JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1985, p. 620-6250095-1137/85/100620-06$02.00/0Copyright C 1985, American Society for Microbiology

H7 Antiserum-Sorbitol Fermentation Medium: a Single TubeScreening Medium for Detecting Escherichia coli 0157:H7

Associated with Hemorrhagic ColitisJ. J. FARMER III* AND BETTY R. DAVIS

Enteric Bacteriology Section, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333

Received 29 April 1985/Accepted 3 July 1985

Escherichia coli serotype 0157:H7 has been isolated from outbreaks and sporadic cases of hemorrhagiccolitis. There is convincing evidence that it can cause this diarrheal disease. Because of the interest inhemorrhagic colitis, it has become desirable to detect this particular strain in human feces, which usuallycontains many other strains of E. coli. Two characteristics of the incriminated E. coli 0157:H7 strain havemade its isolation and identification easier. It does not ferment D-sorbitol rapidly, in contrast to about 95% ofother E. coli strains. In addition, the strain has H antigen 7, but only about 10% of other E. coli strains havethis particular antigen. To screen for E. coli 0157:H7 we devised H7 antiserum-sorbitol fermentation medium(18 g of enteric fermentation base, 10 g of D-sorbitol, 4 g of agar, 10 ml of Andrade indicator, 989 ml of water;all ingredients were mixed, autoclaved, and cooled; 1 ml of E. coli H7 antiserum was then added). Colonies tobe screened were inoculated into this medium. Strains of E. coli 0157:H7 gave a characteristic pattern; theydid not ferment sorbitol and were immobilized in the semisolid medium because of the reaction of their flagellawith the flagella antiserum. Almost all other strains of E. coli gave a different pattern; they fermented sorbitolor were not immobilized by the H7 serum or both. Strains which were presumptive positives (sorbitol negative,H7 positive) were then tested in E. coli 0157 serum by slide or tube agglutination. The number of strains whichwere presumptive positive by H7-sorbitol medium but then were not found to be 0157 was less than 1%. Asecond approach has been helpful in deciding which colonies,to screen in H7-sorbitol medium. MacConkey-sorbitol agar (22.2 g of MacConkey agar base [which contains no sugar], 10 g of D-sorbitol, 1,000 ml of water)was designed as a plating medium. Stools were plated on MacConkey agar to estimate the number of E. colicolonies and also plated on MacConkey-sorbitol agar to estimate the number of sorbitol-negative colonies of E.coli. These two approaches have proved useful for isolating and identifying E. coli 0157:H7 from human fecesand from feces of animals infected in the laboratory with this strain. The results suggest that media may beformulated in a similar fashion for detecting other specific strains of E. coli.

In 1982, there were two outbreaks of an unusual form ofbloody diarrhea called hemorrhagic colitis (10). This diseasewas characterized by the sudden onset of severe abdominalpain with cramps, followed within 24 h by watery diarrhea,which subsequently became bloody. The stools were de-scribed by some of the patients as "all blood and no stool."Recovery was usually within a few days without complica-tion or specific therapy. Originally, the stool specimens werenegative for all the usual enteric pathogens, but eventually astrain of Escherichia coli 0157:H7 was implicated as thecausative agent in both outbreaks (10, 12). This rare E. colistrain was also isolated from raw hamburger which was theincriminated source in each outbreak (12), and was shown tocause diarrhea in the infant rabbit animal model for diarrhea(4). E. coli 0157:H7 also produces a toxin which is cytotoxicto the Vero cell culture line (5, 7). This toxin is very similaror identical to the "Shiga toxin" which is produced byShigella dysenteriae 1, other strains of the genus Shigella,and E. coli (7). Toxin production in these strains is associ-ated with lysogeny by one of two closely related bacterio-phages (8). The production of this shiga-like toxin may be an

important aspect of pathogenesis (8).Interest in E. coli 0157:H7 increased when it was also

found in sporadic cases of hemorrhagic colitis and in cases of

* Corresponding author.

hemolytic uremic syndrome (9). Experiments are being donein animals to study the pathogenic mechanisms of these E.coli strains. In both of these diseases and in animal experi-ments, it would be helpful to detect E. coli 0157:H7 in thepresence of other E. coli strains which normally inhabit theintestine.The purpose of this study was to devise a simple proce-

dure to screen for E. coli 0157:H7. We took advantage oftwo of its properties that are absent in most other strains ofE. coli: its flagellar antigen H7 and its inability to fermentD-sorbitol after overnight incubation. In this paper, wedescribe a new screening medium for E. coli 0157:H7 andshow how this general approach can be used to devise othersingle-tube media for detecting specific strains of E. coli inepidemic investigations or special studies. In addition, wedescribe a plating medium with added sorbitol that is usefulin detecting colonies of E. coli 0157:H7 in the presence ofother E. coli colonies.

MATERIALS AND METHODS

General procedures. Unless exceptions are given, thefollowing statements hold throughout this paper: thetemperature of incubation was 36 ± 1°C; commercial mediawere used whenever possible (the terms "from individualingredients" or "was made with" appear if a commercialmedium was not used); media were sterilized in an autoclave

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FIG. 1. At left, reaction in H7-sorbitol medium of the epidemicstrain of E. coli 0157:H7; the strain is immobilized. At right, a strainof E. coli which does not have H7 antigen: it is not immobilized.

at 121°C for 15 min; and refrigeration was at a temperature of5 + 10C.

Strains. Biochemical reactions (3) were tested on 64 dif-ferent cultures of E. (0/li 0157:H7 from stock cultures,outbreaks, and sporadic cases. The tube test for D-sorbitolfermentation was incubated for 7 days before it was dis-carded as negative. E. coli 0157:H7 A8187-M5 (Michiganoutbreak) was used in all the animal experiments. Strains ofE. coli from control subjects without diarrhea were alsoused. The distribution of different H (flagella) antigensamong E. coli strains was based on 573 strains (388 motile;185 nonmotile) which had been serotyped at the EntericBacteriology Section, Centers for Disease Control, Atlanta,Ga., between 1972 and 1982. Duplicate isolates of the same

strain were excluded before these data were tabulated. Thedata for the fermentation reactions of E. (coli were based on

over 1,000 strains which had been tested between 1972 and1982 (some strains were not tested for each one of thesugars). A number of other strains were isolated from adultRhesus monkeys and infant rabbits (5 to 21 days old) withand without diarrhea.

Sera. All sera for determining the 0 and H antigens of E.coli were obtained from the Biological Products Division,Centers for Disease Control. They had been produced inrabbits by standard methods (2) and were preserved with an

equal volume of glycerol (these sera were defined to be a 1:2dilution because of the added glycerol). The individual sera

used were E. (oli 0157 serum and E. coli H7 serum.

Agglutination in E. coli 0157 serum. A colony was touchedwith a sterile loop and used to heavily inoculate 3 ml ofTrypticase soy broth. This was grown for 5 to 6 h, duringwhich time the culture became very turbid and went into thestationary phase of growth. The broth culture was thenheated for 1 h at 1000C (in live steam in an Arnold sterilizer

or in a beaker of boiling water). The heated suspension wasthen diluted by adding 5 volumes of formalinized saline (5 gof NaCl, 5 ml of commercial Formalin, 995 ml of water) toone volume of heated culture. This is referred to as theheated antigen. For the actual agglutination reaction, thefollowing were mixed in a disposable glass tube (100 by 13mm): 0.15 ml of heated antigen, 0.75 ml of formalinizedsaline, and 0.1 ml of a 1:100 dilution of E. c oli 0157 serum.This mixture was incubated in a 48°C water bath overnightand then read for visible agglutination when compared witha control tube (0.15 ml of heated antigen, 0.85 ml offormalinized saline).

Slide agglutination. Slide agglutination was used to testcolonies directly from plating media such as MacConkeyagar, MacConkey-sorbitol agar, sheep blood agar, and Tryp-ticase soy agar. A wooden applicator stick was used to touchthe colony and remove as much of the growth as possible.The cells sticking to the bottom of the stick were thensuspended in a small drop (about 0.025 ml) of saline. Anequal-sized drop of E. coli 0157 antiserum was added, thedrops were mixed with applicator stick, and the slide wasrocked back and forth for 1 min. The epidemic strain of E.coli 0157:H7 agglutinated rapidly and completely with E.coli 0157 serum.H antigen determination by immobilization. Immobilization

for H antigen determination was described many years agoby Orskov (as quoted in reference 6) but has not been usedvery often in serotyping E. c(li. This technique is based onthe fact that a motile strain of E. coli will migrate through acolumn of semisolid agar (0.4% agar) but will be immobilized(Fig. 1) if an antiserum to its flagella (H antiserum) isincorporated into the medium.

Titration of E. coli H7 serum by H immobilization. Thetitration was done in Enteric Fermentation Base (DifcoLaboratories, Detroit, Mich.) with 0.4% agar added. Themedium was made, and 6.3 ml was dispensed into glass tubes(13 by 100 mm). These were autoclaved and cooled to 45°C,and 0.1-ml portions of various dilutions of H7 serum wereadded to give final serum dilutions of 1:64 to 1:65,536. Thetubes were mixed and then placed in the refrigerator for 1 hto harden. They were removed from the refrigerator andallowed to warm to room temperature. A straight needle wasused to touch the growth of E. coli 0157:H7 and to inoculatethe center of each tube to a depth of about 0.3 cm. The tubeswere then incubated overnight and compared with a controltube which contained no serum (Fig. 1). For each tube, werecorded the distance the culture migrated from the inocu-lation stab line. The tubes with the highest concentrations ofserum strongly prevented motility; in contrast, the culturemigrated to the bottom of the control tube without serum(Fig. 2).

MacConkey-sorbitol agar. The epidemic strain of E. coli0157:H7 fermented lactose but not D-sorbitol after overnightincubation. Thus, on lactose-containing media such as Mac-Conkey agar, the epidemic strain is indistinguishable frommost fecal strains of E. (oli which were also lactose positive.However, there is a commercial medium, MacConkey agarbase (Difco), which is identical to MacConkey agar exceptthat it does not contain lactose. E. coli 0157:H7 does notferment sorbitol (at 24 h), but about 95% of E. (0li strains do.Thus, MacConkey agar base plus 1% D-sorbitol (Mac-Conkey-sorbitol agar) was formulated to detect sorbitol-negative colonies of the epidemic strain in the presence ofsorbitol-positive colonies of other E. c oli strains. Mac-Conkey-sorbitol agar was prepared by adding the followingingredients to a 2-liter flask: 22.2 g of MacConkey agar base,

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10 g of D-sorbitol (Sigma Chemical Co., St. Louis, Mo.), and1,000 ml of water. A magnetic stirring bar (2 in. [5.08 cm])was also added. The medium was autoclaved at 121°C for 20min (to prevent boiling over, the autoclave door was openedabout 3 min after the buzzer sounded), placed on a magneticstirrer to mix the melted agar, cooled to 45 to 50°C, and thenpoured into plastic disposable petri dishes (25 ml each).After the dishes had cooled overnight, they were dried(laminar flow cabinet), placed in sterile bags, sealed, andthen stored in the refrigerator until used.H7 antiserum-sorbitol fermentation medium (H7-sorbitol

medium). This medium was designed as a single-tube screen-ing test for the strain of E. coli 0157:H7. It containsD-sorbitol and E. coli H7 antiserum. In H7-sorbitol medium,the strain of 0157:H7 does not ferment sorbitol within 24 hand is immobilized; most other strains of E. coli are notimmobilized because they have a flagella antigen other thanH7 and they ferment sorbitol. H7-sorbitol medium contained18 g of enteric fermentation base, which contains 10 g ofpeptone, 3 g of meat extract, and 5 g of NaCl. These wereadded to a 2-liter flask, and 100 ml of distilled water wasadded to make a wet paste. An additional 890 ml of waterwas added, a 2-inch magnetic stirring bar was put into theflask, and the mixture was placed on a magnetic stirrer untilall the ingredients had dissolved (about 1 min); 4 g of agar, 10g of D-sorbitol, and 10 ml of Andrade indicator (0.2 g of acidfuchsin, 100 ml of distilled water, 16 ml of 1 N NaOH) werethen added. The medium was autoclaved at 121°C for 20 min,stirred on the magnetic stirrer about 1 min to evenly distrib-ute the melted agar, and then cooled to 45°C for about 30 minin a 45°C water bath. One milliliter of glycantiserum was added. The final mediuminto several different containers for the inAbout 2.7 ml was dispensed into each of

1:64

1:4096

Dm

1:128

1:8192

1:256

1:16,384

1:512

1:32,768

FIG. 2. Titration of H7 antiserum by Hnumbers represent the final dilution of H7(control) contained no antiserum. The cross-hwhere E. coli 0157:H7 migrated away frcinoculation.

3 ml) of a plastic tissue culture dish (such as Linbro no.76-063-05 from Flow Laboratories, McLean, Va., or Costarno. 3424 from Costar, Cambridge, Mass.). The medium wasallowed to solidify (about 1 h) and then was stored in sterileplastic bags at 4°C until used. When many colonies were tobe screened, we preferred to use the 24-well plates, but wehave also dispensed the medium into sterile glass tubes (13by 100 mm) at 3 ml per tube. The medium works best whenthe surface is thoroughly dry. This can be accomplished inthree ways: (i) by using it after storage at 4°C for 2 to 3weeks, (ii) by drying it under a stream of sterile air in avertical laminar flow safety cabinet for 2 to 3 h, or (iii) bykeeping the tubes in a dry 36°C incubator for several days.

Use of H7-sorbitol screening medium. A colony to bescreened as a possible E. coli 0157:H7 was touched with athin wire needle which was stabbed about 5 mm into thecenter of dry H7-sorbitol medium (in either a 24-well dispos-able plastic tray or a test tube). The medium was incubatedat 36°C for 18 to 24 h and read for sorbitol fermentation andimmobilization by the H7 serum. A reference strain of E. coli0157:H7, which does not ferment sorbitol and contains H7antigen, was included in each run along with a negativecontrol strain of E. coli. Sorbitol fermentation was indicatedby a change in the indicator from colorless to dark pink orred, often with the appearance of gas bubbles. Immobiliza-tion in H7 serum was indicated by the failure of the teststrain to migrate from the stab line (see Fig. 1 and 2).

RESULTS

erinated E. coli H7 D-Sorbitol fermentation by E. coli 0157:H7. A total of 64can be dispensed isolates were studied for their biochemical properties. Onlynmobilization test. one strain of E. coli 0157:H7 fermented sorbitol after24 wells (capacity, overnight incubation. The strain was from a Canadian case

of hemorrhagic colitis, and its history in regard to exposureto sorbitol (which selects sorbitol-positive mutants) wasunknown. All the other cultures of E. coli 0157:H7 required3 or more days of incubation to become sorbitol positive.Sorbitol fermentation in E. coli 0157:H7 did occur uponfurther incubation. Strain A8187-M5 was streaked ontoMacConkey-sorbitol agar; after about 7 days, sorbitol-positive (pink) daughter colonies appeared (Fig. 3) on sorbi-tol-negative (colorless) colonies. Tubes of sorbitol fermen-tation broth were incubated with growth from these papillae,and sorbitol was fermented with acid and gas production at48 h. The experiment above is an example of artificialselection; however, 63 of 64 naturally occurring cultures of

1:1024 1:2048 E. coli 0157:H7 were sorbitol negative after 2 days ofincubation.Other fermentation reactions of E. coli. Table 1 gives the

fermentation reaction of our collection of E. coli strains for20 carbohydrates and polyhydroxyl alcohols commonly usedfor identification. These data can be used in helping to decideon a carbohydrate(s) to include in single-tube screeningmedia for detecting other specific strains of E. coli inepidemics or special studies.

E. coli 0157 serum. E. coli 0157 serum seems to be veryspecific for 0157 strains. When 0 antigen 157 was originallyadded to the antigenic schema for E. ccli, a serum wasprepared against its reference strain (strain A2, which has a

1:65,536 CONTROL different H antigen). This serum had a homologous titerimmobilization. The (tube agglutination) of 1:2,560. The serum did not react withserum; the last tube any of the other standard 0 antigen strains except for 0116.atched areas indicate The 0157 serum had a titer of 1:1,280 against this heterolog-m the stab line of ous antigen. However, E. ccli 0116 is a very rare type; no

isolate has been seen in our laboratory since 0116 serum has

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FIG. 3. Delayed sorbitol fermentation of E. coli 0157:H7. Sorbitol-positive papillae (daughter colonies) are present on the colonies whichwere originally sorbitol negative. The six arrows point to sorbitol-positive daughter colonies of various sizes.

been in use. Thus, the cross-reaction with E. coli 0116should not be a problem. E. coli 0157 can also be detectedby slide agglutination with 0157 serum. The 0157 serum hada titer of 1:8 by slide agglutination (2+ agglutination at 1 min;4+ agglutination at 3 min) against E. coli 0157:H7.

Immobilization titration. Figure 2 shows the results of thetitration of E. coli H7 serum against the strain of E. coli0157:H7. The titer was defined to be 1:4,096 (the endpointwas defined as almost complete immobilization). Based onthese results, the in use dilution of H7 serum was set at1:2,000 (1 ml of glycerinated serum per 1,000 ml of medium).Figure 1 shows the immobilization of E. coli 0157:H7 atdifferent concentrations of H7 serum.

TABLE 1. Fermentation reactions of E. coli strains which maybe useful in designing media to detect a particular strain

Sugar or polyhydroxyl % of E. volialcohol strains positive"

D-Adonitol.....................................

L-Arabinose.................................... 99

D-Arabitol ..................................... 8

Cellobiose ..................................... 2

Dulcitol ....................................... 60Erythritol...................................... 1

Lactose ....................................... 90

Maltose ........................................ 95D-Mannitol..................................... 98

D-Mannose .................................... 98Melibiose ...................................... 75

Mucic acid...................................... 95ca-Methyl-D-glucoside ........................... 1

Raffinose ...................................... 50

L-Rhammose ................................... 80

Salicin....................................... 40

D-Sorbitol ..................................... 95

Sucrose........................................ 50Trehalose ...................................... 98

D-Xylose ...................................... 95

"Percentage of strains positive at 1 or 2 days at 36°C; the vast majority are

positive after 24 h of incubation.

Distribution of H antigens among strains of E. coli. Table 2gives the distribution of E. coli cultures into each of the 53 Hantigens currently recognized in the serotyping schema.Certain H antigens were common, but others were rare ornot observed. H7 was the most common, representing 14.2%

TABLE 2. Distribution of H antigens among 388 motile strains ofE. coli

H antigen No. (9%) of H antigen No. (91) ofHantigen ~~strains'" nie strains"~

1 ........... 40 (10.3) 29 ........... 1 (2.1)2 ........... 17 (4.4) 30 ........... 8 (2.1)3 ........... 0 (0) 31 ........... 21 (5.4)4 ........... 25 (6.4) 32 ........... 1 (0.3)5 ........... 21 (5.4) 33 ........... 10 (2.6)6 ........... 27 (6.9) 34 ........... 2 (0.5)7 ........... 55 (14.2) 35 ........... 08 ........... 7(1.8) 36 ........... 09 ........... 11 (2.8) 37 ........... 010 ........... 22 (5.7) 38 ........... 011 ........... 15 (3.9) 39 ........... 1 (0.3)12 ........... 16 (4.1) 40 ........... 6 (1.5)13 ....._...... b 41 ........... 014 ........... 4 (1.0) 42 ........... 1 (0.3)15 ........... 3 (0.8) 43 ........... 016 ........... 9 (2.3) 44 ........... 017 ........... 2 (0.5) 45 ........... 1 (0.3)18 ........... 9 (2.3) 46 ........... 019 ........... 19 (4.9) 47 ........... 3 (0.8)20 ........... 4(1.0) 48 ........... 1 (0.3)21 ........... 30 (7.7) 49 ........... 022 ......_..... h 50 ...........023 ........... 1(0.3) 51 ........... 024 ........... 0(0) 52 ........... 025 ........... 8 (2.1) 53 ........... 026 ........... 1 (0.3) 54 ........... 027 ........... 7 (1.8) 55 ........... 2 (0.5)28 .......... 4 (1.0)

Percent of 388 motile strains.bH antigens 13 and 22 have been deleted from the antigenic schema.

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of the motile cultures and 9.6% of the total number of strainsserotyped, which included 185 nonmotile cultures.

Specificity of the H7-sorbitol medium in detecting the epi-demic strain. During the search for E. coli 0157:H7, over 300colonies from various animal experiments were picked toH7-sorbitol medium. There were only three colonies whichwere found to be false-positive (sorbitol negative and immo-bilized, but not confirmed to be 0157:H7). These were easilyshown not to be E. coli 0157:H7 by tube agglutination in0157 serum but are counted as false-positive results inevaluating H7-sorbitol as a screening medium. In all in-stances, the strain producing the false-positive result was anonfermenter which was nonmotile and had been picked asa sorbitol-negative colony from MacConkey-sorbitol agar.Sorbitol-negative, nontmotile strains of E. coli were notdetected in the animal experiments.

DISCUSSIONMuch of the early work in enteric bacteriology was

directed toward isolating enteric pathogens. The objectivewas to detect Salmonella and Shigella strains in the presenceof normal (aerobic) stool flora, which are predominately E.coli. The most popular approach has been to incorporatelactose as the differential sugar in enteric plating media.Most Salmonella and Shigella strains do not ferment lactose,but E. coli does. Lactose-negative colonies are consideredsuspect and tested further as possible Salmonella or Shigellastrains.

In the last 40 years, strains of E. coli have been shown tocause diarrhea by several different pathogenic mechanisms.The majority of these pathogenic strains ferment lactose andthus cannot be differentiated from nonpathogenic strains ofE. coli which are also lactose positive. Thus, it has beendifficult to distinguish specific pathogenic strains of E. coli.In our study, we wanted to detect E. coli 0157:H7 in thepresence of other E. coli strains.The strain of E. coli associated with hemorrhagic colitis

had two properties that made it easy to differentiate fromother E. coli strains. It did not ferment D-sorbitol within 2days, whereas about 95% of other E. coli did. It had H7antigen, but over 90% of other E. coli strains are eithernonmotile or have a different H antigen. Since sorbitolfermentation and the presence of the H7 antigen can bedetermined in a single tube, the screening method is veryspecific. This was true for all the animal experiments. Indiarrheal stools from humans, there are more false-positiveresults (Joy Wells, personal communication) which must beruled out by agglutination in 0157 antiserum. The false-positive results in the screening medium are easily detectedwith 0157 antiserum in the confirmatory step. The single-tube screening method was particularly useful for screeninga large number of colonies.

This same approach can also be applied in other studieswhen a specific strain of E. coli is being sought. One or moresugars can be incorporated into a screening medium basedon the fermentation reactions of a particular strain. Thesecan be selected by comparing the reactions of the strain withthose of most E. coli given in Table 1. Once the H antigen ofthe strain is known, an H antiserum can also be included ina semisolid to immobilize only strains with that H antigen.The specificity of this approach can be estimated from Table1, which gives the distribution of H antigens in a collection ofE. coli strains.The incorporation of D-sorbitol instead of lactose into

MacConkey agar was also helpful in detecting E. coli0157:H7 when it was mixed with other E. coli. In the animal

studies (4), many MacConkey agar plates had only lactose-positive colonies. However, MacConkey-sorbitol agar con-tained sorbitol-negative colonies among the red sorbitol-positive colonies. The MacConkey-sorbitol plate indicatedthe most likely colonies to test in H7-sorbitol medium. Platesof MacConkey-sorbitol should be picked after 24 h ofincubation to avoid the selection of sorbitol-positive mutantsof E. coli 0157:H7.

E. coli 0157:H7 has been definitely associated with hem-orrhagic colitis in both outbreaks and sporadic cases, and itappears that this strain causes this diarrheal disease (1, 4, 5,7, 9-12). Stool specimens from patients who present withsymptoms typical of the illness or with frank blood in theirstool can be screened for the presence of E. coli 0157:H7.Specimens from less severe diarrhea can also be screened.Sorbitol-negative strains of E. coli can be considered assuspect for 0157:H7 but must be confirmed with antisera to0157 and H7. We have also had success with testingcolonies directly from plates of MacConkey agar with 0157serum in the slide agglutination test. Those colonies whichagglutinate must then be serologically confirmed aftergrowth on a noninhibitory medium to eliminate false-positiveresults which can occur with this method. Both antisera forthese procedures are now commercially available; E. colisera 0157 and H7 can be purchased from R. A. Wilson,Pennsylvania State University, 105 Henning Building, Uni-versity Park, PA 16802, or from Difco Laboratories (contactA. E. Bunner). An alternative is to freeze a stool specimentaken in the acute phase of illness and submit it to areference laboratory. Plating on MacConkey-sorbitol agarand screening in H7-sorbitol medium has been particularlyuseful in specifically identifying E. coli 0157:H7. Futurestudies will be required to determine the frequency of E. coli0157:H7 in the United States and in other countries. Wehope that MacConkey-sorbitol agar and H7-antiserum-sorbitol fermentation medium will assist in the isolation andidentification of this strain.

LITERATURE CITED1. Centers for Disease Control. 1982. Isolation of E. coli 0157:H7

from sporadic cases of hemorrhagic colitis-United States.Morbid. Mortal. Weekly Rep. 31:584-585.

2. Edwards, P. R., and W. H. Ewing. 1972. Identification ofEnterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneap-olis.

3. Farmer, J. J., III, M. A. Asbury, F. W. Hickman, D. J. Brenner,and the Enterobacteriaceae Study Group. 1980. Enterobactersakazakii: a new species of "Enterobacteriaceae" isolated fromclinical specimens. Int. J. Syst. Bacteriol. 30:569-584.

4. Farmer, J. J., III, M. E. Potter, L. W. Riley, T. J. Barrett, P. A.Blake, C. A. Bopp, M. L. Cohen, A. Kaufmann, G. K. Morris,R. S. Remis, B. M. Thomason, and J. G. Wells. 1983. Animalmodels to study Escherichia coli 0157:H7 isolated from patientswith hemorrhagic colitis. Lancet i:702-703.

5. Johnson, W. M., H. Lior, and G. S. Bezanson. 1983. CytotoxinEscherichia coli 0157:H7 associated with hemorrhagic colitis inCanada. Lancet i:76.

6. Kauffman, F. 1954. Enterobacteriaceae. 2nd Munksgaard,Copenhagen.

7. O'Brien, A. D., T. A. Lively, and M. S. Chen. 1983. Escherichiacoli 0157:H7 strains associated with hemorrhagic colitis in theUnited States produce a Shigella dysenteriae 1 (Shiga)-likecytotoxin. Lancet i:702.

8. O'Brien, A. D., J. W. Newland, S. F. Miller, R. K. Holmes,H. W. Smith, and S. M. Formal. 1984. Shiga-like toxin-converting phages from Escherichia coli strains that cause

hemorrhagic colitis or infantile diarrhea. Science 226:694-696.9. Remis, R. S., K. L. MacDonald, L. W. Riley, N. D. Puhr, J. G.

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Wells, B. R. Davis, P. A. Blake, and M. L. Cohen. 1984. Sporadiccases of hemorrhagic colitis associated with Escherichia coli0157:H7. Ann. Intern. Med. 101:624-626.

10. Riley, L. W., R. S. Remis, S. D. Helgerson, H. B. McGee, J. G.Wells, B. R. Davis, R. J. Hebert, E. S. Olcott, L. M. Johnson,N. T. Hargrett, P. A. Blake, and M. L. Cohen. 1983. Outbreaksof hemorrhagic colitis associated with a rare E. coli serotype. N.

Engl. J. Med. 308:681-685.11. Stewart, P. J., and W. Desormeauz. 1983. Hemorrhagic colitis in

a home for the aged-Ontario. Can. Dis. Weekly Rep. 9:29-32.12. Wells, J. G., B. R. Davis, I. K. Wachsmuth, L. W. Riley, R. S.

Remis, R. Sokolow, and G. K. Morris. 1983. Laboratory inves-tigation of hemorrhagic colitis outbreaks associated with a rareEscherichia coli serotype. J. Clin. Microbiol. 18:512-520.

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