FURTHER OBSERVATIONS ON THE PERSISTENCE OF INDIVIDUALSTRAINS OF ESCHERICHIA COLI IN THE INTESTINAL

TRACT OF MAN

H. J. SEARS AND INEZ BROWNLEEDepartment of Bacteriology, University of Oregon Medical School, Portland, Oregon

Received for publication July 17, 1951

In a previous paper Sears, Brownlee, and Uchiyama (1949) recorded the re-sults of a study of the Escherichia coli strain composition of the feces of four in-dividuals which led to the conclusion that the E. coli flora of the intestinal tractat any time usually consists of one or two strains which persist over long periods,together with a small number of other strains whose tenure is limited to a fewdays, a few weeks, or a few months. The long-tenure strains we designated asresidents, the short-tenure ones transients. The method made use of in this earlierstudy consisted merely of plating out fecal specimens at frequent intervals fromthe subject under study, selecting a number of typical E. coli colonies, and classi-fying the cultures so obtained into groups on the basis of their 0 antigens. Thevalidity of the conclusions drawn from these observations was based on the as-sumption that the antigenic diversity of the E. coli strains found in human fecesis so great that when a number of cultures isolated from the same person at shortintervals over a period of time belong to the same 0 group they can be assumedto be all the progeny of the same strain, and that when a new strain is introducedand appears in the feces the chances are very great that it will belong to a differ-ent 0 group. The studies of Kauffmann and Perch (1943), Perch (1944), andWallick and Stuart (1943), though having somewhat different objectives, lendsupport to the above conclusions.

It is the purpose of the present paper to present the results of further observa-tions on this subject. During the past 2 years we have continued the study of twoof the subjects of our former report, A and B, and have added four others, a pairof identical twins, Ki and Kr, 2 weeks old at the beginning of the study; anotherinfant, BH, 10 days old; and another adult, E.The method used has been essentially the same as that employed in the previ-

ous study. Fecal specimens have been collected at various intervals and platedon eosin methylene blue agar, E. coli colonies picked, and stock cultures madeon agar slants. These were stoppered and stored until a convenient time could befound for determining their biochemical and antigenic characteristics. Classifica-tion of the cultures with respect to their 0 antigens was rendered simpler in thisstudy by the possession in our laboratory of 0 serums for most of the 112 coliO groups of the Kauffmann-Knipschildt-Vahlne (KKV) schema. When a num-ber of cultures were accumulated, 24-hour broth cultures were made of each,which were heated in the Arnold sterilizer for one hour and 0.3 per cent formalinadded. Each antigen so prepared was then first tested with mixtures of the groupserums, each mixture containing four or five serums. Each serum was present in

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the mixture in a dilution of 1:160 making a final dilution in the test of 1:320.When an antigen was agglutinated by a given mixture, it was then submittedto test in an equivalent dilution of the individual serums of the mixture and to acomplete titration in those serums which gave agglutination in the single dilu-tion test. The tube method was employed in all agglutination tests and incuba-tion was at 50 C for 18 to 24 hours. When an antigen was agglutinated to withinone dilution of the full titer of the serum for its homologous antigen, it was as-signed to the corresponding 0 group. Deviations from or additions to this stand-ard procedure are noted in connection with the discussion of the results obtainedfrom our individual subjects.

SUBJECT A

The results of the study of 182 fecal specimens from subject A extending overa period of more than 4 years are summarized in table 1. During this entire timetwo strains stand out as the principal residents, one belonging to 0 group 81,designated in our previous paper as A-17, the other to 0 group 28 which wascalled A-18 in the former paper. The duration of the presence of the 0-7 straingives it the standing of a resident also, but its entire tenure was within the periodof residency of the 0-28 strain. The latter, at the time of the writing of this paper,remains the dominant E. coli strain in this subject. This strain was not recog-nized, however, during a period of 40 days following the return of the subjecton June 4, 1949 from an extended absence from home. On the basis of previousexperience it was assumed that it had probably been lost permanently, and itwas decided to attempt its re-establishment as a resident strain. Water suspen-sions of agar cultures were accordingly swallowed by A, as indicated in table 1.Four days after the first ingestion the strain was again found in the proportionof two colonies in ten. From this time to the last date in the table this 0-28 strainmaintained its residency, accounting for 351 of the 456 cultures isolated from 54specimens. It is, of course, impossible to assert, on the basis of the previous ob-servations, that the 0-28 strain had been permanently lost during the period ofthe subject's absence from the city and that it had been reinstated by ingestion.However, we have not found in any of our subjects that a resident strain hasremained entirely undiscovered during a similar period when as thorough a searchfor it was made. Therefore, we are inclined to believe that this was an instance,the only one in many trials, of the establishment of an ingested strain as a resi-dent.The 0-2 strain, with which a completely unsuccessful attempt was made to

displace the 0-28 strain from its residency, was isolated from another person andhad been maintained in laboratory culture for about 2 years. Not a single cultureof this ingested strain was isolated from the subject during or after the period ofingestion.

SUBJECT B

The data of table 2 show that it is a general characteristic of this subject thathis E. coli flora consists preponderantly of strains maintaining relatively long

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tenure, two resident strains often being concurrently present. His first residents,0-2 and 0-75, designated in our previous paper as B-1 and B-2, respectively, weredemonstrated over a period of 485 days and disappeared during an extended

TABLE 10-group distribution of fecal Escherichia coli cultures isolated from adult subject A over a

period of four years

NUMBER OF CULTURES IN SPECIFrED 0 GROUPSNUM- N1Z

PERIOD OF DAYS DER OF..e COMSCOLLECTION IN OF C4UOMR-TOF SPECIMEN PZRIOD | TUES|o| 4-

TUEWS S 00 (% l- 0

1/12/47- 270 62 324 185 1 14 57 6710/5/47

10/9-12/1 54 0 0 Subject ill. Part oftime in hospital.

12/2/47- 520 53 350 302 23 11 144/29/49

4/30/49- 35 0 0 Subject on trip out6/3/49 of state.

6/4/49- 31 6 53 17 16 10 107/4/49

7/5-7/13 9 7 51 50 1 Ingested 108 0-28over period of 3days beginning7/9/49.

7/14-8/28 46 10 92 41 19 4 28 Out of city 8/23-8/27

8/29/49- 633 44 364 310 54 Ingested 5 X 1085/22/51 0-2 over a

period of 8 daysbeginning 9/20/-50.

Totals.... 1598 182 1234 185 654 69 41 17 16 79 173

* Cultures in this column belong to several different 0 groups each of which was foundonly in one or two specimens.

t Serum A-24 was made with a strain, isolated from this subject 6/30/49, which could notbe assigned to any of the KKV 0 groups.

motor trip by the subject. The 0-75 cultures isolated in specimens more than ayear later must be regarded as resulting from the acquisition of a new strain ofthis group rather than as the unrecognized persistence of the previous strain.The 0-28 strain with a total tenure of 137 days was accompanied during most of

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its residency by the 0-7 strain, but the latter outlasted it and persisted to theend of the period of observation, a total of 646 days, sharing its tenure alter-nately for long periods with the strain called B-9 and the new 0-75 strain dis-cussed previously.

Transients were relatively scarce in subject B. Of the 1,311 cultures studied,1,202 or 91.7 per cent clearly belonged to the resident class.The loss of a resident strain or the acquisition of a new one by this subject

often seemed to be related to temporary changes in the subject's environment,

TABLE 20-group distribution of fecal Escherichia coli cultures isolated from adult

three yearsB over a period of

xNuMBR 0o cuLTutES IN SPECIFIED 0 GROUPSPERIOD OF DAYS 01M3Z 0U1ZRlCOLLECTION IN OF OF COMMCEOTSOF SPECIMENS PERIODSPC-C-UENS TURES * 55 0

6 6) 6 6 m

2/11/47- 485 87 904 379 442 67 166/9/48

6/10-7/27 58 0 0 Subject on motortrip duringperiod.

7/28-8/31 35 6 75 63 12

9/1-12/11 102 6 61 27 30 4

12/12/48- 167 8 120 32 885/27/49

5/28/49- 377 14 151 43 98 9 1 On motor trip6/8/50 6/8/49-7/9.

Moved to anothercity 6/8/50.

Totals .... 1224 121 1311 3791485 90 1160 88 76 33

* All the cultures in this column agglutinated to titer with a serum prepared with one ofthem. They could not be definitely assigned to any of the KKV 0 groups.

but in a few instances such changes occurred without any such obvious altera-tion in the subject's experiences.

SUBJECT E

This subject proved to be an exception among the adults we have studied thusfar in that specimens frequently yielded coliform organisms other than trueE. coli. Such strains were not studied further than to rule them out of the categoryin which we were interested. It is seen in table 3 that throughout the entire 2

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years this person harbored cultures belonging to 0 group 1. Of the 890 culturesstudied during the period, 477 belonged to this group. These cultures were dividedbetween a typical E. coli ("Typ") which fermented lactose with acid and gasin 24 hours or less and a mutable type ("Mut") which never gave acid or gason lactose in 1 day but which did ferment this sugar typically after periods of36 hours to 3 days. In all other respects, including the possession of a certainminor antigen present in only about half of our 0-1 strains, these two types ofcultures were identical. We feel justified, therefore, in considering them as vari-ants derived from the same strain.Of the 0-7 cultures from E all but three were of the late lactose fermenting

type. These three were identical antigenically and biochemically with the others

TABLE 30-group distribution of fecal Escherichia coli cultures isolated from adult E over a period of

two years

NUMBER OF CULTURS W SPEaCFIED 0 GROUPSDAYS UBRNME

PERIOD OF COLLECTION AS OF OO SPECIMENS PERIOD SPECI- C. 0-1 0-7 Other Un-MENS TURES Tp utypMtB-9t 0-2 0-45 SS:ecific grue. | PXkWD | Ni TV ZS |Typ |Mut |Typ Mut | 1°ZiSmrgoupe

3/26/49-4/1/49 7 3 20 204/2-5/20 49 0 0 Subject on vacation in Central America.5/21-6/6 17 3 41 1 406/7-9/1 87 14 155 4 54 3 51 3 32 89/2-2/13/50 165 10 103 41 47 7 82/14-3/28 43 5 57 16 40 13/29-4/19 22 0 0 Out of state.4/20-6/3 45 10 94 17 57 2 17 16/4-7/17 44 13 165 31 17 40 56*1 13 1 87/18-7/29 12 0 0 On vacation in Canada.7/30-3/26/51 240 27 255 200 291 14 2 10

Totals............. 732 85 890 296 181 3 51 137 20 56 71 75

* All of these 56 cultures were isolated from specimens obtained during a one day periodof diarrhea induced by castor oil taken 6/12/50.

t Cultures in this column agglutinated to titer with serum B-9 of table 2.

except that they gave acid and gas from lactose with the promptness typical ofE. coli. The 81 days' tenure of these 0-7 cultures began and ended within theperiod of residency of the 0-1 cultures as did also the 135 days' tenure of the B-9strain which followed. The only other single 0 group in the data from subject Ewhich is represented by more than a few cultures is 045. Fifty-six cultures ofthis strain were studied. They all came, however, from four specimens taken onthe same day during a period of diarrhea induced by a dose of castor oil. Thenext day the resident strain 0-1 was again dominant.

In the column of table 3 headed "Other Specific Groups" 13 0 groups are repre-sented, each of which was present in only one, two, or, at most, three specimensduring a single short period. They clearly riepresent transient occupants of the

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bowel. The 74 cultures which for various reasons remained unclassified as totheir 0 groups were distributed irregularly throughout the whole period of 2years and seem best interpreted as composed also of several transient strains.The 20 0-2 cultures were isolated from this subject on three occasions about 8months apart. On each occasion the group was apparently present about 1 month.Since 0-2 has been one of the commonest groups found in this laboratory both innormal feces and in urine specimens from the urology clinic, it seems probablethat these cultures in subject E represent three strains rather than one.

SUBJECT BH

This subject was a baby who was 10 days old at the time the first specimenwas collected. During the period of study the child was cared for at home by itsmother, a young woman who had had training as a medical laboratory technician.There were in the family two older children and the father, who was a graduatestudent in this laboratory. The fecal specimens were collected from the diapersand brought promptly to the laboratory where they were treated as alreadydescribed. Table 4 gives the findings on this subject. Aerobacter cultures werefrequently found in specimens from BH, but these were ignored or, when recog-nized as such only after biochemical study of picked colonies, were discarded.The cultures of table 4 are all true E. coli. A strain of 0 group 2, already wellestablished as a resident at the time of the first specimen, persisted throughoutthe period of study and was joined after about 3 months by a strain belongingto 0 group 4. These two strains dominated the E. coli flora of this subject, con-stituting about 84 per cent of all the cultures studied.Only five strains established definitely as transients were encountered in BH

during the 11 months of study. It appears then that even very young babies mayconform to the pattern that appears to be established for adults, that is, theharboring of certain strains of E. coli for many months and of others for onlyshort periods. This child led a quite uneventful life from the point of view ofintestinal disturbances. It may be significant that the first appearance of the0-4 strain was coincidental with the first feedings of vegetables and fruits, butother changes in the diet seem to have had no effect upon the strain compositionof the bowel.

SUBJECTS KI AND KR

These subjects were a pair of identical twin boys born just 2 weeks before thefirst specimen was examined. They were cared for in a family containing thefather, who was a senior medical student, the mother, and two other children.Thirty-three specimens were collected from each child over a period of approxi-mately 1 year. These children also frequently yielded Aerobacter cultures. Theseare not included in the data of table 5, which shows the results of the study ofE. coli cultures from both children. It seems best to present the data from thetwo in parallel columns since its most striking feature is the remarkable similar-ity of E. coli types in the two subjects at all times. Not only did they harbor thesame resident 0 groups, 0-1 and Ki-2, during the entire period, but it is seen

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that they acquired the same transients and frequently at about the same time.All 0 groups found except three were present at some time in both babies. Thethree exceptions, represented by only 8 cultures, were isolated only from Ki.These 8 cultures are included in the column of table 5 headed "Six Other Groups".

It is another noteworthy feature of the data on these children that the dis-tinction between residents and transients is by no means as sharp as in the data

TABLE 4Data on fecal Escherichia coli cultures isolated from baby BH over a period of 10 months

NUMBER NMBER OF CULTURES IN SPECIFED 0 GROUPDAY OF SPECIMEN Or

CULTURES 0-2 0-4 0 0-51 0-54 Others Ungrouped

1 11 112 12 123 12 12

5 11 116 10 9 19 8 5 3

20 11 1131 9 949 10 4 6

56 10 8 1=0-169 8 3 590 10 5 2 3

107 10 10113 12 12141 9 1 8

165 10 2 4 3 1187 9 6 3224 12 10 2

252 12 1 11285 10 8 2309 12 6 1 1 =0-35 4

Totals....218 118 65 10 6 12 2 5

on our other subjects, including baby BH. The strains 0-1 and Ki-2 appear tohave been resident in both babies during most of the period they were studied,though the relatively long periods when one or the other of these strains wasisolated from only one subject suggest that their residency was being maintainedby a constant interchange between the two children. All the other strains iso-lated were less persistent in their appearance, but, on the other hand, failed toshow the marked transiency characteristic of the transient strains in our other

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TABLE 50-group distribution of Escherichia coli cultures isolated from twin babies Ki and Kr over a

period of one year

NUMEZR OF CULTURES IN SPECIFIED 0 GRtOUPS

or CUL- SixrDAY OF SPECIMEN TURES 0-1 Ki-2* 0-17 0-88 0-46 0-8 0-26 0-3 Other n

GroupsgrueKi Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr

1 10 10 4 10 68 9 10 11 8 4 1411 14 1418 10 10 1 1 3 1 2 7 1 419 10 1 6 3

27 10 9 129 10 1 2 2 535 10 7 2 137 10 3 2 3 4 1 346 8 10 3 5 3 1 6

53 10 81 9 860 10 10 1 1 8 1067 11 2 1 7 168 12 5 5 1 172 10 10

74 10 2 3 682 10 10 1 6 9 1 1 288 12 8 489 11 1 1096 11 11 11 11

103 11 11 3 9 6 2 2119 10 10 2 1 9 1 1 2 4123 10 10 5 1 1 1 1 4 7130 10 10 34 5 7138 10 10 84 2 6

152 10 10 10 10166 10 6 4167 10 10179 1 1180 10 6 3 2

193 310 2 91 1207 10 1 9208 12 9 2 1222 8 10 3 75 1 2236 3 10 310

*The serum used to identify this group was made with a strain isolated from Ki on

10/17/49.

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TABLE 5-(Continued)

NUMBE:R OF CULTURES IN SPECIFD 0 GROUPS

OF CUL- six UnDAY OF SPECIMEN TUREC 01 Ki-2* 0-17 0-88 0-46 0-8 0-26 0-3 Other UoedGroups group

Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr Ki Kr

255 10 10256 10 10272 6 8 3 8 3284 - 6 4 2285 10 10

298 10 3 8 10313 9 6 2326 7 6 1340 10 1 9341 10 10

Totals....2868221651120157 41 71 15 10 82 141 1122 5 121 1433 271 181 15 481 49

TABLE 6Escherichia coli 0 groups isolated from other members of the family of subjects Ki and Kr

SPECIFIC 0 GROUPS DEN)ONSTAEDDAY OF SPECDIMN

Mother Father rother, age 4 Sister, age 2

272 1 1, 75 1 1285 1 1, 75 1, 19 1299 81 75 36 Aerobacter only313 75, 19, 7, H-6 1, 75, 54 6 *340 1, 19 75* 75 85

* One or more cultures of a different but undetermined group.

subjects. Their tenure was marked frequently by long periods of absence, againsuggesting a source of repeated reinfection. It would, of course, be expected thatsubjects as intimately associated as. twin babies would freely share their fecalorganisms. The data of table 5 appear to us to give clear evidence that this wasthe case with Ki and Kr. It occurred to us, however, that a common source in thefamily could be a factor. Accordingly, we procured fecal specimens from the otherfour members of the family. Five specimens were examined from each over a pe-riod of 2 months, with results shown in table 6. In this table the "Day of Speci-men" gives the time relationships of these specimens to those of table 5. About10 cultures were examined from each specimen, but only the actual numbers ofthe KKV 0 groups isolated are given in the table. It is seen that while 0 group1 was found in all members of this family, none of the other groups isolated fromKi and Kr was found in any of the other four. This appears to indicate that thefecal E. coli strains of the twins were not primarily obtained from other membersof the family.

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OBSERVATIONS ON THE MANNER IN WHICH ESCHERICHIA COLI STRAINSARE ACQUIRED AND LOST

All strains of E. coli have their natural habitat in the intestinal tracts of manand animals. It may be presumed that when a new strain appears in the feces ofan individual it was living and multiplying, a few days or a few weeks previously,in the intestinal tract of another person or animal. It has presumably reached theintestinal tract of its new host through ingestion of food or water. It can hardlybe doubted that many new strains are thus ingested by most persons every day.That few of these ingested strains succeed in multiplying sufficiently to be recog-nized in the feces by the techniques used in our study is shown by the results ofour previous study as well as of the present one. In only one instance, by deliber-ate ingestion of a culture of known 0 group, have we obtained evidence that astrain can be established in this manner as a resident. Our subject A, as previouslystated, apparently re-established a former resident in his intestinal tract. Itmust be admitted, however, that the evidence that this strain had permanentlydisappeared from the subject before ingestion is not entirely conclusive. That aningested organism may completely fail to multiply in the bowel is indicated by alater attempt by subject A to establish in his intestinal tract a strain of 0 group2 isolated from another individual. In our previous paper we reported the intro-duction by ingestion of several strains as transients. It is not surprising that theresults of such experiments are variable since there are many and variable fac-tors that probably have some influence in determining whether ingested strainswill survive and multiply sufficiently to be recognized in the stool. The numbersingested, the extent to which they may be destroyed by the variable conditionsin the stomach, the changing nature of the intestinal contents that serve as foodmaterial, the rate of their movement, and the character of the accompanyingflora and fauna may all exert some influence. Whether there are peculiarities ofthe individual which favor the multiplication of one strain more than anothercan at present be only a matter of conjecture.The occasional loss by an individual of a resident strain likewise cannot as

yet be ascribed to any single factor or simple set of factors. That it has frequentlyfollowed a complete change of environment of the subject is shown in the suddenloss of 0-28 by our subject A, of 0-2 and 0-75 by subject B, and of B-9 by sub-ject E. On the other hand subject E retained his 0-1 strain for 2 years in spiteof several complete changes of environment.

It has been suggested that a thorough emptying of the bowel, either throughfortuitous attacks of diarrhea or by artificial purges, might be a factor in thedisplacement of a resident strain. As will be seen in several of our tables, some ofour subjects suffered short but severe diarrheic attacks without any markedchange in the fecal E. coli strains. In an attempt to test this factor more care-fully, our subject E on two different occasions took doses of castor oil which pro-duced several liquid stools over periods of 12 to 24 hours. In the first trial, re-ported before, the actual diarrheic stools yielded none of the resident strain0-1 but only a new strain belonging to 0 group 45. The next day, however, the0-1 strain reappeared and remained dominant. At the second trial even thediarrheic stools contained the 0-1 resident as the dominant strain.

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SUlMMARY

In this paper we present further evidence in support of our previously statedthesis that the normal human individual harbors two kinds of strains of Escheri-chia coli, residents and transients, the former comprised typically of one or twostrains at any one time and the latter often absent and rarely consisting of morethan three or four strains. It is further demonstrated that this pattern of bowelE. coli may be shown by very young babies as well as adults, resident strainsbecoming well established as early as 10 days after birth. Babies as intimatelyassociated as twins are likely to harbor the same residents as well as freely sharetheir transient strains.The conditions under which resident strains are lost and new ones acquired

cannot be accurately defined, but evidence is adduced that a complete change ofenvironment is often, but not always, accompanied by such changes. Diarrheicattacks of short duration and artificial purges do not by themselves usuallybring about changes in resident strains.

REFERENCESKAUFFMANN, F., AND PERCH, B. 1943 tVber die Coliflora des gesunden Menschen. Acta

Path. Microbiol. Scand., 20, 201-220.PERCH, B. 1944 Weitere Untersuchungen ilber die Coliflora des gesunden Menschen.

Acta Path. Microbiol. Scand., 21, 239-247.SEARS, H. J., BROWNLEE, I., AND UCHIYAMA, J. K. 1949 Persistence of individual strains

of Escherichia coli in the intestinal tract of man. J. Bact., 69, 293-301.WALLICK, H., AND STUART, C. A. 1943 Antigenic relationships of Escherichia coli iso-

lated from one individual. J. Bact., 46, 121-126.

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