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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;
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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.
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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.
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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#h
L.:.. _ v i. _ ....Z....,
FIG. 4. Lysis patterns of a smooth colony (left) and a rough
colony of the same strain (right).
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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).
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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.
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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
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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.
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