JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1977, p. 66-74Copyright X 1977 American Society for Microbiology

Vol. 5, No. 1Printed in U.S.A.

Bacteriophage Typing of Shigella sonneiRUDY C. PRUNEDA AND J. J. FARMER III*

Department of Parasitology and Laboratory Practice School of Public Health University of North CarolinaChapel Hill, North Carolina 27514, and Bacteriophage-Bacteriocin Laboratory, Center for Disease Control,

Atlanta, Georgia 30333*

Received for publication 22 September 1976

A bacteriophage-typing schema was developed for differentiating strains ofShigella sonnei. Sixty-seven bacteriophages were obtained from other collec-tions, and 36 bacteriophages were isolated from sewage. From these 103 bacteri-ophages, a provisional set of 12 was chosen by computer analysis as being themost sensitive in differentiating strains of S. sonnei isolated in the UnitedStates. The provisional schema was used to type 265 strains from differentgeographical areas. It divided them into 87 different lysis patterns, and all 265strains were typable. Smooth and rough colonial variants of the same strain haddifferent lysis patterns, so the technique was standardized to type rough coloniesonly. Reproducibility was difficult to obtain until all conditions were carefullystandardized. Changes in results were noted even on different lot numbers ofTrypticase soy agar, which was defined as the standard medium. So that themedium would not be a variable, 100 pounds (ca. 453.5 kg) of the same lotnumber was purchased. Bacteriophage typing was very useful in differentiatingstrains, and work should continue on establishing a standardized schema.

In many parts of the world, Shigella sonneihas become the predominant species of Shi-gella. In the United States, S. sonnei accountsfor about 85% of all Shigella isolates, S. flex-neri accounts for about 14%, and S. dysenteriaeand S. boydii account for only about 0.5% each(7). S. sonnei is found in all regions of theUnited States and is often endemic in institu-tions for the mentally ill, on Indian reserva-tions, and in day-care centers and communitiesof lower socioeconomic status (10).Each species of Shigella except S. sonnei can

be divided into serotypes (9), and serologicalsubdivision is usually sufficient for tracing theepidemiology of S. dysenteriae, S. Flexneri,and S. boydii (7). However, other methodsmust be used for the epidemiological finger-printing of S. sonnei. Szturm-Rubinstein (29)used beta-galactosidase, xylose, and rhamnoseas markers in biotyping. Abbott and Shannon(1) developed a method based on colicin produc-tion (colicins are antibiotic substances pro-duced by strains of Escherichia coli, Shigella,and related species that kill other strains ofthese same species), and this typing methodhas often been used as an epidemiologicalmarker (16, 26). Resisto-typing (growth inhibi-tion by organic and inorganic chemicals) hasbeen used by Elek et al. (11, 23). Antibiograms(4, 8) and phage typing (19, 22, 28) have alsobeen used to differentiate strains of S. sonnei,

and combinations of several typing methodshave also been used (4, 14, 20).

In the United States, colicin production (16)has been the most common method for typingS. sonnei. Unfortunately, in recent years, 40%of the S. sonnei isolates from outbreaks havenot produced colicins that kill any of the colicinindicator strains and thus have been "untypa-ble" (23). A similar problem exists in England;Gillies (17) found that 79.5% of the S. sonneicultures were untypable in 1963 and 92.3% wereuntypable in 1964.Of all the typing methods, bacteriophage typ-

ing appears to be the most sensitive; therefore,the purpose of this study was to evaluatephage-typing schemas that others have usedand to select the most useful phages for a provi-sional schema. A future article compares bacte-riophage typing, colicin typing, and antibio-grams as epidemiological markers in the sur-veillance of outbreaks due to S. sonnei.

MATERIALS AND METHODSMedia. Trypticase soy agar (TSA), Trypticase soy

broth (TSB), and Mueller-Hinton agar were ob-tained from Bioquest (Div. of Becton, Dickinson &Co., Cockeysville, Md.) and were prepared accord-ing to the manufacturer's instructions. Soft agarfor overlays contained 0.4% (wt/vol) Oxoid Ionagarno. 2 (Colab Laboratories, Inc., Chicago, Ill.). Phageagar contained nutrient broth (Difco Laboratories,Detroit, Mich.), 20 g; NaCl, 7.5 g; agar-agar, 20 g;

66

PHAGE-TYPING SCHEMA FOR S. SONNEI STRAINS

and distilled water, 1,000 ml. All other media werefrom commercial sources and were prepared by theMedia Unit at the Center for Disease Control(CDC). All dilutions of bacteriophages in hoststrains were in either phage broth or TSB. All in-cubations were at 36 + 1°C, and all phages werestored at 4°C.

Bacterial strains. Three hundred isolates of S.sonnei, 15 isolates of S. boydii, 15 isolates of S.flexneri, and 5 isolates of S. dysenteriae were ob-tained from the Enteric Section, CDC; these sampleshad been sent to CDC from throughout the UnitedStates. Species and serotypes for all cultures hadbeen identified by the WHO Collaborating Centrefor Shigella. Additional cultures were obtained fromthe Enteric Section, CDC; S. sonnei strains werefrom the Medical Bacteriology Unit, Texas HealthResources, Austin, Tex., and a number of Entero-bacteriaceae came from the stock culture collectionof the Parasitology and Laboratory Practice Depart-ment, School of Public Health, University of NorthCarolina, Chapel Hill. All cultures were stored insealed test tubes at room temperature in the dark.

Bacteriophages. Sixty-seven bacteriophages wereobtained from the following collections: 13 phagesused in typing Escherichia coli (25) from J. T. Parisi,Department of Microbiology, University of Mis-souri, Columbia, Mo.; 12 phages used in typing S..flexneri from C. Ciufecu, Bucharest, Romania; 10phages used in a provisional typing schema for S.sonnei (28) from S. Slopek, Polish Academy of Sci-ences, Wroclaw, Poland; and 32 phages from theBacteriophage-Bacteriocin Laboratory, CDC.

Isolation of bacteriophages from sewage. Samplesof raw sewage were collected from the sewage treat-ment plant, Chapel Hill, N.C., and pooled. Bacterio-phages in the sewage were isolated by the enrich-ment method of Adams (2), modified as follows. (i)Each host strain was inoculated into TSB and grown(overnight) to the stationary phase. Then 0.1 ml ofthe culture and 2 to 4 ml of the raw sewage wereadded to 9 ml of TSB in an 18- by 15-mm tube and

incubated for 6 to 7 h at 35°C. (ii) A 0.3-ml amount ofchloroform was added to 3 ml of the host-sewagemixture; the tube was vigorously shaken with aVortex mixer, and the chloroform was allowed tosettle for about 1 h at 4°C. (iii) A 0.3-ml portion ofthe top layer was removed into a petri dish, whichwas placed in a hood with laminar air flow to removethe residual chloroform. (iv) Serial 100-fold dilutionswere made of the enrichment and dropped onto alawn of the host on TSA. After the drops had dried,the plates were incubated overnight and observedfor bacteriophage plaques. (v) Single isolatedplaques were picked and added to 106 cells ofthe hoststrain in TSB. This was necessary to obtain a stockwith a titer of more than 108 plaque-forming units(PFU)/ml. (vi) This mixture was then treated withchloroform, as in step iii, and stored.

Thirty-six bacteriophages were isolated from sew-age: 8 on S. sonnei; 9 on S. flexneri; 3 on S. dysenter-iae; and 16 on E. coli. Each of the 36 host strains wasfrom a different source; thus, diversity of the phageswas insured.RTD. Serial 10-fold dilutions were made of each of

the final phage preparations, and each dilution wastested against the host strain on TSA. Based on thistitration, a tube of phage that contained 106 PFU/mlwas made, and this was called the routine test dilu-tion (RTD) tube. Our standard syringes were filledfrom the RTD tubes. Since each syringe deliversdrops of 0.01 ml, each drop contains 104 PFU ofphage. We define our RTD to be 104 PFU of phage(106 PFU/ml x 0.01 ml = 104 PFU/test), thus aban-doning less precise definitions previously used forRTD such as "that producing confluent or semi-confluent lysis" (3). Two titrations are shown in Fig.1.

Bacteriophage typing. Initially, whole cultureswere used for the phage-typing procedure; however,we soon learned that the lysis patterns were depend-ent on the ratio of smooth to rough colonies presentin the culture.Lawns for phage typing were prepared as "flood

- 4.Op

A

___- _..dFIG. 1. Tenfold dilutions ofphage F7 (left) and F9 (right) on their host strains.

67VOL. 5, 1977

68 PRUNEDA AND FARMER

plates" from rough colonies. TSA plates were driedeither for 30 min under laminar air flow or with thetops on in room air for 72 h. The dry plates wereflooded with a broth culture that had been adjustedto the standard turbidity (optical density at 650 nm= 0.10, with a light path of 1.3 cm). This turbidity isalmost identical to a 0.5 MacFarland standard usedin antimicrobial sensitivity testing. The excess fluidwas removed with a safety pipette and discardedinto 0.5% (wt/vol) Amphyl (National Laboratories,Toledo, Ohio). The plates were dried with the tops offfor 10 min; the phages (at RTD) were then appliedsimultaneously with the applicator shown in Fig. 2(Johnny Brown Machine Shop, Tuscaloosa, Ala.).This applicator delivers 61 uniform drops in a singleoperation. After the drops had dried, the plates wereincubated overnight and observed by indirect light-ing with a model C100 electronic colony counter(New Brunswick Scientific, New Brunswick Co.,N.J.) for lysis. An area of lysis with 20 or moreplaques was defined as positive; however, all resultswere recorded as confluent lysis or semiconfluentlysis and as to size and number of plaques seen perdrop. The nomenclature was that of Anderson andWilliams (3). The lysis patterns were converted tonumbers by using the simplified notation describedby Farmer (12) (Table 1). Thus, the phage type inour provisional schema consisted of a four-digitnumber and represented its reaction against ourbest 12 bacteriophages.

Selection of standard set of bacteriophages. Thebest bacteriophages were selected on the basis of acomputer analysis as described by Farmer (13) anddeveloped by Milton Hutson and John Zakanycz,Computer Honors Program, University of Alabama,Tuscaloosa, Ala. Computer analysis from 340 iso-lates of S. sonnei indicated that the best set forroutine typing consisted of 12 phages; this set was

defined as the provisional set in the typing proce-dure.Smooth and rough colony types. Cultures of S.

sonnei were streaked on TSA or Tergitol-7 (Difco)agar plates and incubated overnight; isolated colo-nies were observed under a dissecting microscopewith oblique lighting. The smooth and rough colo-nies observed were similar to those described byBaker et al. (5) (see Fig. 3). The lysis patterns of thesmooth and rough colonies were compared as de-scribed previously (12). Serological typing was donewith S. sonnei antisera (Bioquest, Cockeysville,Md.) from growth taken from TSA plates. Acrifla-vine was also used to detect roughness (6). Smoothcolonies were streaked on TSA plates, incubatedovernight, and observed for both rough and smoothcolonies. Both colony types were then phage typed.Comparison of the Slopek phage set and new set.

Two hundred and sixty-five isolates of S. sonnei

TABLE 1. Simplified notation for reportingbacteriophage types a

Results of three tests Representation

+++ 1++- 2+-+ 3_++ 4+-- 5_+_ 6__+ 7

8

aIf the number of tests is not evenly divisible by3, a second (++ = A, +- = B, -+ = C, -- = D)and third (+ = E, - = F) code can be used torepresent those results remaining after division by3.

&.IS

-:....

FIG. 2. Bacteriophage applicator with syringes containing the bacteriophages at RTD.

J. CLIN. MICROBIOL.

PHAGE-TYPING SCHEMA FOR S. SONNEI STRAINS 69

were obtained from the CDC collection. These cul-tures were from throughout the United States andwere submitted over a period of 20 years. The strainswere then typed with our provisional set of 12phages and the Slopek set of 10 phages (28).

RESULTSEffect of colonial variation on typing re-

sults. Figure 3 shows the two types of colonies(defined to be rough [r] or smooth [s]) usuallyseen in plates streaked for isolation. Thesmooth colonies were round and moist; therough colonies were dry, granular, and flat andhad irregular borders. Both of these colonytypes had characteristic agglutination reac-tions (Fig. 3) in commercial S. sonnei antisera.

This type of agglutination was not observed inacriflavine or in antisera to S. sonnei (phage Ior II) prepared at CDC.

Figure 4 shows the lysis patterns of roughand smooth colony types derived from the samestrain (m7480). The first 12 phages are the pro-visional set described in this paper. The roughcolony type is lysed by 18 phages, but thesmooth colony type is lysed by only 6. Thus, thelysis patterns of smooth and rough colonieswere different even though they were derivedfrom the same strain. This is why the techniquewas standardized for typing only the rough col-onies. When the whole culture (no selection forrough or smooth) of S. sonnei m7480 was phagetyped, it had a lysis pattern intermediate be-

FIG. 3. Smooth (s) and rough (r) colonies ofS. sonnei (left) and agglutination ofsmooth (S) and rough (R)colonies with antisera for S. sonnei (right).

_'' * 'v'

Y e r e * *O -uS-v PPC 1

*- ----t#hL.:.. _ v i. _ ....Z....,

FIG. 4. Lysis patterns of a smooth colony (left) and a rough colony of the same strain (right).

VOL. 5, 1977

i_5 a_

70 PRUNEDA AND FARMER

tween rough and smooth, which probably re-flected the proportions of rough and smoothcolonies in the culture.

Effect of media on typing results. Figure 5shows different lysis patterns of Slopek's strain1393 of S. sonnei on phage agar and TSA. Sixphage reactions were media dependent. Aftertrying both media, we decided to use TSA instandardizing the provisional typing schema,for the following reasons: (i) TSA was availablecommercially, but phage agar was not; (ii) TSAwas easier to prepare and pour; and (iii) dropsof phage did not spread or run together on TSA,as they often did on phage agar.

Selection of the best 12 bacteriophages. Onehundred and three bacteriophages were evalu-ated. Many of these were eliminated for one ormore of the following reasons: (i) they lysedonly a small percentage of the S. sonnei strains;(ii) they gave reactions that were difflcult toread and reproduce; or (iii) they gave lysis pat-terns too similar to those of other, more usefulphages. Many of the phages were eliminated byimpartial computer analysis. An example ofhow the computer program works is shown inFig. 6. Here, 145 isolates had been testedagainst 72 phages which yielded over 10,000reactions. The first step in the program is tochoose the phage that best divides the 145 iso-lates into two groups, with half being lysed andhalf not being lysed. None of the phages madethis division exactly, but phage P1 came theclosest (75+, 70-); thus, it was chosen first.The program then chose the phage that bestdivided the two groups formed by the firstphage. In this case, P2 was chosen because itdivided the two groups with the most equal

r

subdivisions. With P1 and P2, the 145 strainswould now be divided into four groups (+ +, 40strains; + -, 35 strains, - +, 36 strains; --, 34strains). The program then continues to selectphages that best subdivide the groups formedby the previous selections. Figure 6 shows theactual analysis for the first three phages chosenand how they divided the 145 strains into eightdifferent "plus-minus" patterns. The programcontinues to choose the best phages until a level(set by user) of sensitivity (or number ofphages) is reached. Table 2 shows the 12 phageschosen by computer analysis as being the mostsensitive. Of these 12 phages, four had beenused in Slopek's typing schema for S. sonnei,two came from a typing set for S. flexneri, threecame from the CDC collection, and three hadbeen isolated from sewage.Table 3 shows that the phages divided 265

isolates of S. sonnei into 88 different lysis pat-terns. In this table, the phage type consists of afour-digit number that represents the 12 phagereactions. Type 1111 was the most common andcomprised 20% of all isolates. Types 2111, 2211,

145 Isolates

+ Phage 1 -

75 70

++ +-Phage 2 --40 35 36 34

+++ ++_ +-+ +-- Phage 3 -++ - -+ ---

17 23 7 28 19 17 12 22

FIG. 6. Example of how the computer programchose the first three phages and divided the 145strains into different patterns.

.¢,,W aEZI I\

o

.0-0

FIG. 5. Lysis pattern of S. sonnei Slopek 1393 on TSA (left) and phage agar (right).

J. CLIN. MICROBIOL.

Ni--.,. Ik

t

II

PHAGE-TYPING SCHEMA FOR S. SONNEI STRAINS

TABLE 2. Bacteriophages selected for final typingsystem

New Old Lysis ofphage phage Source strains

designa- designa- lysed (%)tion tion

P1 SS4a Sewage 52P2 18i Enteric collection 53P3 F7 Slopek 35P4 34b Enteric collection 57P5 EC5 Sewage 63P6 19c Enteric collection 45P7 F4 Slopek 69P8 R9 Ciufecu 84P9 F3 Slopek 80PlO SD2 Sewage 90P11 Fl Slopek 89P12 R7 Ciufecu 77

4111, and 4511 were also common, but the re-maining types were rare. All 265 strains werelysed by at least one phage, so all were typable.

Slopek's phage set (28) was also used to typethese same 265 isolates (Table 4). Both setstyped all of the isolates; however, our provi-sional phage set was more sensitive. It dividedthe isolates into 88 types, as compared to 34 forSlopek's set, and also considerably reduced thesize of the most common types (Table 4).

Colicin typing of the 265 isolates. The 265isolates that were phage typed were also colicintyped by CDC's Epidemiologic InvestigationLaboratory Branch. Colicin typing showed that35.7% were untypable. The next largest groupswere types 7 and 9, with 12.7 and 12.3%, respec-tively. These were followed by colicin types 2(11.5%), 12 (9.12%), and 6 (7.9%). Twelvegroups were differentiated by this method.

DISCUSSIONShigellosis due to S. sonnei is a problem in

the United States. People on Indian reserva-tions and in mental institutions and those inlow-income areas are most susceptible, becausethey are often undernourished, lack proper san-itary facilities, and generally practice poor hy-giene (15). To better understand the epidemiol-ogy of these infections, a sensitive typing sys-tem is needed. This could help pinpoint thesources of infection and reduce the likelihood offurther spread. Such a system would give theepidemiologists reliable, sensitive, and fast re-sults, especially when an outbreak is in prog-ress.

Recent work by Slopek et al. (28) has indi-cated that bacteriophage typing could becomethe method of choice for studying outbreaksfrom S. sonnei. They selected their 10 phagesfrom those that had been previously used for

TABLE 3. Distribution of265 isolates of S. sonneiinto phage types

Phage type

11111113112311411311132113521411163221112132221121412232224123212432261126132643266128112821285256135614563258115832586261116141621161616314634165416561761176137711778678117862782178537861

No. of iso-lates

5411521111

121

14214133211312131111

133311211111111111

Phage type

3111312341114211431143124321432343244332443346134811484151115132514152115214521652445311561156138211861288228841884288448845884688618862886488668872887588768882

No. of iso-lates

811212141121111811

153111511121111125115421

typing S. sonnei (18, 19, 27). Thus, in establish-ing their system, they used the best phagesfrom these previous typing streams. All of theS. sonnei strains that they tested were typable,and the phage set divided the 2,064 isolatestested into 100 types with their provisionalphage set one and into 85 types with provisionalset two. Because of Slopek's excellent studies,we decided to evaluate this typing set with 265isolates of S. sonnei from the United States.

VOL. 5, 1977 71

72 PRUNEDA AND FARMER

TABLE 4. Sensitivity of two typing schemas in differentiating 265 isolates of S. sonnei

No. of dif- % of isolates in common phage types:Phage set fernNo.ofun-ttypable phage types 1st most 2nd most 3rd most 4th most 5th most 6th most

common common common common common common

Slopek 0 34 34 26 6 4 3 3

This study 0 88 20 5 5 4 4 3

One criterion for a good typing system is thatthe phages should divide the isolates into asufficient number of phage types (3). Our 265 S.sonnei isolates were divided into 88 phagestypes. In comparison, Slopek's phage set di-vided these same isolates into only 34 phagetypes. In addition, the percentage of the iso-lates in the most common phage types weremuch smaller with our typing system than withSlopek's. With our new phage set, the largestphage type comprised 20%, which is the mostsensitive reported to date (2, 18, 21, 22).The second criterion is that the technique

should be simple and give clear-cut results. Theprocedure for making lawns and the automatedapplication of bacteriophages have eliminatedthe cumbersome process of applying individualphages (3). Clear-cut areas of lysis were usuallyobtained with the new phage set, especiallywhen rough colonies were used. Another factorthat influenced the readings was the end pointused for designating a reaction as positive. Forthe new typing set, an RTD of 10,000 PFU/testwas used, and a positive reaction was defined tobe the presence of 20 or more bacteriophageplaques. These two criteria may be modified inthe future if reproducibility proves to be betterwith a different end point or RTD. This pointshould be investigated further before a stand-ardized method is proposed. The criterion cho-sen for defining a phage type was broad com-pared with that selected by Slopek et al. (28). Inthe Slopek system, the difference between somephage types was based on very small variationsin lysis between the reactions of the samephage. For this reason, it was difficult to com-pare the phage types seen in the United Stateswith those seen in Poland. Several modificatonscould be tried to improve the test results. Onemodification would be to use a media such asTergitol agar to convert all smooth strains intorough strains, thus insuring uniformity of col-ony types used for typing. This, along withchanges in the RTD and end point, could im-prove the results.The third criterion is that the typing re-

agents should be stable. There was very littledrop in titer of any of the typing phages, even

though some had been stored at 4°C for as longas 6 months.The fourth criterion is that the results should

be available quickly. The method of makinglawns and applying phages with an applicatorhas eliminated many of the time-consumingprocedures used previously. Phage typing ismuch quicker than colicin typing for these rea-sons. Colicin typing is best suited for typing alarge group of cultures together, because of themethodology and quality control required. Incontrast, phage typing is equally suited for typ-ing 1 or 100 strains in one run, since only one ortwo control strains are needed to indicatewhether the phages are at the proper RTD. Ourresults were often available within 8 h of re-ceipt of the culture.The fifth criterion is the most important; it is

that typing results should agree with epidemio-logical findings. In the past, this importantcriterion has not been satisfied with many typ-ing systems. Abbott and Shannon (1) describedtyping by colicin sensitivity along with typingby colicin production; however, they rejectedcolicin sensitivity as a typing tool because of itspoor agreement with epidemiological findings.Any new typing method should be comparedwith other systems to verify its accuracy andreliability. For this reason, phage typing wascompared with colicin typing and antibio-grams. These data appear in a companion pa-per (in preparation), but it can be said thatthere was usually excellent agreement betweenphage-typing results and epidemiological data.Rough and smooth forms of the same culture

greatly influenced the typing results. This hasoften been a problem in phage typing (3). "Vi-positive" and "Vi-negative" cultures of Salmo-nella typhi react differently to typing phages(3), as do different colony types ofPseudomonasaeruginosa (30). When the first S. sonnei iso-lates were phage typed, whole cultures wereused without regard to possible colony type var-iation; however, it became apparent that thiswould not work. Rough colony types producedthe best lysis; other investigators typing S. son-nei (18, 19) and still others doing colicin typing(29) have noticed this. The degree of lysis by

J. CLIN. MICROBIOL.

PHAGE-TYPING SCHEMA FOR S. SONNEI STRAINS

whole cultures depended on the proportion ofrough or smooth colonies found. Thus, the cul-ture must be streaked and rough colonies mustbe picked for typing. Most cultures, however,have reverted to the rough form by the timethey reach us, so whole cultures can be used ifspeed is essential to an epidemiological investi-gation. However, these results should be con-firmed with rough colonies.Medium was also an important variable,

since quite different results were obtained withTSA and phage agar. A final decision to useTSA was based on a number of practical consid-erations. A single lot number of this medium isnow used in all phage typing at the NationalCenter for Enteric Phage Typing at CDC. Thisis because quite different results were obtainedwith batches of TSA made from agar-agar withdifferent lot numbers. For these reasons, werecommend that others buy many bottles of thesame lot number and do all typing on thisstandardized medium. This would eliminatemedia as a variable. When the variables ofcolony types and media were eliminated, thetyping results became reproducible.

Like Slopek (28), we selected our provisionalset of phages from a large number that wereevaluated. By comparing the Slopek set withour new set, we showed that the latter is moresensitive; it was capable of dividing the largegroups formed with the Slopek set into smallergroups, thus revealing its greater sensitivity.Another factor that could limit the epidemio-

logical usefulness of phage typing is that 20% ofthe 265 isolates were of one bacteriophage type(Table 3). Laszlo and Kerekes (20) recom-mended that such groups be divided by colicintyping, Rishe (27) suggested that adding tem-perate phages to the phage set might break upthese large groups. Antibiograms may also byuseful in subdividing these groups; however, Rfactor acquistion by an isolate can easilychange the resistance pattern. Therefore, iso-lates with different antibiograms are not neces-sarily different strains.The new phage-typing system may prove

useful for typing other members of the generaShigella and Escherichia. With the addition ofother bacteriophages from collections or sew-age, strains of certain serotypes that predomi-nate in certain areas ofthe country can perhapsbe differentiated. Possibly, phages could be se-lected that are specific for that particular genusor species. For this reason, a reevaluation couldshow phages that might be used in the prophy-lactic treatment of shigellosis, such as thoseMulezyk and Slopek (24) used in Poland. Theseworkers have used phages that lyse most of the

S. sonnei strains in their country. With the aidof sodium bicarbonate to neutralize stomachacidity, a phage solution has been given orallywith good success. This approach is theoreti-cally possible in any country.Our results suggest that a standardized

phage typing system for S. sonnei can be estab-lished in the United States. Since this phage setis composed of the best phages from other colec-tions, a typing set with worldwide coverage canalso be established. Typing results of strainsthroughout the country indicate that regionalcenters could do surveillance. Such a systemhas been established at designated state labora-tories and at CDC for S. typhi. We hope that asimilar system for S. sonnei can be imple-mented.

ACKNOWLEDGMENTSThis research was performed as a part of the Laboratory

Practice Training Program, School of Public Health, Uni-versity of North Carolina, in cooperation with the Bureau ofLaboratories, Center for Disease Control, Atlanta, Ga., andwas supported by research grant CC 00606 from the Centerfor Disease Control.We thank S. Slopek, J. T. Parisi, and C. Ciufecu for their

bacteriophage sets, J. P. Zakanycz for his computer analy-sis, and Joy Wells and Lynn Matsen for colicin typing.

LITERATURE CITED

1. Abbott, J. D., and R. Shannon. 1958. A method oftyping Shigella sonnei using colicine production as amarker. J. Clin. Pathol. 11:71-77.

2. Adams, M. H. 1959. Bacteriophages. John Wiley andSons, Inc., New York.

3. Anderson, E. S., and R. E. 0. Williams. 1956. Bacterio-phage typing of enteric pathogens and staphlococciand its use in epidemiology. J. Clin. Pathol. 9:94-127.

4. Aoki, Y. 1868. Colicin type, biochemical type, and drug-resistance patterns of Shigella sonnei isolated in Ja-pan and its neighboring countries. Arch. Immunol.Ther. Exp. 16:303-313.

5. Baker, E. E., W. E. Goebel, and E. Periman. 1948. Thespecific antigens of variants of Shigella sonnei. J.Exp. Med. 89:325-338.

6. Braun, W., and A. E. Bonestell. 1947. Independentvariation of characteristics in Burcella abortus var-iants and their detection. Am. J. Vet. Res. 8:386-390.

7. Center for Disease Control. 1975. Shigella surveillancereport no. 36, 1st and 2nd quarters. Center for Dis-ease Control, Atlanta.

8. Cruickshank, R. 1960. Laboratory uses of antibiotics.Br. Med. Bull. 16:79-81.

9. Edwards, P. R., and W. H. Ewing. 1972. Identificationof Enterobacteriaceae, 3rd ed. Burgess PublishingCo., Minneapolis.

10. Eichner, E. R., E. J. Gangarosa, and J. B. Goldsby.1968. The current status of shigellosis in the UnitedStates. Am. J. Public Health 58:753-763.

11. Elek, S. D., J. R. Davies, and R. Miles. 1973. Resisto-typing of Shigella sonnei. J. Med. Microbiol. 6:329-345.

12. Farmer, J. J., III. 1970. Mnemonic for reporting bacte-riocin and bacteriophage types. Lancet ii:96.

13. Farmer, J. J., III. 1972. Epidemiological differentiationof Serratia marcescens: typing by bacteriocin produc-tion. Appl. Microbiol. 23:218-225.

14. Farrant, W. H., and A. J. H. Tomlinson. 1966. Some

73VOL. 5, 1977

74 PRUNEDA AND FARMER

studies on the epidemiology of Sonne dysentery.Changes in colicin type and antibiotic resistance be-tween 1956-1965. J. Hyg. 64:287-303.

15. Gangarosa, E. J., and H. L. DuPont. 1973. Bacillarydysentery, p. 203-204. In H. F. Conn and R. B. Conn.,Jr., (ed.), Current diagnosis. W. B. Saunders Pub-lishing Co., Philadelphia.

16. Gillies, R. R. 1964. Colicine production as an epidemio-. logical marker of Shigella sonnei. J. Hyg. 62:1-9.

17. Gillies, R. R. 1965. Fermentation studies on Shigellasonnei. J. Pathol. Bacteriol. 90:345-348.

18. Gromkova, R., and A. Trifonova. 1967. Attempts atphage typing ofShigella sonnei. Zentralbl. Bakteriol.Parasitenkd. Infektionskr. 204:212-215.

19. Hammarstrom, E. 1949. Phage typing of Shigella son-nei. Acta Med. Scand. 133(Suppl. 223):1-132.

20. Laszlo, V. G., and L. Kerekes. 1969. Phage and colicintyping ofShigella sonnei. Acta. Microbiol. Acad. Sci.Hung. 16:309-317.

21. Ludford, C. G. 1953. Bacteriophage types of Shigellasonnei in Queensland, Austral. J. Exp. Biol. 31:545-552.

22. Mayr-Harting, A. 1952. The phage typing of Shigellasonnei, and the limits oftype stability. J. Gen. Micro-biol. 7:382-396.

J. CLIN. MICROBIOL.

23. Morris, G. K., and J. G. Wells. 1974. Colicin typing ofShigella sonnei. Appl. Microbiol. 27:312-316.

24. Mulezyk, M., and S. S!opek. 1973. Use of a new phagepreparation in prophylaxis and treatment of shigel-losis. Acta. Microbiol. Acad. Sci. Hung. 21:115-119.

25. Parisi, J. T., J. C. Russel, and R. J. Merlo. 1969. Bacte-riophage typing as an epidemiological tool for urinaryEscherichia coli. Appl. Microbiol. 17:721-725.

26. Reller, L. B. 1971. Colicin typing as an epidemiologictool in investigations of outbreaks ofShigella sonnei.Appl. Microbiol. 21:21-26.

27. Rishe, H. 1968. Phage typing ofShigella sonnei accord-ing to Hammarstrom's current experiences and com-pletion of the method by temperature phages. Arch.Immuno. Ther. Exp. (Warsz) 16:392-401.

28. Slopek, S. I., Dublakowa, A. Kucharewiczkrub-kowska, and T. Krzywy. 1973. Phage typing of Shi-gella sonnei. Arch. Immunol. Ther. Exp. (Warsz)21(l):1-161.

29. Szturm-Rubinstein, S. 1968. Determination of biotype,phage type, and colicinogen character ofShigella son-nei, and its epidemiologic importance. Arch. Immu-nol. Ther. Exp. 16:421-428.

30. Zierdt, C. H., and P. J. Schmidt. 1964. Dissociation inPseudomonas aeruginosa. J. Bacteriol. 87:1003-1010.