Acta Pzdiatr Scand 66: 349-354, 1977

ASYMPTOMATIC BACTERIURIA I N SCHOOLGIRLS

V I . The Correlation between Urinary and Faecal Escherichia coli. Relation to the Duration of the Bacteriuria and the Sampling Technique G. LIDIN-JANSON and U. LINDBERG From the Departments of Clinical Bacteriology and Immunology, Institute of Medical Microbiology, and Departments of Infectious Diseases and Pediatrics, University of Goteborg, Giiteborg, Sweden

ABSTRACT. Lidin-Janson, G. and Lindherg, U. (Departments of Clinical Bacteriology and Immunology, Institute of Medical Microbiology, and Departments of Infectious Diseases and Pediatrics, University of Gotehorg, Gotehorg, Sweden). Asymptomatic hacteriuria in schoolgirls. VI. The correlation between urinary and faecal Escherichia coli. Relation to the duration of the hacteriuria and the sampling technique. Acta Paediatr Scand, 6 6 349, 1977.The occurrence of the urinary strain in the anus, rectum and faeces was investigated in 27 girls with asymptomatic hacteriuria (ABU). In patients with bacteriuria of relatively short duration 46 % of the faecal isolates were of the urinary strain as compared to only 18 % in patients with bacteriuria of relatively long duration. In general the correlation between the urinary and faecal flora is striking at the time of establishment of ABU but dimiriishes with time. The diminished correlation may he due to two factors: firstly, the composition of the faecal flora changes with time. Secondly, the correlation may he obscured by complex changes in the properties of bacterial strains established in the urinary tract. Contamination by the infected urine did not seem to be a serious problem when the rectal mucosa was swabbed proximal to the anal canal.

KEY WORDS: Asymptomatic hacteriuria, E coli. faecal flora

Escherichia coli infecting the urinary tract usually originate in the patient’s own bowel flora (17). Analyses of the correlation between the urinary and the faecal flora may be of importance for an understanding of the hostparasite relationship in urinary tract infection (15). Such analyses have to take into account the two possibilities of contamination of the faecal specimen by the infected urine, and of colonization of the gut by a strain already established in the urinary tract (3, 7). A longitudinal study of schoolgirls with asymptomatic bacteriuria (9) offered an opportunity to study the problems of sampling the faecal flora in bacteriuria and to analyse the relationship between the urinary and faecal flora. 23-772873

MATERIAL AND METHODS Patients The patients were all included among the schoolgirls with asymptomatic bacteriuria (ABU) described and prospectively followed up, treated or untreated, by Lindberg (9, 10). Twenty-seven bacteriuric children attending the clinic for their scheduled appointments during a five-month period took part in this study. Patients harbouring 0 typable urinary strains were preferred. The 27 patients were grouped according to the duration of their ABU. Seventeen patients were classified as having bacteriuria of relatively long duration. They were either still bacteriuric with the strain originally found on screening, or had a reinfection strain carried for at least one year. Two of these patients had new infections during the study period. Together with ten others, they were then classified as having ABU of relatively short duration, that is reinfections of 0-1 1 months duration. Sampling Without preliminary cleansing, a cottonwool swab was rotated superficially in the anus. After that, two swab Acru Pzdiurr .S(wtid 66

350

G . Lidin-Janson and U . Lindberg

Table 1. Occurrence in swabs from the anus ( A ) and the rectum (R) of twenty-two urinary strains not recovered among thirty randomly selected colonies from the innermost part of the faeces One rectal swab ( R 2 )was taken through a sterile tube inserted into the anal canal Pattern of occurrence of the urinary strain A-R,-RZA+R,+R2+ A +R , - R 2 A +R ,- R 2 + A - R , andlorR,+

No. of cases 11 3 7 1

urinary strain with respect to 0 agglutination pattern, haemolytic capacity and antibiogram were preserved as deep agar stab cultures under sterile paraffin oil, usually one colony from each positive site. Urinary strains were preserved in the same manner. The preserved strains were later subjected to a limited K antigen typing with the serum agar technique ( 2 ) . Strains giving no precipitates around the colony on any of the plates were designated KNt. 0 and K nontypable strains found in the urine and the faeces of the same patient were biotyped according to their pattern of fermentation of 16 carbohydrates as described by Bettelheim & Taylor ( I ) . Statistical calculations The sign test (4) and Fisher's permutation test (14) were used.

0

RESULTS specimens were taken from the rectum, the stick inserted to a mark 40 mm from the tip. In endeavour to minimize contamination from the anal canal, one of the swabs was inserted through a small tube. The tubes were made from heat sterilizable nylon. They were 30 mm long, with an internal diameter of 8 mm, a rounded inner tip and a flange at the outer end. Sterile vaseline was applied to the tip and the tube was passed through the anal canal, the flange being firmly pressed against the skin. The order of taking the two rectal swabs in each case was randomized. Each swab was put into a tube with transport medium (13) and kept at +4"C until cultured, usually later the same day. Material for the collection of faecal specimens a t home was dispensed and careful instructions were given. The girl was asked first to urinate into the lavatory basin. After wiping herself carefully, she was then to deliver the faeces directly into a large plastic bag, placed in a suitable container. The bag was to be sealed and put into an insulating box with cold accumulators. The boxes were collected from the patients' homes, usually within twelve hours of defaecation. Bacteriological procedure The faecal specimens, which were all formed, were cut into pieces using a red-hot metal rod. At each of three roughly equidistant points, one swab was bored into the centre of one cut surface. There were thus six swabs from each patient: one anal, two rectal and three faecal swabs. Each swab was inoculated ten times on a modified Conradi-Drigalski agar. The inocula were spread with a platinum loop. The plates were incubated aerobically at 37°C overnight. The last colony growing from each of the ten cultures was then subcultured, as were representatives of any colony types not included among these. The subcultures were typed as previously described (8). The procedure included a simplified E. coli 0 grouping using mono- and multivalent antisera against 69 0 groups in all. Strains that agglutinated spontaneously were designated Sa. Haemolysin production was assessed in plates with 5 % washed horse erythrocytes. Antibiotic sensitivity was tested using a disc diffusion method ( 5 ) . Strains conforming to the Actu

Pzdiatr S c u d 66

Thirty-nine sets of specimens were obtained from the 27 patients. All but two of the anal swabs and five of the swabs from two faecal specimens yielded Enterobacteriaceae from all ten spread out cultures. Altogether 2 279 colonies were randomly selected and typed. Out of these, 649 isolates (28.5%) were found to concord with the urinary strain present. The rates of concordance at the different sampling sites were 40.2 % for the anus, 3 1.8 % for the rectum when the tube was not used and 30.3 % when it was. The overall figure for the faeces proper was 22.7% and the range between the faeces swabs I , I1 and 111 was from 19.4 to 24.9%. In twenty-two episodes of bacteriuria the urinary strain was not found on at least one occasion among thirty randomly selected isolates from the innermost part of the faeces. The reTable 2. Proportion of faecal isolates conforming to the urinary strain in twelve episodes of bacteriuria of short duration (A) and in seventeen episodes of long duration (B) Two girls had two episodes each Number of concordant colonies among 60 randomly selected colonies

Bacteriuria group A

B

&20 2140 4140

5 1 6

15 2

35 1

Asymptomatic bacteriuria in schoolgirls. VZ

Table 3 . Comparison between urinary and faecal E . coli on two or three occasions in nine episodes of asymptomatic bacteriuria

Girl

Duration of bac teriuria with present strain, months 0-2 2- 4

Number of colonies from each site corresponding to urinary strain Faeces, swab no.

Urinary strain

Anus

Rectum, tube not used

9 10

9 10

8 6

10 0

10 0

10 0

10 0

10 0

10 0

10

0

Rectum, tubeused

ME ME EL EL

4- 8'

025 K N t H f a 0 2 5 KNtH + a 0 7 K1 0 7 K1

MH MH

0-6 6-1 1

0 6 KNtH+ 0 6 KNtH+

EA EA TS TS KK KK

12-18 13-19" 13-18 15-20

01 K51 0 1 K51 01 KI 01 K1

0 0 0 0

3 0 0 0

0 0 0 0

> 8 > 10

016 (SafK 1 -H + 016 (Sa)Kl-H+

9

4

10 2

10 8

AKW AKW AKW MJ MJ

> 8 > 9 >I1 >I1 >I2

0 1 7 KNt 0 1 7 KNt 017 KNt 0 2 5 KNtH+ 0 2 5 KNtH+

9 8 3 1 0

7 3 0 0+' 0

8 5 0

AS AS

> 19 >21

0 7 5 KNt 0 7 5 KNt

1 013

2 8

0-4

I

I1

111

8 0

-d 5 10 0

10

8

10

0

0

0

0 7

0 8

2 6

0 -

0 0

0 -

9 0

10 0

10 0

8

10

10

0120 1

416'

2

6 0 O+ 0

4

0

3 0 O+ 0

2 4

0 O+

1

2

0 2

Ot

0 O+ 0

H+=haemolysis in solid medium. Only two out of ten cultures grew enterobacteria. Neither of two colonies corresponded to the urinary strain. Only six out of ten cultures grew enterobacteria. Four out of six colonies corresponded to the urinary strain. None out of ten cultures grew enterobacteria. After a ten-day course of nitrofurantoin, one negative urine culture obtained, in E L 4 months and in EA 3 weeks before indicated specimen. O+=very small amount of strain found by selection for colony morphology.

a

'

covery of the urinary strains from the swabs from the anus and rectum is summarized in Table 1. In no instance was the urinary strain recovered from the rectum but not from the anus. In eight instances it was found in the anus but not in the plain rectal swab, and in seven cases in the anus but not in the rectal swab taken through the tube. In these specimens the urinary strain thus occurred more often in the anal swab than in either of the rectal swabs (Sign test). The overall proportion of stool isolates conforming with the urinary strain present was 399/875 isolates (45.6 %) in the group with bacteriuria of short duration and 250/1404 (17.8%) in bacteriuria of long duration. The proportions of isolates corresponding to the

urinary strain found among 60 randomly selected colonies were compared for the twelve episodes of bacteriuria of short duration ( A ) and the seventeen episodes of long duration ( B ) , Table 2. In group A , 5 patients had 0-20 concordant colonies and 6 had 4 1 4 0 colonies. In group B , 15 girls had only 0-20 colonies conforming to the urinary strain. The two distributions are different at the 5% level of significance (Fisher's permutation test). The same bacteriuria strain was found more than once in nine girls. The characteristics of the urinary strains and the numbers of colonies from each sampling site-anus, rectum and faeces-that corresponded to the urinary strain of each patient, are shown in Table 3 . Three girls (ME, EL and MH) had recent re-

352

G . Lidin-Janson and U.Lindberg

currences of asymptomatic bacteriuria with a strain not present at the latest check-up two to six months earlier. In all of these there was a strong correlation between urinary and faecal E . coli. At the next control examination, two to five months later, the correlation had disappeared in two of the girls, EL and MH. A similar pattern was observed in KK and AKW, both of whom had entered the ABU-study eight months earlier when their bacteriuria was detected at screening. In KK, on the second occasion the strain was still found in the anal and rectal swabs, but not in the faeces proper. In one girl (MJ), only selection for colony morphology showed the urinary strain to be present in the rectum and faeces. On the first sampling occasion, three girls (EA, TS and AS) harboured a urinary strain which had been present for at least one year. In TS, the strain was not found in any of the swabs, and in AS it occurred in varying proportions at all sites. In EA, three colonies conforming to the urinary strain were found in the rectum and two in the faeces proper on the first occasion. On the second occasion, more than half of the colonies from the innermost part of the faeces but none of the isolates from the anus or rectum were of the same strain. In this girl, as in EL, one negative urine culture had been obtained after a short course of nitrofurantoin, but bacteriuria with the same strain had recurred. One girl EJ, not included in Table 3 , had originally harboured a urinary E. coli 0 18 K1 biotype A (1). At the time of the study, the urinary strain was SaK1, not fermenting salicin but otherwise with the same fermentation pattern. In one rectal and one faecal swab one isolate indistinguishable from the original bacteriuria strain was found. DISCUSSION One of the aims of the present study was to study the methodological problems involved in sampling the faecal flora in bacteriuria. In a preliminary study (unpublished), 33 girls with Actu Pzdiutr

Scurrd 66

asymptomatic bacteriuria had their anuses swabbed before and on the morning after a bladder wash-out with neomycin solution (6, 12). In 20 cases, a negative urinary culture was obtained at the same time as the second and swab. A strain corresponding to the urinary E. coli was found in 12 girls both before and after the wash-out, in 13 patients neither before nor after, and in 8 before but not after the wash-out. In these cases the recovery probably represented passive contamination of the anal region and not true colonization. In endeavour to obtain a “clean catch” faecal specimen, one approach was to analyze the interior of a specimen of faeces delivered, as far as possible, separately from urination. This should represent the gut flora oral to the rectum. Another approach was to sample the flora of the rectal ampulla. The swab then had to pass through the probably contaminated anus. We tried to assess the importance of this by analyzing duplicate rectal swabs, one being introduced through a small tube. The tip of the tube may have carried some urinary bacteria, so the shielding was not expected to be perfect. The variation between the results of the two rectal swabs in each patient was, however, strikingly small and very similar to that between different swabs from the same specimen of faeces proper. When the urinary strain corresponded to less than one in thirty colonies from the innermost part of the faeces, correspondence was greater in the anal swabs than in the rectal swabs of either kind (Table 1). Thus, when the rectal mucosa was swabbed proximal to the anal canal, superficial contamination did not seem to be of any great importance. The apparent degree of correlation between urinary and faecal flora in patients with bacteriuria may thus be influenced by the sampling technique. Apart from this, an important difference was revealed when the girls of the present study were divided into two groups depending on the duration of their bacteriuria with the present strain. Bacteriuria of short

Asymptomatic bacteriuria in schoolgirls. VI

duration was defined as a fairly recent recurrence, established within 0-1 1 months. Bacteriuria of long duration was defined as either persistence of the strain harboured when bacteriuria was first detected at screening or of a recurrence established at least one year earlier. In the first group 46% of the pooled faecal isolates corresponded to the urinary strain, compared to 18 % in the second group. In the individual patients, the proportions of the urinary strains found among 60 faecal isolates were differently distributed in the short-duration and the long-duration bacteriuria group (Table 2). In the former group, there was a bimodal distribution, and in the latter there was a shift towards a low proportion of the urinary strain in the faecal flora. The development was clearly illustrated by some of the girls in whom the same bacteriuria strain was found more than once (Table 3). These findings corroborate the interpretation given by Roberts et al. (15) to their results from a study of bacteriuria in non-pregnant women. The correspondence between urinary and faecal E . coli was lower in Al3U than in symptomatic infection. Since bacteria infecting the urinary tract commonly originate in the patient’s faecal coliform flora (7, 17, 18) and since the faecal flora changes at intervals (16), it was concluded that when symptoms develop they probably do so in most cases within a short time of the infection becoming established. In most cases of mu, on the other hand, the infection will have been established long enough for the faecal flora to have changed (15). The possibility of colonization of the gut from the urinary tract (3, 15, 18) might explain the divergent findings in the rectal swabs and the faeces proper in a few cases, e.g. EA and KK, Table 3. It may well be that urine, containing at times millions of viable E. coli per millilitre, exerts a stabilizing influence on the normal flora of either mainly the rectum, via true retrograde colonization, or also of the faeces proper, via the retrograde or the oral route. Such a mechanism might ex-

353

plain some recurrences with strains identical to that found at the preceding infection, as in the girls EA and EL (Table 3). Generally, however, the dynamics of the faecal flora will in time eliminate the strain that once became established in the urinary tract. In the urinary tract, on the other hand, the strain may undergo complex changes of its antigenic and biochemical properties (1, 11). In one girl (EJ) the present urinary E. coli SaKl was considered to be a degraded variant of the 0 18 K 1 originally found in her urine and still a minority strain in her faecal flora. Thus, a strain may sometimes remain stable in the faecal flora while the clone established in the urinary tract changes under selective pressures exerted in this milieu. ACKNOWLEDGEMENTS This work was supported by grants from the Medical Faculty of Goteborg, the Goteborg Medical Association, the First of May Flower Annual Campaign for Children’s Health and the Swedish Medical Research Council (Project no. 215). The skilful technical assistance of Helena Lomberg, Ingela Delgado, Lilian Nilsson and Stina Wallenkrans is very much appreciated.

REFERENCES 1. Bettelheim, K. A. & Taylor, J . J.: A study ofEscherichia coli isolated from chronic urinary infections. J Med Microbiol, 2 : 225, 1969. 2 Bradshaw, M. W., Parke, J . C., Jr, Schneerson, R. & Robbins. J. B.: Bacterial antigen cross-reactive with the capsular polysaccharide of Haemophilus influenzae type b. Lancet, I : 1095, 1971. 3. Cooke, E. M.: Escherichia coli and man. ChurchillLivingstone, Edinburgh 1974, p. 39. 4. Docurnenta Geighy, 6th ed., Basle 1962, p. 105. 5 . Ericsson, H. M. & Sherris, J. C.: Antibiotic sensitivity testing. Acta Pathol Microbiol Scand, Suppl. 217, 1971. 6. Fairley, K . G . ,Grounds, A. D., Carson, N . E., Laird, E. C., Gutch, R. C., McCallum, P. H. G., Leighton, P . , Sleeman, R. L. & OKeefe, C. M.: Site of infection in acute urinary tract infection in general practice. Lancer, 11:615, 1971. 7. Griineberg, R. N., Leigh, D. A. & Brurnfitt, W.: Escherichia coli serotypes in urinary tract infection: studies in domiciliary, antenatal and hospital practice. In F. OGrady & W. Brurnfitt (eds.): Proceedings of the first national symposium on urinary tract infection. London 1968, p. 68. Actti

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8. Lidin-Janson, G., Falsen, E., Jodal, U., Kaijser, B . & Lincoln, K.: Characteristics of antibiotic-resistant Escherichia coli in the rectum of healthy school-children. J Med Microbiol, in press. 9. Lindberg, U.: Asymptomatic bacteriuria in schoolgirls. V. The clinical course and response to treatment. Acta Paediatr Scand, 64:718, 1975. 10. Lindberg, U., Claesson, I., Hanson, L. A. & Jodal, U.: Asymptomatic bacteriuria in schoolgirls. I . Clinical and laboratory findings. Acta Paediatr Scand, 64: 425, 1975. 11. Lindberg, U., Hanson, L. A., Jodal, U., Lidin-Janson, G., Lincoln, K. & Olling, S.: Asymptomatic bacteriuria in schoolgirls. 11. Differences in Escherichia coli causing asymptomatic and symptomatic bacteriuria. Acta Paediatr Scand, 64:432, 1975. 12. Lindberg, U., Jodal, U., Hanson, L. A. & Kaijser, B.: Asymptomatic bacteriuria in schoolgirls. IV. Difficulties of level diagnosis and the possible relation to the character of infecting bacteria. Acta Paediatr Scand, 64: 574, 1975. 13. Moiler, A.: Microbiological examination of root canals and periapical tissues of human teeth. Goteborg 1%6, p. 365. 14. Oden, A. & Wedel, H.: Arguments for Fisher’s permutation test. Ann Sfatistics,3: 518, 1975.

15. Roberts, A. P., Linton, J. D., Waterman, A. M., Gower, P. E. & Koutsaimanis, K. G.: Urinary and faecal Escherichia coli 0-serogroups in symptomatic urinary-tract infection and asymptomatic bacteriuria. J M e d Microbiol, 8:311, 1974. 16. Sears, H. J., Brownlee, I. & Uchiyama, J. K.: Persistence of individual strains ofEscherichia coli in the intestinal tract of man. J Bacteriol, 59: 293, 1950. 17. Turck, M., Petersdorf, R. G. & Fournier, M. R.: The epidemiology of non-enteric Escherichia coli infections: prevalence of serological groups. J Clin Invest, 41: 1760, 1%2. 18. Vosti, K. L., Goldberg, L. M., Monto, A. S. & Rantz, L. A.: Host parasite interaction in patients with infections due to Escherichia coli. I. The serogrouping of E . coli from intestinal and extraintestinal sources. J Clin Invest, 43: 2377, 1964. Submitted Aug. 12, 1976 Accepted Nov. 16, 1976 (G. L. J.) Dept. of Infectious Diseases East Hospital S41685 Goteborg Sweden