Embed Size (px)
Citation preview
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2817-28230095-1137/91/122817-07$02.00/0Copyright © 1991, American Society for Microbiology
Vol. 29, No. 12
Phage Typing of Salmonella enteritidis in the United StatesF. W. HICKMAN-BRENNER,l* A. D. STUBBS,2 AND J. J. FARMER I1l'
Enteric Bacteriology Section, Enteric Diseases Branch, Division of Bacterial and Mycotic Diseases,National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333,1 and
Southeast Regional Laboratory, Food and Drug Administration, Atlanta, Georgia 303092
Received 17 May 1991/Accepted 18 September 1991
The number of reported isolates of Salmonella enteritidis has increased dramatically in the last 10 years. Formany years phage typing has been a useful epidemiologic tool for studying outbreaks of S. typhi and S.typhimurium. In 1987, Ward et al. (L. R. Ward, J. De Sa, and B. Rowe, Epidemiol. Infect. 99:291-294, 1987)described a phage typing scheme for S. enteritidis. This system differentiated 27 phage types by use of 10 typingphages. With these phages, we typed 573 strains of S. enteritidis from humans (42 outbreaks), animals, food,and the environment. Ninety-six percent of the strains were typeable. The most common phage types were 8(48.2%), 13a (20.1%), 13 (7.8%), and 14b (7.8%). Most of the strains were specifically collected fromegg-related outbreaks in the northeastern United States in 1988 and 1989, probably accounting for thedistribution of the four most common types in this sample. This system was particularly useful fordifferentiating a group of animal strains that had a number of diverse phage types. For 49 animal strains typed,16 different patterns were obtained. Phage type 8 represented 32% of these strains, but no other phage typerepresented more than 8% of these strains. One-half of the 16 animal strains that were phage type 8 were frompoultry. This phage typing system will be useful for comparing phage types found in the United States withthose types encountered worldwide and for determining whether virulent strains of phage type 4 are enteringthe United States. Additional phage typing systems as well as molecular techniques are being studied todetermine whether they can differentiate strains of phage types 8 and 13a.
The rate of isolation of Salmonella serotype Enteritidishas been increasing dramatically in the last 10 years (partic-ularly in the northeastern United States), and this serotypehas been the second most common one reported in thatcountry (S. typhimurium has been the most common one). In1989, 8,340 isolates of S. enteritidis were reported (4).
Detailed information concerning reported outbreaks of S.enteritidis was collected at the Centers for Disease Controlfrom January 1985 through May 1987. For 65 outbreaksreported, a food vehicle was established for 35. For 27 of the35, or 77%, the food vehicle was grade A shell eggs. Eggswere traced to 13 different farms or distributors in sevenstates. In nursing homes, 10 of 130 infected persons died, afatality rate of 8%, compared with an overall fatality rate of0.5% (18). Since phage typing has been a useful laboratorytool for investigating outbreaks of S. typhi and S. typhimu-rium, the phage typing scheme for S. enteritidis, described in1987 by Ward et al. (21), was obtained from the WorldHealth Organization (WHO) International Center for EntericPhage Typing, London, United Kingdom. Since the increasein S. enteritidis appears to be worldwide (17), we alsowanted to compare the phage types found internationallywith those found in the United States. This system was usedto type 573 strains of S. enteritidis isolated in the UnitedStates. The data obtained and our experience with thissystem are presented here. This system is also being used atthe U.S. Department of Agriculture (USDA), the Food andDrug Administration, and many national centers throughoutthe world. In the future there will be reports on the nationaland worldwide distributions of phage types. We hope thatour experience with this phage typing system will benefitthose reading such reports.
* Corresponding author.
MATERIALS AND METHODS
Media. The media used for phage typing have been previ-ously described (2, 7, 12). Modified phage agar contained1.3% agar instead of the 2% previously used. Blood agarconsisting of Trypticase soy agar and 5% sheep blood (BBLMicrobiology Systems, Cockeysville, Md.) was used as theplating medium. Isolates were stored on semisolid Trypti-case soy agar containing 15 g of Trypticase peptone (BBL),5 g of Phytone peptone, 5 g of sodium chloride, 4 g of agar,and 1,000 ml of distilled water.
Processing of diagnostic strains. Isolates of S. enteritidiswith accompanying epidemiologic data were sent by statehealth departments and other laboratories for phage typing.Isolated colonies were tested. Incubation was done at 361°C unless otherwise noted. All stocks were stored at roomtemperature. For this study we made no distinction betweenthe terms "isolate" and "strain."
Preparation of phage agar plates for phage typing. Plateswere prepared as previously described (7, 12). Modifiedphage agar plates (15 by 100 mm) contained 30 ml ofmedium.
Preparation of broth cultures. Strains to be phage typedwere inoculated from 24-h cultures on blood agar into 3 ml ofphage broth (2) in screw-cap tubes (16 by 125 mm) to yield a
heavy suspension (turbidity equal to a no. 1 MacFarlandstandard [11]). The cultures were grown for 2 h on a rollerdrum (20 rpm) located in a walk-in incubator. After 2 h, thebroth was very turbid (equal to a no. 5 or 6 MacFarlandstandard).
Preparation of lawns. Each broth, prepared as describedabove, was poured directly onto a dry modified phage agarplate. After the excess fluid was removed, the plates were
ready for phage application.Application of phages. A phage applicator was used to
deliver the 10 phages in the system (see Table 1) to the lawns
2817
on June 16, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
2818 HICKMAN-BRENNER ET AL.
TABLE 1. Titration of the S. enteritidis phages of Ward et al.(21) with the phage type 1 strain as the host
Result at dilution: PFU/0.1-mlPhage RTD drop in the
10-1 1V-2 1O-3 10-4 io-0 final RTD
1 CL CL SOL iooa _b 2 x 10-3 2,0002 OL SCL 250 25 - 10-2 2,5003 OL OL SOL 50 5 10-2 5,0004 OL OL 200 20 - 2 x 10-2 4,0005 OL OL SOL <SCL 25 10-3 2,5006 SOL 4+ 150 15 - 2 x 10-2 3,0007 OL SOL 25 3 - 3X 10-3 758 OL SOL 80 8 - 2 x 10-2 1,6009 OL OL 75 75 - 2 x 10-2 1,50010 OL SOL 200 20 - 10-2 2,000
a Number of individual plaques.b not done.
prepared as described above (7, 12). The plates were incu-bated overnight.Reading of the plates. Areas of lysis were observed with
oblique lighting. The various degrees of lysis were recordedas follows: -, no lysis; +, 6 to 20 plaques; +, 21 to 40plaques; 2+, 41 to 80 plaques; 3+, 81 to 100 plaques; 4+,>100 distinct plaques, too numerous to count, but notconfluent; SCL, semiconfluent lysis; SOL, semiopaque ly-sis; CL, confluent lysis; OL, opaque lysis; and <SCL and<CL, intermediate degrees of lysis.
Interpretation of results. The phage lysis pattern of eachculture was compared with those determined for the typestrains in our laboratory and with the published patterns (seeTable 2). Strains that were lysed by a number of the phagesbut had phage lysis patterns that did not conform exactly toany of the phage lysis patterns of the known type strainswere designated RDNC, for "reacted, but did not conform(21)." Strains that were reported as RDNC representedmany different phage lysis patterns.
S. enteritidis phage typing system of Ward et al. (21). The 10phages that were described by Ward et al. (21) were obtainedfrom the WHO International Center for Enteric Phage Typ-ing. They were sent unrefrigerated at 100 times the routinetest dilution (100x RTD). Of the 10 phages in the system, 4were lysogenic phages, 5 were adapted phages, and 1 wasisolated from sewage (21).Phage titrations. The phages were titrated on the phage
type 1 strain, which is lysed by all the phages, as recom-mended by the WHO International Center for Enteric PhageTyping, although all the phages were not propagated on thisstrain. All phages were titrated by applying 10-fold dilutions(made in phage broth) to lawns of the phage type 1 strain onmodified phage agar. The lawns were prepared in the same
manner as that used for phage typing. The RTD for eachphage has been defined by the WHO International Center forEnteric Phage Typing as the highest dilution yielding con-
fluent lysis or semiconfluent lysis of the host strain (2). Table1 also gives an operational definition (PFU/0.01-ml drop) foreach RTD.Phage type strains of S. enteritidis. The S. enteritidis type
strains for phage types 1 to 25 are listed in Table 2.Additional phage types have been identified with this sys-
tem, but they are not listed in Table 2 because they have notbeen published. The reactions for these new phage types are
given in a chart provided with the typing system. There are
now at least 44 phage types that can be differentiated with
the 10 original phages. Among the new types are 13a and14b, two of the most common types in our sample (Table 3).Fermentation of melibiose. Salmonella strains typically
ferment melibiose; however, it was observed that many ofthe S. enteritidis strains were melibiose negative at 7 days.For this reason, we routinely included the melibiose fermen-tation test to determine whether it could be used as anepidemiologic marker. The test was done with enteric fer-mentation medium containing 0.5% melibiose (autoclavedwith the basal medium) and was read at 1, 2, 3 to 5, and 7days (6).
S. enteritidis strains that were phage typed. First, a group of73 egg-related strains chosen for a pilot study was typed.These strains had been phage typed by the WHO Interna-tional Center for Enteric Phage Typing, and plasmid profileshad been obtained.
Second, a group of 49 animal strains sent by the USDA,Ames, Iowa, was typed (Table 4). This was a diverse groupof strains from many different animals, including poultry(chicken, turkey, and duck), cattle, swine, sheep, horses,dogs, cats, a rodent, a mink, a monkey, and other zooanimals.The largest group of strains (approximately 250) that was
typed was from 42 documented outbreaks that occurred inthe United States in 1988 and 1989. These outbreaks, theirsources, and the numbers of strains typed are listed in Table5. For outbreaks involving large numbers of strains isolatedfrom patients, a representative number from each outbreakwas typed.
Various miscellaneous strains were also phage typed.These included 19 strains selected because they had adistinct or unique plasmid profile, strains from farms in thenortheastern United States, and collections of other, well-characterized strains typed at the request of various re-searchers.
Strains that did not fit a defined pattern or had patterns notseen previously at CDC were first confirmed serologicallyand then sent to the WHO International Center for EntericPhage Typing.
RESULTS AND DISCUSSION
Setting up the phage typing system. (i). Preparation ofRTDs. Table 1 lists the RTDs used for each of the phages andgives the number of PFU present in each 0.01-ml drop.Although the phages were sent at 100x RTD, it was notalways possible to use a 1:100 dilution because the phageswere sent unrefrigerated and the titers of some of the phageshad dropped. In addition, the conditions used at the WHOInternational Center for Enteric Phage Typing could not beduplicated, although that organization's procedures werefollowed as closely as possible. With some phages we wereable to use a more dilute suspension, probably also becauseof differences in conditions, such as variations in the lots ofagar or broth. Phage 7 had very large plaques; therefore,there was a smaller number of PFU per drop in its RTD.
(ii) Phage typing of type strains. The lysis patterns obtainedin our laboratory are given in Table 2 along with the lysispatterns published by the WHO International Center forEnteric Phage Typing. Five types, 7a, 9a, 9b, 13a, and 14b,were added to the scheme after it was published by Ward etal. (21). The phage lysis patterns of the type strains of 9a and19, as described by Ward et al. (21), could not be duplicatedin our laboratory (Table 2); however, these two phage typesare rarely encountered. There were other slight differencesin a few of the patterns that we obtained for the type strains
J. CLIN. MICROBIOL.
on June 16, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
PHAGE TYPING OF S. ENTERITIDIS FROM THE UNITED STATES 2819
TABLE 2. Reactions of the S. enteritidis type strains with the typing phages and comparison with published reactions (21)
p'hage Source oftype results
1 U.SU.K.
2 U.S.U.K.
3 U.S.U.K.
4 U.S.U.K.
4a U.S.U.K.
5 U.S.U.K.
6 U.S.U.K.
6a U.S.U.K.
7 U.S.U.K.
8 U.S.U.K.
9 U.S.U.K.
10 U.S.U.K.
11 U.S.U.K.
12 U.S.U.K.
13 U.S.U.K.
14 U.S.U.K.
15 U.S.U.K.
16 U.S.U.K.
17 U.S.U.K.
18 U.S.U.K.
19 U.S.U.K.
20 U.S.U.K.
21 U.S.U.K.
Reaction for phage:
1 2 3 4 5 6 7 8 9 10
OL SOL OL SOL OL SCL <OL SOL OL OLOL SCL CL OL OL SCL CL SCL OL OL
CLOL
CLOL
- CL <SOL CL SCL <SOL- CL OL CL SCL SCL
2+
- SCL- SCL
- CL- SCL
- <SCL- SCL
- <SOL- OL
- SCL- SCL
- <CL- SCL
_ 4+- OL
SCL OLCL OL
CL OLCL OL
2+ <SOL1+ OL
- SOL- OL
- SCL- SCL
- OL- OL
SOL <SOLSCL OL
CL -OL -
- SCL- OL
CL -2+ -
<SOL SOL1+ OL
- 4+- OL
3+SCL
_ 4+- SCL
SOL SOL3+ OL
- <SCL- SCL
OLOL
OLOL
<SOL1+
OLSCL
2+
CLSCL
SCLSCL
OLOL
2+
OLOL
CL <SCL <SCLCL SCL CL
CL 2+ CLCL SCL CL
3+ <SCL 1+CL SCL 1+
- SOL -- OL -
- SCL -- SCL -
+ _
<SCL <SCL 3+CL SCL SCL
CL - SCLOL - OL
3+ SCL +2+ SCL -
CLCL
<SOL3+
+f-
- <SOL- SCL
- <SCL -- SCL -
- <SCL 3+- SCL 1+
<SOL <SCL -SCL SCL -
2+- SCL -
SCL - SOL3+ - SCL
OL <SCL <OLCL SCL CL
- 2+ -
2+ SCL 1+
CL - 1+CL - 1+
<SCL -
OL -
CLOL
CLOL
OL OLOL OL
- CL- OL
OL <SOLSCL OL
- 3+- OL
4+ OL2+ OL
OL OLOL OL
- OL- OL
- OL- OL
<OL OLSCL OL
- OL- OL
OL -SCL -
- OL- OL
_ Qn]T
<SOLOL
- or 2+
<SOL3+
OLOL
OLOL
OLCL
- OL -
OL - OLSCL OL OL
4+ - 4+SCL - 3+
<OL - OLSCL - OL
SOL SOL OL1+ OL 3+
OL 2+ SCLSCL - OL
<SOL - SOLOL 1+ OL
OL - OLCL - OL
OL OL <OLSCL OL OL
Continued on following page
VOL. 29, 1991
on June 16, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
2820 HICKMAN-BRENNER ET AL.
TABLE 2-Continued
Pliage Source of Reaction for phage:type results 1 2 3 4 5 6 7 8 9 10
22 U.S. <OL - - SOL - SCL - OL OL OLU. K. OL - - SCL - OL - OL OL OL
23 U. S. - - - SOL - 1 + 2+ - OL -U.K. - - - SOL - - - - OL -
24 U.S. - - - - - - - - OL -
U.K. - - - - - - - - OL -
25 U.S. - - - - - - - + - OLU.K. - - - - - - - 1+ - OL
U.S., results were obtained in our laboratory; U.K., published results.
(Table 2). Overall, however, our results correlated well withthe published results. Phage types 7 and 23 are sometimesrough; therefore, it is important to pick smooth colonies.Phage types 24 (Table 2) and 28 (not on the published list, buton a chart provided for those using this system) are some-times drug resistant, a property which could also help withdifferentiation. Our definition of phage type 28 had to bechanged because, although the lysis pattern was recogniz-able, it was unlike the lysis pattern given for phage type 28on the chart provided. These differences could also havebeen due to slightly different conditions.Four new phages have now been added to the 10 original
phages that were included in this system. With the additionof these new phages, at least four new phage types have beendescribed. One of these is phage type 29, which is not on thepublished chart and which requires the four additionalphages for recognition. One of our outbreaks involved
TABLE 3. Phage types of 573 S. enteritidis strains isolatedin the United States
Phagetype ~~~~~~~~~~No.(%) ofPliage type strains
i....................................... 1 (0.2)2....................................... 8 (1.4)3 (Variant)' .......... ............................ 1(0.2)4...................................... 5 (0.9)4 (Variant)' .......... ............................ 3 (0.5)7a ...................................... 1 (0.2)8...................................... 276 (48.2)9...................................... 1 (0.2)9a ...................................... 3 (0.5)9b................... 6 (1.0)13 ...................................... 45 (7.8)13a...................................... 115 (20.1)14b...................................... 45 (7.8)23 ....................................... 5 (0.9)23 (Variant) ...................................... 1 (0.2)24 ....................................... 3 (0.5)28 ...................................... 4 (0.7)29b .............. ...................................... 6 (1.0)34 ................................................................ 3 (0.5)RDNCc ...................................... 20 (3.5)Untypeable ...................................... 21 (3.7)
strains of phage type 29 (Table 5). These strains wereidentified as phage type 29 by the WHO International Centerfor Enteric Phage Typing because we did not have the fournew phages (these strains were called RDNC in our labora-tory). These strains were only lysed by phage 5 (of the 10original phages), with a + reaction. These strains were alsounusual because of their antigenic formula. The antigenicformula for S. enteritidis is normally 9,12:g,m:-, but theformula for these outbreak strains was 9,12:g,m,p:-. Thepresence of H factor p is rare in S. enteritidis, but it doesoccur.
Distribution of phage types in the United States. Table 3lists the phage types of the 573 strains tested. Ninety-sixpercent of the strains were typeable. There were nineteenphage types in the total sample, and there were also strainsthat were reported as RDNC, which represents many pat-terns.
Forty-eight percent of the strains were phage type 8.Sixty-eight percent of the strains were either phage type 8 orphage type 13a. These results may not give a true picture ofthe distribution of the phage types in the United States fortwo reasons. First, we concentrated on strains that werefrom the northeastern United States and that were associ-ated with eggs because this area had the most dramaticincrease in S. enteritidis. Studies are currently being done to
TABLE 4. Phage types of 49 animal strains of S. enteritidis
No. (%) ofPliage type strains
2....... 1 (2)3 (Variant)....... 1(2)4....... 1 (2)4 (Variant)....... 3 (6)8....... 16" (32)9....... 1 (2)9b....... 4 (8)13....... 2 (4)13a....... 4 (8)23....... 1 (2)23 (Variant)....... 1(2)24....... 3 (6)28....... 3 (6)34....... 1 (2)RDNC....... 5 (10)Untypeable....... 2 (4)
' 8 from poultry, 3 from swine, 2 from cattle, 1 from sheep, 1 from a giraffe,and 1 from feed.
" These animal strains were from the USDA. Variants have atypicalreactions (see Table 4).
b These isolates were typed by the WHO International Center for EntericPhage Typing (see the text).
' These 20 strains had many lysis patterns (see the text).
J. CLIN. MICROBIOL.
on June 16, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
PHAGE TYPING OF S. ENTERITIDIS FROM THE UNITED STATES 2821
TABLE 5. Sources of 42 outbreaks studied in 1988 and 1989and numbers of strains tested
No. of outbreaks (no. of strains)Phage type
Egg related Unknown Other
8 24 (163) 1 (8), Tuna13a 3 (7) 2 (13) 2 (9), Chicken salad and
sandwich bar13 2 (20) 2 (14), Chicken14b 1 (3)2 1 (3), Fish4 1 (3)29 1 (4), Turkey34 1 (1)Untypeable 1 (3)
survey phage types in other parts of the United States.Second, all strains that were tested from a particular out-break were included in our tabulation; if we had onlyincluded one strain per outbreak, the percentages may havebeen different.The group of 73 strains chosen for the pilot study was
typed first, since these strains had already been typed at theWHO International Center for Enteric Phage Typing. Weobtained the same results as that organization, except for aphage type 9a strain that we could not recognize and thatwas not on the published chart and a phage type 23 strainthat had a pattern that was somewhat different from thepattern obtained in our laboratory. Of the 73 strains typed,35 were type 13a, 28 were type 8, 4 were type 14b, and 1 eachwas type 4, 9a, 23, and 28, RDNC, and untypeable.The second set of strains typed was the group of USDA
strains from animals. The phage types of these strains werediverse. Table 4 gives the distribution of phage types in theseanimal strains. Sixteen different patterns were obtained forthe 49 strains. Sixteen of these strains were phage type 8,and 8 of these 16 were from poultry. Variants (strains thathad atypical reactions for that phage type) of types 3, 4, and23 (Table 4) were found in this group of animal strains. Thephage typing results obtained with these animal strains mayprovide a more accurate reflection of the value of the phagetyping system used and of phage typing as a technique ingeneral because, unlike the egg-related strains, the animalstrains were more diverse. In addition, other than phage type8, which represented 32% of the animal strains, no otherphage type represented more than 8% of these strains(RNDC represented many of the strains, as stated above).
After typing this diverse group of animal strains as well asthe strains from the pilot study, we typed the outbreakstrains. Forty-two outbreaks were studied (Table 5). Thestrains from 25 outbreaks were phage type 8; 24 of these(involving 163 of the 250 strains tested) were associated witheggs. The outbreak involving phage type 2 occurred inPuerto Rico, and the one involving phage type 4 occurred ona European ship that had docked in Mobile Bay. Phage type14b was associated with only one outbreak, although itrepresented 7.8% of the total number of strains typed. Mostof these strains were from farms in the northeastern UnitedStates.Twenty-one of the outbreaks occurred in the northeast, 11
occurred in the south (including 5 in Maryland), 4 occurredin the District of Columbia, 3 occurred in the west or
northwest, 2 occurred in the midwest, and 1 occurred inPuerto Rico. Of the 24 egg-related outbreaks involving phage
type 8, 14 were in the northeast, 3 were in Maryland, 3 werein Virginia, 2 were in Illinois, 1 was in the District ofColumbia, and 1 was in Tennessee.
S. enteritidis is an important cause of infections in theUnited Kingdom, and the number of isolates has risendramatically there also. Phage type 4 is the most commontype; over one-half (54%) of the more than 16,000 strainstyped in the United Kingdom between 1981 and 1986 weretype 4 (21). Phage type 8 was the second most common(approximately 30%). Since strains of phage type 4 report-edly have increased virulence in poultry (17) and entireflocks infected with strains of this phage type have had to bedestroyed in the United Kingdom, S. enteritidis strains(particularly from animals) are being monitored to determinewhether strains of this phage type are entering the UnitedStates. The only U.S. outbreak involving phage type 4strains (Table 5) was associated with Europe, as were theother two phage type 4 strains (Table 3). The strain of phagetype 4 from the USDA was isolated from a chicken fromSpain. The USDA has also obtained this phage typingsystem and is monitoring animal strains.
Stability of phage types. Some of the phage types appearedto be more stable than others. Strains that acquire a plasmidor a phage can change phage types. Frost et al. (8) reportedthat the acquisition of a resistance plasmid resulted in theconversion of a phage type 4 strain to phage type 24. Weencountered two strains that appeared to have reverted tophage type 8. A strain in the pilot study that was found to bephage type 23 (culture number B8506) when typed by theWHO International Center for Enteric Phage Typing hadreverted to phage type 8 when we typed it. A second strainwas originally found to be phage type 14b (B8400), but whenit was streaked and four colonies were typed, two werefound to be phage type 14b and two were found to be phagetype 8. Chart et al. (5) reported a conversion of a phage type4 strain to phage type 7; this conversion involved a loss oflipopolysaccharide and an accompanying loss of virulence.
Fermentation of melibiose. Some correlation was seenbetween phage type and melibiose fermentation. Ninety-eight percent of the phage type 8 strains and 93% of thephage type 13 strains were melibiose positive in 1 to 2 days,but only 38% of the phage type 14b strains and 30% of thephage type 13a strains were melibiose positive. There was nofirm correlation, however, within specific outbreaks (melibi-ose-positive and -negative strains of the same phage typeoccurred within one outbreak). In addition, in two instanceswhen five tubes with the same inoculum source were tested,one or two of the five tubes were positive in 3 days, while theothers were negative. Melibiose-negative strains were appar-ently reverting to the wild type (melibiose positive).
Conclusions. The S. enteritidis phage typing system ofWard et al. (21) was successfully used to type 573 strains. Itwas less tedious than the S. typhi and S. typhimuriumsystems because only small plates and 10 phages were usedand the cultures did not have to be diluted for routine typing.An operational definition for the RTDs based on PFU perdrop was made to standardize our use of these phages. TheWHO International Center for Enteric Phage Typing hasmade the decision to send each phage at 1,OOOx RTD in thefuture so that laboratories will have to be resupplied lessoften.The phage lysis patterns obtained were reproducible. The
fact that a few strains appeared to revert`to more stablephage types could pose a problem. Further studies are
needed to assess this problem.It may be advantageous to add more phages to this system
VOL. 29, 1991
on June 16, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
2822 HICKMAN-BRENNER ET AL.
to subdivide phage type 8 strains as well as strains of theother common types. Three other phage typing systems arecurrently being evaluated. These are a U.S. system de-scribed by Gershman (9, 10), a Hungarian system describedby Laszlo et al. (14), and a system provided by FrancineGrimont at the Pasteur Institute, Paris, France (20). Thesestudies will be addressed in a subsequent publication.At first glance, this typing system may appear to be of
limited value because, although 96% of the strains weretypeable, 68% fell into two types (types 8 and 13a) and 84%fell into four types. As stated previously, however, thisdistribution may have been due to our concentration onnortheastern U.S. strains associated with eggs and ourinclusion of all of the strains tested in each outbreak in ourtabulations. Molecular methods and additional phages arecurrently being used in our laboratory to subdivide the phagetype 8 strains. Preliminary results show that the phage type8 strains associated with eggs appear to be a clone (15, 19)that has spread throughout the northeastern United Statesand is spreading to Maryland, Virginia, and other adjoiningstates. These studies will be addressed in a subsequentpublication.Phage typing is still the method of choice for subdividing
Salmonella strains in most countries. The phage typingsystem that was evaluated in this study offers an opportunityto compare results from all over the world. The WHOInternational Center for Enteric Phage Typing providesphages to national centers; therefore, no personnel time isneeded for the propagation of phages and standardization isensured. Phage typing systems have the advantage of pro-viding results easily and rapidly (within 24 h) once thesystems are set up. A minimum of personnel time is re-quired, as 60 to 75 strains can easily by typed in 1 day, incontrast to time-consuming molecular techniques. Phagetyping of S. enteritidis strains is a service offered by theCenters for Disease Control to state health laboratories andothers who request it. It is also used by USDA for animalisolates and by the Food and Drug Administration for foodisolates. Many countries now have national phage typingcenters and have obtained these phages.
Other subtyping methods, such as ribotyping (polymor-phisms in genes containing ribosomal operons), plasmidprofile analysis, and multilocus enzyme electrophoresis,have been used for subdividing Salmonella serotypes andphage types (1, 3, 13, 15, 16). These methods are verylabor-intensive and have not proven to be more sensitivethan phage typing. With the exception of plasmid profileanalysis, these molecular methods have no internationalstandards at this time to provide an opportunity to compareresults obtained in different laboratories or other countries.They may be useful for laboratories that do not have accessto the phages and that are set up to do these proceduresroutinely. Because they are so labor-intensive, it would beimpossible for reference laboratories to use them for differ-entiating thousands of animal and human isolates of S.enteritidis. Such differentiation seems practical only withphage typing.
ACKNOWLEDGMENTS
We thank Linda Ward of the WHO International Center forEnteric Phage Typing for supplying the phages and type strains andfor many helpful discussions. We also thank those in the WHOInternational Center who typed many of our strains.
ADDENDUM
Since this manuscript was prepared, we phage typed asmall number of strains from the western United States andfound that phage type 4 was present. We also found phagetype 4 in a small sample from Mexico. The introduction ofphage type 4 in the United States may have serious implica-tions and will be covered in a subsequent publication.
REFERENCES
1. Altwegg, M., F. W. Hickman-Brenner, and J. J. Farmer III.1989. Ribosomal RNA gene restriction patterns provide in-creased sensitivity for typing Salmonella typhi strains. J. Infect.Dis. 160:145-149.
2. Anderson, E. S., and R. E. O. Williams. 1956. Bacteriophagetyping of enteric pathogens and staphylococci and its use inepidemiology. J. Clin. Pathol. 9:94-127.
3. Beltran, P., J. M. Musser, R. Helmuth, J. J. Farmer III, W. R.Frerichs, I. K. Wachsmuth, K. Ferris, A. C. McWhorter, J. G.Wells, A. Cravioto, and R. K. Selander. 1988. Toward a popu-lation genetic analysis of Salmonella: genetic diversity andrelationships among strains of serotypes S. choleraesuis, S.derby, S. dublin, S. enteritidis, S. heidelberg, S. infantis, S.newport, and S. typhimurium. Proc. Natl. Acad. Sci. USA85:7753-7757.
4. Centers for Disease Control. 1990. Salmonella surveillance re-port, 1989. Centers for Disease Control, Atlanta.
5. Chart, H., B. Rowe, E. J. Threlfall, and L. R. Ward. 1989.Conversion of Salmonella enteritidis phage type 4 to phage type7 involves loss of lipopolysaccharide with concomitant loss ofvirulence. FEMS Microbiol. Lett. 60:37-40.
6. Edwards, P. R., and W. H. Ewing. 1972. Identification ofEnterobacteriaceae, 3rd ed., p. 337-354. Burgess PublishingCo., Minneapolis.
7. Farmer, J. J., III, F. W. Hickman, and J. V. Sikes. 1975.Automation of Salmonella typhi phage typing. Lancet ii:787-790.
8. Frost, J. A., L. R. Ward, and B. Rowe. 1989. Acquisition of adrug resistance plasmid converts Salmonella enteritidis phagetype 4 to phage type 24. Epidemiol. Infect. 103:243-248.
9. Gershman, M. 1976. Phage typing system for Salmonella enter-itidis. Apple. Environ. Microbiol. 32:190-191.
10. Gershman, M. 1977. Single phage-typing set for differentiatingsalmonella. J. Clin. Microbiol. 5:302-314.
11. Hendrickson, D. A. 1985. Reagents and stains, p. 1093-1107. InE. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J.Shadomy (ed.), Manual of clinical microbiology, 4th ed. Amer-ican Society for Microbiology, Washington, D.C.
12. Hickman, F. W., and J. J. Farmer III. 1978. Salmonella typhi:identification, antibiograms, serology, and bacteriophage typ-ing. Am. J. Med. Technol. 44:1149-1159.
13. Holmberg, S. D., I. K. Wachsmuth, F. W. Hickman-Brenner,and M. L. Cohen. 1984. Comparison of plasmid profile analysis,phage typing, and antimicrobial susceptibility testing in charac-terizing Salmonella typhimurium isolates from outbreaks. J.Clin. Microbiol. 19:100-104.
14. Laszlo, V. G., E. S. Csorian, and J. Paszti. 1985. Phage typesand epidemiologic significance of Salmonella enteritidis strainsin Hungary between 1976 and 1983. Acta Microbiol. Hung.32:321-340.
15. Morris, J. G., Jr., D. M. Dwyer, C. W. Hoge, A. D. Stubbs, D.Tilghman, C. Groves, E. Israel, and J. P. Libonati. Submitted forpublication.
16. Reeves, M. W., G. M. Evins, A. A. Heiba, B. D. Plikaytis, andJ. J. Farmer III. 1989. Clonal nature of Salmonella typhi and itsgenetic relatedness to other salmonella as shown by multilocusenzyme electrophoresis, and proposal of Salmonella bongoricomb. nov. J. Clin. Microbiol. 27:313-320.
17. Rodrigue, D. C., R. V. Tauxe, and B. Rowe. 1990. Internationalincrease in Salmonella enteritidis: a new pandemic? Epidemiol.Infect. 105:21-27.
J. CLIN. MICROBIOL.
on June 16, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
PHAGE TYPING OF S. ENTERITIDIS FROM THE UNITED STATES 2823
18. St. Louis, M. E., D. L. Morse, M. E. Potter, T. M. DeMelfi, J. J.Guzewich, R. V. Tauxe, P. A. Blake, and the Salmonella enteri-tidis Working Group. 1988. The emergence of grade A eggs as amajor source of Salmonella enteritidis infections: new implica-tions for the control of salmonellosis. J. Am. Med. Assoc.259:321-340.
19. Stubbs, A. D., F. W. Hickman-Brenner, and J. J. Farmer III.1990. Subdivision of S. enteritidis phage type 8 by plasmidprofiles and additional typing phages, abstr. C-355, p. 403.
Abstr. 90th Annu. Meet. Am. Soc. Microbiol. 1990. AmericanSociety for Microbiology, Washington, D.C.
20. Vieu, J. F., S. Jeanjean, B. Tournier, and B. Klein. 1990.Application d'une serie unique de bacteriophages a la lysotypiede Salmonella serovar Dublin et de Salmonella serovar Enter-itidis. Med. Mal. Infect. 20:1-6.
21. Ward, L. R., J. De Sa, and B. Rowe. 1987. A phage-typingscheme for Salmonella enteritidis. Epidemiol. Infect. 99:291-294.
VOL. 29, 1991
on June 16, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
