sulfamethoxazole-trimethoprim by abroth microdilution procedure. Curr. Microbiol. 1:189-193.

20. Swenson, J. M., C. Thornsberry, L. M. McCroskey,C. L. Hatheway, and V. R. Dowell, Jr. 1980. Suscep-tibility of Clostridium botulinum to thirteen antimicro-bial agents. Antimicrob. Agents Chemother. 18:13-19.

21. Thornsberry, C., and J. M. Swenson. 1978. Antimicro-bial susceptibility testing of anaerobes. Lab. Med. 9:43-48.

22. Wilcke, B. W., Jr., T. F. Midura, and S. S. Arnon.1980. Quantitative evidence of intestinal colonization byClostridium botulinum in four cases of infant botulism.J. Infect. Dis. 141:419-423.

23. Willey, S. H., and J. G. Bartlett. 1979. Cultures forClostridium difficile in stools containing a cytotoxinneutralized by Clostridium sordellii antitoxin. J. Clin.Microbiol. 10:880-884.

VOL. 13, 1981

on January 29, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from