Characterisation of Escherichia coli strains associated with canine urinary tract infections

Research in Veterinary Science /987, 42, 404-406

Characterisation of Escherichia coli strains associated with canine urinary tract infections B. WESTERLUND, A. PERE, T. K. KORHONEN, Department of General Microbiology, University of Helsinki, SF-00280, Helsinki, Finland, A-K. JARVINEN, Department of Medicine, College of Veterinary Medicine, Helsinki, Finland, A. SIITONEN, National Public Health Laboratory, Helsinki, Finland, P. H. WILLIAMS, Department of Genetics, University of Leicester, Leicester, England

Of 33 Escherichia coli strains isolated from canine urinary tract infections, 22 were haemolytic and 27 were classified into 0 serogroups, the most common being 04, 06, 02 and 083, P-fimbriated strains were haemolytic and belonged mainly to serogroups 04 and 06. Twenty-nine strains possessed type-I fimbriae but only small numbers possessed S fimbriae, type-Ie fimbriae, X adhesins or the aerobactin system. It is postulated that P fimbriae and haemolysin production contribute to bacterial virulence in canine pyelonephritis and cystitis.

ESCHERICHIA coli is the most common causative agent of urinary tract infections in dogs (Grindlay et al1973, Kivisto et a11978) as well as in man. Certain E coli strains predominate in these infections. They belong to a limited number of 0 serogroups, notably to 02, 04, 06 and 075 (Grind lay et al 1973, Wilkinson 1974), and adhere more frequently to canine uroepithelium than do strains isolated from faeces of healthy dogs (Kivisto et al 1978). The ability to adhere to uroepithelium is also an important factor in human pyelonephritis (Svanborg Eden et al 1976). Adhesion is mediated by so-called P fimbriae (Korhonen et a11982) which bind to P-blood group-specific glycosphingolipids on human cells. Besides the P fimbriae, E coli strains associated with human urinary tract infections possess other adhesins whose role in pathogenesis remains unclear. They include S fimbriae which bind to sialyl galactosides (Korhonen et al 1984), type-I fimbriae which recognise D-mannosides and are found on most enterobacterial strains regardless of their origin (Duguid and Old 1980), type-I C fimbriae whose binding properties are unknown (Pere et al 1985) and a number of socalled X adhesins which recognise unidentified receptors on human erythrocytes (Vaisanen-Rhen et al 1984). Other recognised virulence factors in E coli from the human urinary tract are haemolytic activity (Minshew et al 1978), acidic capsules, especially the

KI capsular antigen (Kajser et al 1977), and iron scavenging systems of which the aerobactin system has received most attention (Carbonetti and Williams 1985). An important conclusion from analysis of human strains is that these factors, together with P fimbriae, occur in the same strains, which probably have a common evolutionary origin (Vaisanen-Rhen etaI1984). Putative virulence factors have not been described on E coli strains associated with canine urinary tract infections. As a step towards understanding the pathogenesis of these infections, 33 E coli strains from canine urine were screened for the presence of factors that possibly contribute to bacterial virulence.

Materials and methods The 33 E coli strains were isolated between 1981 and 1984 from dogs with significant bacteriuria (over I ()5 bacteria ml- I) as described previously (KivistO et al 1978). Twenty-six of the strains were isolated from female and nine from male dogs. The strains were stored in nutrient agar stabs containing 15 per cent glycerol at - 70°C until use. Haemolytic activity was assayed on sheep blood agar plates. P fimbriae and X adhesins were detected by specific haemagglutination tests (Vaisanen-Rhen et al 1984), type-I fimbriae by the mannose-sensitive agglutination of yeast cells (Korhonen 1979) and S fimbriae by colony blotting with a specific anti-S-fimbriae antiserum (Korhonen et al 1985). Type-I C fimbriae were detected by bacterial agglutination with specific monoclonal antibodies (Pere et aI1985). Secretion of aerobactin by bacterial isolates was determined in a bioassay using an E coli K-12 indicator strain designated LG 1522, as described previously (Carbonetti and Williams 1985). A confirmatory test (Carbonetti et a11986) used two restriction endonuclease fragments from within the defined

404

E coli from canine urinary lraci infeclions

405

TABLE 1: Characteristics of Ecoli strains from canine urinary tract infections compared with strains from human pyelonephritis and from faeces of healthy children

o group 02 04 06 08 025 050 075 083 0100 Rough Not typable*

Number of strains 3 8 6 2 2 1 1 3 1 3 3

P fimbriae 8 4

Type-l fimbriae

S

fimbriae

2 8 6 2

1 2

1 1 3 1 3 2

2 1 1 1

Number of strains with type-1C X fimbriae adhesin Haemolysin

2

2 8 6

1 1

1 2

Aerobactin

Kl capsule 2

Total

33

16

29

6

6

2

22

4

3

Human pyelonephritist Human faecest

67 50

51 8

61 38

5 2

18 0

10 3

40 5

50 16

21 11

* Not typable with antisera to the 0 types mentioned above, nor to 01,07,09,016,018,022,077,085,086 or 0119 t Data compiled from Viiisiinen-Rhen et al (1984), Pere et aI(1985), Korhonen et al (1985) and Carbonetti et aI(1986)

aerobactin gene cluster (Carbonetti and Williams 1984) as DNA probes in colony hybridisation (Grunstein and Hogness 1975) to detect the presence of the aerobactin genetic determinants. There was complete agreement between the two tests on the 33 canine isolates. 0 grouping of the strains was performed in microtitre plates with 19 anti-O sera (Table I). K I antigen was detected by a latex agglutination test (Leinonen and Sivonen 1979). Results The 27 typable strains belonged to nine different 0 groups (Table I), three strains were rough and the remail)ing three could not be typed with the anti-O sera available. The most common serogroups were 04, 06, 02 and 083. Sixteen of the strains possessed P fimbriae and 29 had type-I fimbriae. The other adhesins were, less frequent (Table I). Twenty-two strains were haemolytic but only four possessed the aerobactin system. These factors occurred with similar frequency on strains from females and males (not shown). The three KI-capsulated strains were isolated from female dogs. P fimbriae occurred on all strains of 04 serogroup and were also detected in serogroups of 06 and 075, and on rough and non-typable strains (Table I). All the 04 and 06 strains were haemolytic, but haemolysis occurred with only a few strains in the other 0 groups. It is noteworthy that the P-fimbriated strains were alsq haemolytic. The other adhesin types and the possession of an aerobactin system showed no significant association with particular 0 serogroups.

Discussion Previous reports (Grind lay et al 1973, Wilkinson 1974) have shown that E coli strains associated with canine urinary tract infection or pyometra belong to a limited number of 0 groups, in particular to 02, 04, 06 and 075. These serogroups, especially 04 and 06, were also the major serogroups in the present study. Their common occurrence may be explained by the fact that P fimbriae and haemolytic activity were frequent in these serolypes. Thus, 17 of the 22 haemolytic strains in our material belonged to serogroups 02,04,06 and 075, and 13 of the 16 P-fimbriated strains were either 04, 06 or 075. As previously noted for strains from human urinary tract infections (Vaisanen-Rhen et al 1984), P fimbriae and haemolysin largely occurred together. In the human subject, P fimbriae contribute to virulence by attaching the bacteria to uroepithelium, which enables them to resist the cleansing action of urine flow. P fimbriae, or other adhesins, have not been recognised on E coli strains from faeces of healthy dogs, but those from canine urinary tract infections are known to differ in that they adhere efficiently to canine urinary tract epithelium (Kivisto et al 1978), and it may be that P fimbriae are involved in this process. Haemolysin is known to cause tissue damage (Cavalieri and Snyder 1982) and to increase the amount of free iron available for bacterial growth (Linggood and Ingram 1982). About 80 per cent of all E coli strains (Duguid and Old 1980, Vaisanen-Rhen et aI1984), including those in the present study, possess mannose-binding type-I fimbriae which probably function as colonisation

406

B. Westerlund, A. Pere, T. K. Korhonen, A-K. Jarvinen, A. Siitonen, P. H. Williams

factors in the intestine. Type-I fimbriae are known to bind to phagocytic cells, a property which enhances their elimination from parenteral sites in the animal body (Leunk and Moon 1982). The frequencies of type-Ie and S fimbriae and of X adhesins on the canine strains were low and comparable with those on the E coli strains from human urinary tract infections. The aerobactin system occurred in only four of the canine strains (Table I). In this respect they differed markedly from human pyelonephritis and cystitis strains. It may be that the canine strains utilise systems other than aerobactin for iron scavenging or that iron is not a growth-limiting factor for E coli in the canine urinary tract. Also, the frequency of the KI capsular antigen was fairly low in these strains. Some similarities between E coli strains associated with canine urinary tract infections and those causing pyelonephritis in humans have been demonstrated, indicating that the pathogenetic mechanisms in the two hosts may be similar. The similarity between human and canine pyelonephritogenic strains may in fact be higher than these numbers indicate since in the present study it was not possible to differentiate between pyelonephritis and cystitis. The findings should help in the detailed study of the pathogenesis of canine urinary tract infections as well as in the design of methods for their prevention. The data also suggest that dogs are potential carriers of P-fimbriated and haemolytic E coli strains within human communities. Acknowledgements This study was supported by the Academy of Finland. We thank Tuula Taskinen for skilled technical assistance.

CARBONETTI, N. H. & WILLIAMS. P. H. (1984) Injection and Immunity 46,7-12 CARBONETTI, N. H. & WILLIAMS, P. H. (1985) The Virulence of Escherichia coli. Ed M. Sussman. New York, Academic Press. pp 419-424 CAVALIERI, S. J. & SNYDER. I. S. (1982) Journa! oj Medica! Microbio!ogy 15,11-21 DUGUID, J. P. & OLD, D. C (1980) Bacterial Adherence. Ed E. H. Beachey. London, Chapman and Hall. pp 185-217 GRINDLAY, M., RENTON, J. P. & RAMSAY, D. H. (1973) Research in Veterinary Science 14, 75-77 GRUNSTEIN, M. & HOG NESS, D. S. (1975) Proceedings oj the Nationa! Academy oj Sciences USA 72, 3961-3965 KAJSER, B., HANSON, L. A., JODAL, U., L1D1N-JANSON, G. & ROBBINS. J. B. (1977) Lancel i, 663-664 KIVISTO, A-K., VASENIUS, H., LINDBERG, L-A. & SANDHOLM, M. (1978) Investigative Uro!ogy 15,412-415 KORHONEN, T. K. (1979) fEMS Microbio!ogy Letters 6, 421-425 KORHONEN, T. K., vAISANEN-RHEN, V., RHEN, M., PERE, A .. PARKKINEN, J. & FINNE, J. (1984) Journal oj Bacteriology 159, 762-766 KORHONEN, T. K., VAISA.NEN, V., SAXEN, H., HULTBERG, H. & SVENSON, S. B. (1982) Injection and Immunity 37, 286-291 KORHONEN, T. K., VALTONEN, M. V., PARKKINEN, J., VAISANEN-RHEN, V., FINNE, J., ORSKOV, F., ORSKOV,I., SVENSON, S. B. & MAKELA, P. H. (1985) Infection and !mmunily 48, 486-491 LEINONEN, M. & SIVONEN, A. (1979) Journa! ojClinica! Microbio!ogy 10, 404-408 LEUNK, R. D. & MOON, R. J. (1982) "(tection and Immunity 36, 1168-1174 L1NGGOOD, M. A. & INGRAM, P. L. (1982) Journa! of Medica! Microbio!ogy 15, 23-30 MINSHEW, B. H., JORGENSEN, J., COUNTS, G. W. & FALKOW, S. (1978) Injection and Immllnity 20, 50-54 PERE, A., LEINONEN, M., VAISA.NEN-RHEN, V., RHEN, M. & KORHONEN, T. K. (1985) Journa! oj Genera! Microbio!ogy 131, 1705-1711 SVANBORG EDEN, C, HANSON, l.. A .. JODAL, U., I.INDBERG, U. & SOHL-AKERLUND, A. (1976) Lancet ii, 490-492 VAISA.NEN-RHEN, V.. ELO, J., VA.ISA.NEN, V., SIITONEN, A., ORSKOV, I., ORSKOV, F., SVENSON, S. B., MAKELA, P. H. & KORHONEN, T. K. (1984) In/ection and Immllnity 43, 149-155 WILKINSON, G. T. (1974) Veterinary Re('()rd 94, 105

References CARBONETTI. N. H .• BOONCHAI, S., PARRY, S. H .• VA.ISA.NEN-RHEN, V., KORHONEN, T. K. & WILLIAMS, P. H. (1986) Injection and Immunity 51, 966-968

Accepled April/8, /986