INFECTIONS WITH MULTIPLE TYPES OF SALMONELLAE
ARNOLD P. JUENKER, B.S.
Salmonella Typing Station, Division of Laboratories, Michigan Department
of Health, Lansing, Michigan
From our experience5 and that of others it is known that man and animals are
occasionally infected with more than 1 type of Salmonella organism. Hormaeche
and his associates4 (in a 6-year study) found 32 instances of associated infections:
with 2 types, 28; with 3 types, 2; with 4 types, 1; and with 5 types, 1. Greifinger
and Silberstein3 published an account of an outbreak of gastroenteritis in military
personnel in which 3 types of Salmonellae were involved. S. oranienburg and S.
typhimurium were isolated from 16 patients; S. oranienburg and S. anatum were
isolated simultaneously from 33; all 3 types were isolated from 6 patients.
Weiner and Liebler6 studied a small outbreak in a Veterans Administration
hospital in which 7 types of Salmonellae were eventually recovered from 20
patients. In 10 of the instances, 2 or 3 separate types were found. Gordon and Ms
associates2 report the isolation of $. typhosa and S. montevideo from aspirated
fluid in serous arthritis of the knee joint of a child. Numerous instances of
multiple infections in animals have also been described.
I t is our impression that most physicians and laboratory workers are unaware
of the fact that human beings are occasionally infected with more than 1 type
of Salmonella organism. The reason for this may be that the usual laboratory
procedure does not disclose infections by multiple types. The. common practice
of submitting a single isolate of a Salmonella-like culture for identification to a
typing laboratory tends to make the discovery of multiple types one of chance.
In this laboratory we have observed numerous instances of multiple-type
infection in individual patients. Such infections usually have been discovered in
1 of 2 ways: (1) by examination of a series of fecal specimens from the same
person, or (2) by simultaneous isolation of different types from members of the
same family, thereby leading to a thorough re-examination of the individual
specimens. In the work described in this paper, an intensive study was made with
feces from patients who were excreting Salmonellae, in order to determine the
percentage of persons who had multiple types. I t was observed that an unexpectedly large percentage of the patients were infected with more than 1 serotype
of Salmonella organism.
METHODS
Arrangements were made with our diagnostic enteric bacteriology unit to
forward to the Salmonella Typing Station all of the specimens of stools from
which Salmonella organisms had been isolated. When the specimens were received in the typing laboratory, the feces were cultured in tetrathionate broth
for 20 hr. A series of S S and MacConkey agar plates were inoculated from
Received, December 11, 1956; accepted for publication March, 11 1957.
Mr. Juenker is Chief, Salmonella Typing Station.
646
June 1957
SALMONELLAE INFECTIONS
647
the tetrathionate growth in a manner that ensured numerous isolated colonies.
The first step following growth of lactose-nonfermenting colonies was to obtain
an indication of the presence of Salmonellae other than the type that was initally
isolated from the fecal specimen. This was accomplished by means of inoculating
numerous lactose-nonfermenting colonies into modified Craigie tubes that contained semisolid agar mixed with antiserum that was specific for the flagellar
antigens of the Salmonella initially isolated. If no motility occurred, it was assumed that only 1 type of Salmonella was present in the specimen. If motility
occurred, the motile organism was isolated and identified. Specimens that contained Proteus organisms were obviously unsuitable for multiple-type examination and these were not included in the study.
Owing to the numerous reports describing induction of antigenic changes in
the flagellar antigens by the use of antiserums, it was considered necessary to
isolate the types of Salmonellae from single colonies, without use of serum, in
order to verify their natural occurrence. When multiple types in a specimen were
indicated by methods referred to in the preceding paragraph, typical single
colonies were transferred from S S and MacConkey agar mediums to infusion
agar plates. In transferring the colonies, a straight wire was used and inoculations
were made by a single vertical stab in the agar plate. Sixty to 100 colonies were
transferred in this way to a single agar plate that was termed the "master plate."
Inoculations were made in an orderly pattern so that duplication of the pattern
on replicate plates would be possible. In order to determine whether or not each
of the colonies on the master plate was the same, or different from the type of
Salmonella originally identified from the specimen of origin, the technic with
antiserums in semisolid agar was used. This time the semisolid agar-serum
mixture was poured into a petri dish and chilled in the refrigerator. After incubation, each of the colonies from the master plate was transferred by vertical stab
into the semisolid agar in such a way that the pattern of arrangement of colonies
on the master plate was reproduced exactly. If a colony on semisolid agar manifested evidence of motility, the duplicate colony on the master plate was completely identified serologically. In this way, the criticism related to "serum
induction" was avoided.
The presence of 2, 3, or more types of Salmonellae in a single fecal specimen
could be verified by the use of the combination of (1) a master plate and (2) a
replicate plate of semisolid agar that contained the necessary combination of
flagellar serums. Usually, at least 2 plates were replicated for each specimen. If
conditions indicated, and isolated colonies were available, additional plates
were made for some specimens. The average number of colonies picked was 176
per specimen, the least number 55, and the greatest number 489.
RESULTS
A total of 157 fecal specimens was examined from patients who were known to
be excreting Salmonella organisms. It is emphasized that no special effort was
made to select specimens from known excreters of multiple types; instead,
specimens were forwarded to the Salmonella Typing Station in the order in
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JUENKElt
TABLE 1
ISOLATIONS OP SALMONELLAE FROM PATIENTS WITH MULTIPLE TYPES OF THE ORGANISMS
Patient
1. B. S. P.
Total
Number
of Fecal
Age Specimens
Examined
years
2
2
2. F. C.
55
1
3. S. S.
3
5
4. G. J. F.
2
3
5. M. M. F.
23
2
6. J. H.
6
2
7. D. J. J.
If
7
8. N. L. E.
9. B. D.
"b
1
2
3
10. C M . G.
63
2
11. W. A. L.
5S
2
12. P. M.
15
1
13. M. J.
5
1
Routine
Identifications
Number of
Specimen
of Feces
Isolations from
Master Plates
S. bredeney
S. montevideo
S. oranienburg
S. worthington
S. montevideo
S. muenchen
S. worthington
S. montevideo
S. montevideo F-109
S. tennessee
F-183, S. muenchen
S. newport
S. kentucky
F-442
S. worthington
S. kentucky
S. oranienburg
F-374
S. kentucky
S. typhimurium F-472, S. typhimurium
F-1149 S. montevideo
S. bareilly
F-1428 S. typhimurium
S. montevideo
S. bareilly
S. muenchen
S. montevideo F-1148, S. typhimurium
F-1429 S. montevideo
S. bareilly
S. muenchen
F-50
S. muenchen
S. montevideo
S. mo7itevideo F-156
S. oranienburg
S. oranienburg
S. infantis
S. infantis
F-1474 S. montevideo
S. infantis
S. montevideo F-2
S. montevideo
S. bareilly
S. bareilly
S. infantis
F-1159, S. infantis
F-1260, S. muenchen
S. muenchen
and
F-1711
S. muenchen
F-1212 S. muencheii
S. tennessee
S. thompson
F-1723 S. thompson
S. muenchen
S. typhimurium F-2010 S. typhimurium
S. bareilly
S. bareilly
F-1256 S. montevideo
S. anatum
S. worthington
S. typhimurium F-735
S. bredeney
S. montevideo
S. oranienburg
S. muenchen
F-1410
S. bareilly
Number of
^ Colonies Studied
Unknown
7S
100
68 (F-1S3)
55 (F-442)
349
179 (F-472)
141 (F-1149)
132
143 (F-1148)
136 (F-1429)
240
136
103
343
119 (F-1159)
202 (F-1260)
202 (F-1711)
113
187
194
489
which they were recognized as positive. The number of specimens received varied
from 1 to 7 per patient. Twenty-seven specimens contained organisms of the
Proteus group and these were discarded. The remaining 130 specimens were
from 75 persons. Multiple types of Salmonellae were isolated from 13 of the 75
patients (17.3 per cent). The results of multiple isolations from the 13 patients
are indicated in Table 1. It should be noted that examination of fecal specimens
June 1957
SALMONELLAE INFECTIONS
649
from patients listed in Table 1 did not alwa}'S result in the isolation of multiple
types. Only specimens that yielded growth of multiple types are listed.
I t may be observed (under "Routine Identifications") that the routine
diagnostic enteric laboratory found evidence of infection with multiple types of
Salmonellae in several patients. This, of course, was the result of examining a
series of specimens from the same person. An outstanding example may be
observed in the instance of Patient 1 (B. S. P.), from whom the diagnostic enteric
laboratory isolated 6 different types of Salmonellae. This was the result of random
sampling, inasmuch as only a single colony of a Salmonella-like culture was
submitted to the Salmonella Typing Station from each specimen. Random sampling (from the series of specimens) in the routine diagnostic laboratory yielded 2
types (S. typhimurium and S. bareilly) that were not found by special technics.
On the other hand, 1 type (S. worthington) was found by the latter method and
not during the study in the diagnostic enteric laboratory. I t would seem, therefore, that this patient was infected by 7 different types of Salmonellae during
the time that specimens were received in the laboratory. While 7 different types
of Salmonellae from 1 patient is unusual, it is by no means a record. Hormaeche
and his associates4 reported the isolation of 10 different types from a single
patient on several examinations.
In 2 instances, Salmonellae recovered from the preliminary Craigie tubes were
different from those eventually identified from the master plates. Examples are
Patients 2 and 3, from whom S. analum and S. bareilly, respectively, were
isolated by the technic with Craigie tubes. The same organisms could not be
demonstrated from single colonies. From Patient 13, we were unable to recover
the same type of Salmonella (S. bareilly) as that originally sent to us by the
diagnostic enteric laboratory, even though 489 colonies were studied. It may
be observed that the examination of the master plate revealed 3 additional types
of Salmonellae from this patient. I t is of interest that still another serotype, S.
typhimurium, was isolated from 2 members of the family of Patient 13, but this
organism was not found in the patient.
I t was also interesting to note the irregularity with which multiple types
seemed to be present in the stools of some patients. An example of this irregularity
is Patient 3, from whom a total of 5 fecal specimens was examined. In Table 1 it
may be observed that examination of stool specimens F-183 and F-442 revealed
types S. muenchen, S. kentucky, and S. worthington by the technic with master
plates. From the next 2 fecal specimens, not recorded in Table 1, S. worthington
was obtained from the Craigie tubes, but only S. kentucky seemed to be present in
single colonies on the master plate. A total of 1.1.1 colonies was examined from 1
specimen and 129 from the other. From the fifth stool (F-374), S. oranienburg
and S. bareilly were isolated from Craigie tubes, although only S. oranienburg
and S. kentucky were identified from single colonies on the master plate.
Another example of irregular excretion types of Salmonellae was found in
Patient 7. Prior to beginning this study, our diagnostic enteric laboratory isolated
S. monlevideo, S. oranienburg, and S. infanlis from this patient on various oc-
650
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JUENKER
TABLE 2
ADDITIONAL PATIENTS WITH EVIDENCE OF MULTIPLE INFECTIONS BY SALMONELLAE
Patient
14. J. s.
15. D p. K.
16. G L F.
Age
months
10
15
36
Routine Identification
S.
S.
S.
S.
S.
montevideo
thompson
muenchen
muenchen
muenchen
Number of Number of
Specimen
Colonies
Studied
of Feces
F-1477
F-1465
F-1431
F-471
F-1147
343
322
137
189
134
Isolation from
Craigie Tube
S.
S.
S.
S.
S.
infantis
tennessee
montevideo
montevideo
montevideo
casions. During the course of the study, 7 additional specimens were examined.
From the first 4 specimens, it seemed that only S. montevideo was present. From a
fifth specimen, S. infantis was demonstrated from a Craigie tube, but every one
of 120 single colonies examined seemed to be S. montevideo. From the last 2
specimens (F-156 and F-1474 in Table 1), *S. infantis was again found by the
technic with a Craigie tube, whereas the master plates yielded S. montevideo, S.
oranienburg, and S. infantis from specimen F-156, and S. Montevideo and S.
infantis from specimen F-1474.
In 3 additional instances (Patients 14, 15, and 16), there was evidence of
multiple-type infections according to results of the technic with Craigie tubes, but
single-colony isolations proved to be unsuccessful. Data for these patients are
summarized in Table 2. Patient 16 is a member of the same family as Patients 4
and 5 in Table 1.
DISCUSSION
Results of this study suggest that infections with multiple types of Salmonellae
are more prevalent than would be expected. I t is possible, of course, that the
sampling taken at the time of this study is not representative of Salmonella
infections in general. On the other hand, one might be tempted to explain their
occurrence by making reference to the laboratory transduction of types of
Salmonellae by means of phage lysates. This explanation seems unlikely in view
of the proved epidemiologic value of determinations of serotypes in outbreaks of
Salmonella infections. Edwards, Davis, and Cherry 1 emphasized that S. typhosa,
S. schottmuelleri (para B), and S. typhimurium have retained their particular
antigenic characteristics through the years, although some of these antigenic
characteristics are readily changed by laboratory methods.
Infection acquired as a result of ingestion of food products that are contaminated with multiple types of Salmonellae is, of course, a possibility. I t is well
known that poultry, and especially eggs, are frequently contaminated with
Salmonellae. Infections traced to dried egg powder have occasionally occurred,
and other foodstuffs, especially animal products, have been implicated. Salmonella organisms have been isolated from a number of different types of market
meats. The findings described in this paper suggest that a more thorough laboratory and epidemiologic study of the source of these infections would be advan-
June 1957
SALMONELLAE INFECTIONS
651
tageous. Previous methods for isolating multiple types of Salmonellae from the
same patient have been relatively slow and tedious. The use of plates of semisolid agar with combinations of antiserums would provide a more practicable
means of performing a thorough study.
SUMMARY
This paper deals with the results of a special study of 130 fecal specimens from
75 persons who were known to be excreting Salmonella organisms. The special
technics revealed that multiple types of Salmonellae were present in the stools
of 13 of the 75 persons (17.3 per cent).
SUMMARIO IN INTERLINGUA
Iste articulo presenta le resultatos de un studio special de 130 specimens fecal
ab 75 personas qui cognoscitemente excerneva organismos del genere Salmonella.
Le technicas special revelava que multiple typos de salmonellas esseva presente
in le feces de 13 del 75 subjectos (17.3 pro cento).
REFERENCES
1. EDWARDS, P. R., DAVIS, B. R., AND CHERRY, W. B.: Transfer of antigens by phage
lysates with particular reference to the l,w antigens of Salmonella. J. Bact., 70:
279-284, 1955.
2. GORDON, H. S., HOFFMAN, S. J., SCHULTZ, A., AND LOMBERG, F.: Serous arthritis of the
knee joint. J. A. M. A., 141: 460-461, 1949.
3. GRBIFINGER, W., AND SILBERSTEIN, J. K.: Salmonella food infection in military personnel. J. Lab. & Clin. Med., 29: 1042-1053, 1944.
4. HORMAECHE, E., SURRACO, N. L., PELUFFO, C A., AND ALEPPO, P. L.: Causes of infan-
tile summer diarrhea. Am. J. Dis. Child., 66: 539-551, 1943.
5. JUENKER, A. P.: The isolation of four Salmonella types from one carrier. J. Bact.,
50: 673-674, 1945.
6. WEINER, H. A., AND LIEBLER, J. B.: Infections with multiple Salmonella types, including S. braenderup. J. A. M. A., 145: 802-806, 1951.