T H E FIMBRIAL A N D NON-FIMBRIAL HAEMAGGLUTININS O F ESCHERICHIA COLI
J. P. DUGUID,S. CLEGGAND MARGARET I. WILSON Bacteriology Department, University of Dundee Medical School, Ninewells Hospital, Dundee, DDI 9s Y, Scotland
HAEMAGGLUTINATION by Escherichia coli was first described by Guyot (1908) who found that 12 of 18 strains agglutinated the red blood cells from one or more of 13 species of animals. Each strain gave the same reactions with the cells from different animals of the same species. Rosenthal(l943) showed that haemagglutinating cultures also agglutinated leucocytes, sperms, yeasts and pollens, and Kauffmann (1948) found that 78 of 112 strains in 0 serogroups 1-25 were haemagglutinating. Duguid et al. (1955) distinguished three groups of E. coli strains with different patterns of haemagglutinating activity against the red cells of different animal species and a fourth group that was non-haemagglutinating. The haemagglutinating bacteria adhered to the red cells and bound them together. All strains in group I showed the same pattern of activity, agglutinating most species of red cells strongly, human cells moderately strongly, sheep and goat cells weakly, and ox cells not at all. Their haemagglutinating activity was associated with the possession of filamentous appendages, named " fimbriae ", which were best developed in stationary-phase cultures grown serially in static liquid media. It was wholly inhibited in the presence of 0.5 % (w/v) D-mannose or methyl-alpha-D-mannoside (Duguid and Gillies, 1957) and the fimbriae with this mannose-sensitive (MS) activity were later classified as " type 1 " by Duguid, Anderson and Campbell (1966), Duguid (1968) and Ottow (1975). The strains of groups I1 and I11 of Duguid et al. (1955) showed a variety of patterns of reactions with different red cells; e.g., some strains strongly agglutinated ox, sheep, and human cells, and one strain agglutinated only human cells out of 18 species tested. Unlike the haemagglutinating property of the group-I strains, that of groups I1 and I11 was best developed in cultures grown at 37°C on agar and best demonstrated in tests mixed at 3 4 ° C . The reactions were unaffected by the presence of D-mannose (Duguid and Gillies, 1957) and when the agglutinated mixtures were warmed to lo", 20", 30", 40", or 50"C, the bacteria eluted from the red cells and the clumps dispersed. This kind of haemagglutinin was therefore described as mannose-resistant and eluting (MRE) by Duguid (1964). Groups I1 and I11 differed from one another in that strains of the former possessed fimbriae, whilst those of the latter did not. Because only a few groupI1 strains had by then been studied, their fimbriae were not included in the classification of fimbrial types by Duguid et al. (1966). For the present, they will be called " type MRE ". Received 10 Oct. 1978; accepted 30 Oct. 1978. J. MED. MICROBIOL.-VOL.
12 (1979)
213
214
J. P . DUGUID, S . CLEGG AND MARGARET I. WILSON
Interest in the haemagglutinating properties of E. coli has been re-awakened by discoveries associating them with the presence of particular K antigens, adhesiveness for intestinal epithelium, ability to colonise the upper intestine, and enteropathogenicity in man (Duguid, 1964; Evans et al., 1975; McNeish .et al., 1975; Evans, Evans and Tjoa, 1977; 0rskov and 0rskov, 1977), pigs (Stirm et al., 1967; Jones and Rutter, 1972,1974; Hohmann and Wilson, 1975; Isaacson, Nagy and Moon, 1977; Parry and Porter, 1978) and in calves {Burrows, Sellwood and Gibbons, 1976). Because different strains of E. coli may possess one or more kinds of haemagglutinin that require different cultural conditions for their development and different techniques and species of erythrocytes for their demonstration, and because most published studies have been made with only a few strains and a restricted range of tests, we thought it valuable to describe the haemagglutinating properties of a wide range of strains from different serotypes and sources. MATERIALS AND METHODS Bacteria We examined 387 strains of E. coli, 177 of which were from the National Collection of Type Cultures (NCTC) and represented 0 serogroups 1-162 inclusive, except 67,72,94, 122, 159,160 and 161. Other strains were 47 described by Duguid et al. (1955), 25 in 0 serogroups 1-25 received from Dr M. Mulczyk, 10 in 0 serogroups 8, 141, 147 and 149, which contained K88 antigen, received from Dr H. Williams Smith, and 83 isolated from children with gastroenteritis in Dundee and elsewhere, including 14 isolated in Birmingham by Dr K. B. Rogers and 9 isolated in Glasgow by Dr Constance Ross. Another 45 strains were from patients with urinary-tract infections in Edinburgh. All strains were stored on Dorset egg slopes at ambient temperature until subcultured for testing. Culture media Nutrient broth ( p H 7.3) was 1 % (w/v) Oxoid Bacteriological Peptone, 1% (w/v) Oxoid Lab-Lemco beef extract and 0.5 % (w/v) NaCI, or Oxoid Nutrient Broth No. 2, used in 8-10ml volumes in test-tubes (150 x 15 mm) stoppered with cotton-wool. Phosphate-buffered nutrient agar (PB agar) was this nutrient broth buffered at pH 7.0 by the addition of 0.35g KH2P04 and 0.65 g Na2HP04/100 ml and solidified by the addition of Davis New Zealand Agar, 1% (w/v). It was used in 20-ml volumes in petri dishes 85 mm in diameter. Red blood cells Bloods were obtained from laboratory breeds of guinea-pigs, rabbits, rats, mice, toads #( Xeitopus laevis), frogs (Rana temporaria)and domesticated fowls (hens), and from unselected breeds of oxen, sheep, pigs and dogs after slaughter at the abattoir or during veterinary care. Horse blood was Oxoid Defibrinated Horse Blood; rhesus-monkey blood in Alsever’s solution was obtained from Flow Laboratories. Human blood used with all strains was group 0, obtained from the transfusion service, but bloods from individuals of other blood groups were tested with some strains. The red cells were separated by centrifugation from citrated, heparinised, or defibrinated blood, washed twice or thrice in 0.85% (w/v) NaCl solution (saline) and made up to a 3 % (v/v) suspension in fresh saline (e.g., guinea-pig cells to c. 5 x 10*/ml). They were used immediately, or after only a few days’ storage at 3-5°C. MS-haemagglutination tests Guinea-pig red cells were used in the standard test for mannose-sensitive (MS) haemagglutinating activity. Cultures of each strain were grown serially in tubes of broth incubated
FIMBRIAL AND NON-FIMBRIAL HAEMAGGLUTININS OF E. COLI
215
statically at 37°C for 48 h, and up to six cultures of each series were tested. The culture was centrifuged and the supernatant fluid drained from the tube. The bacillary deposit was resuspended in the small amount of residual fluid (c. 1.5 x 1011bacteria/ml) and one drop was added to one drop of red-cell suspension and one drop of saline in a circular depression, 25 mm in diameter, on a white glazed porcelain tile. The tile was rocked at ambient temperature (c. 20°C) by tilting it to and fro to an angle of 30" below the horizontal 30 times/min. for 10 min. Haemagglutination was read as strongly positive (+ +) or moderately strong (+ +) when coarse clumping of the red cells took place within, respectively, c. 20 s or c. 2 min., and weakly positive (+) when fine clumping became perceptible to the unaided eye within 10 min. Absence of haemagglutination in a parallel test in which a drop of 1.5 or 2.0 % (w/v) D-mannose was substituted for the drop of saline in the mixture showed that the activity was mannose sensitive.
+
MRE-haemagglutination tests Tests for mannose-resistant eluting (MRE) haemagglutination were made with bacteria from cultures grown for 18 h at 37°C on plates of PB agar and with the different species of red blood cells shown in table 1. Rocked-tile method. This was the standard method used with all strains. Growth from the PB agar was made up to a dense suspension in saline (c. 1012 bacteria/ml). One drop of this suspension was mixed with one drop of red blood cells and one drop of 1.5 or 2.0% D-mannose in a depression on a tile, and the tile was rocked for 5 min. at c. 20°C and then for 15 min. in a cold room at 3-5°C before reading. In an alternative method, which gave similar results, a loopful of confluent bacterial growth from the agar was suspended directly in a mixture of one drop each of red cells, mannose and saline on a tile at c. 20°C; the tile was chilled to c. 0°C in the freezing compartment of a refrigerator and the test was read within 30 s of the resumption of mixing by rocking in air at c. 20°C. Elution of the bacteria from the red cells and the resultant disappearance of agglutination were demonstrated when the tile, with continued rocking, was gently warmed to between 20°C and 50°C. Settling method. This method was used for a selection of strains, including those with K88 antigen. We followed the " microhaemagglutination" procedure of Jones and Rutter (1974) in which the bacteria and red cells are allowed to settle for 1 h in the wells of a plastic tray, but modified in that we grew the bacteria on PB agar without added glucose, used methyl-alpha-D-mannoside, which is not fermented by E. coli, instead of D-mannose as the inhibitor of MS activity, tested red-cell suspensions at 1 % and at 3 % (v/v), and held the tests at 3-5°C instead of 0-3°C. Electron microscopy Broth cultures were examined for type-1 fimbriae, and broth and PB agar cultures for typeMRE fimbriae, with a transmission electron microscope at a gun voltage of 60 kV. The bacteria, washed in distilled water, were either shadow-cast with gold-palladium alloy at an angle of 15" or negatively stained with a 2 % (w/v) solution of phosphotungstic acid (pH 7.0). Serological agglutination tests Antifimbrial sera were prepared and agglutination tests done as by Gillies and Duguid ( 1 958).
J. P. DUGUID,S. CLEGGAND MARGARET I. WILSON Bacteriology Department, University of Dundee Medical School, Ninewells Hospital, Dundee, DDI 9s Y, Scotland
HAEMAGGLUTINATION by Escherichia coli was first described by Guyot (1908) who found that 12 of 18 strains agglutinated the red blood cells from one or more of 13 species of animals. Each strain gave the same reactions with the cells from different animals of the same species. Rosenthal(l943) showed that haemagglutinating cultures also agglutinated leucocytes, sperms, yeasts and pollens, and Kauffmann (1948) found that 78 of 112 strains in 0 serogroups 1-25 were haemagglutinating. Duguid et al. (1955) distinguished three groups of E. coli strains with different patterns of haemagglutinating activity against the red cells of different animal species and a fourth group that was non-haemagglutinating. The haemagglutinating bacteria adhered to the red cells and bound them together. All strains in group I showed the same pattern of activity, agglutinating most species of red cells strongly, human cells moderately strongly, sheep and goat cells weakly, and ox cells not at all. Their haemagglutinating activity was associated with the possession of filamentous appendages, named " fimbriae ", which were best developed in stationary-phase cultures grown serially in static liquid media. It was wholly inhibited in the presence of 0.5 % (w/v) D-mannose or methyl-alpha-D-mannoside (Duguid and Gillies, 1957) and the fimbriae with this mannose-sensitive (MS) activity were later classified as " type 1 " by Duguid, Anderson and Campbell (1966), Duguid (1968) and Ottow (1975). The strains of groups I1 and I11 of Duguid et al. (1955) showed a variety of patterns of reactions with different red cells; e.g., some strains strongly agglutinated ox, sheep, and human cells, and one strain agglutinated only human cells out of 18 species tested. Unlike the haemagglutinating property of the group-I strains, that of groups I1 and I11 was best developed in cultures grown at 37°C on agar and best demonstrated in tests mixed at 3 4 ° C . The reactions were unaffected by the presence of D-mannose (Duguid and Gillies, 1957) and when the agglutinated mixtures were warmed to lo", 20", 30", 40", or 50"C, the bacteria eluted from the red cells and the clumps dispersed. This kind of haemagglutinin was therefore described as mannose-resistant and eluting (MRE) by Duguid (1964). Groups I1 and I11 differed from one another in that strains of the former possessed fimbriae, whilst those of the latter did not. Because only a few groupI1 strains had by then been studied, their fimbriae were not included in the classification of fimbrial types by Duguid et al. (1966). For the present, they will be called " type MRE ". Received 10 Oct. 1978; accepted 30 Oct. 1978. J. MED. MICROBIOL.-VOL.
12 (1979)
213
214
J. P . DUGUID, S . CLEGG AND MARGARET I. WILSON
Interest in the haemagglutinating properties of E. coli has been re-awakened by discoveries associating them with the presence of particular K antigens, adhesiveness for intestinal epithelium, ability to colonise the upper intestine, and enteropathogenicity in man (Duguid, 1964; Evans et al., 1975; McNeish .et al., 1975; Evans, Evans and Tjoa, 1977; 0rskov and 0rskov, 1977), pigs (Stirm et al., 1967; Jones and Rutter, 1972,1974; Hohmann and Wilson, 1975; Isaacson, Nagy and Moon, 1977; Parry and Porter, 1978) and in calves {Burrows, Sellwood and Gibbons, 1976). Because different strains of E. coli may possess one or more kinds of haemagglutinin that require different cultural conditions for their development and different techniques and species of erythrocytes for their demonstration, and because most published studies have been made with only a few strains and a restricted range of tests, we thought it valuable to describe the haemagglutinating properties of a wide range of strains from different serotypes and sources. MATERIALS AND METHODS Bacteria We examined 387 strains of E. coli, 177 of which were from the National Collection of Type Cultures (NCTC) and represented 0 serogroups 1-162 inclusive, except 67,72,94, 122, 159,160 and 161. Other strains were 47 described by Duguid et al. (1955), 25 in 0 serogroups 1-25 received from Dr M. Mulczyk, 10 in 0 serogroups 8, 141, 147 and 149, which contained K88 antigen, received from Dr H. Williams Smith, and 83 isolated from children with gastroenteritis in Dundee and elsewhere, including 14 isolated in Birmingham by Dr K. B. Rogers and 9 isolated in Glasgow by Dr Constance Ross. Another 45 strains were from patients with urinary-tract infections in Edinburgh. All strains were stored on Dorset egg slopes at ambient temperature until subcultured for testing. Culture media Nutrient broth ( p H 7.3) was 1 % (w/v) Oxoid Bacteriological Peptone, 1% (w/v) Oxoid Lab-Lemco beef extract and 0.5 % (w/v) NaCI, or Oxoid Nutrient Broth No. 2, used in 8-10ml volumes in test-tubes (150 x 15 mm) stoppered with cotton-wool. Phosphate-buffered nutrient agar (PB agar) was this nutrient broth buffered at pH 7.0 by the addition of 0.35g KH2P04 and 0.65 g Na2HP04/100 ml and solidified by the addition of Davis New Zealand Agar, 1% (w/v). It was used in 20-ml volumes in petri dishes 85 mm in diameter. Red blood cells Bloods were obtained from laboratory breeds of guinea-pigs, rabbits, rats, mice, toads #( Xeitopus laevis), frogs (Rana temporaria)and domesticated fowls (hens), and from unselected breeds of oxen, sheep, pigs and dogs after slaughter at the abattoir or during veterinary care. Horse blood was Oxoid Defibrinated Horse Blood; rhesus-monkey blood in Alsever’s solution was obtained from Flow Laboratories. Human blood used with all strains was group 0, obtained from the transfusion service, but bloods from individuals of other blood groups were tested with some strains. The red cells were separated by centrifugation from citrated, heparinised, or defibrinated blood, washed twice or thrice in 0.85% (w/v) NaCl solution (saline) and made up to a 3 % (v/v) suspension in fresh saline (e.g., guinea-pig cells to c. 5 x 10*/ml). They were used immediately, or after only a few days’ storage at 3-5°C. MS-haemagglutination tests Guinea-pig red cells were used in the standard test for mannose-sensitive (MS) haemagglutinating activity. Cultures of each strain were grown serially in tubes of broth incubated
FIMBRIAL AND NON-FIMBRIAL HAEMAGGLUTININS OF E. COLI
215
statically at 37°C for 48 h, and up to six cultures of each series were tested. The culture was centrifuged and the supernatant fluid drained from the tube. The bacillary deposit was resuspended in the small amount of residual fluid (c. 1.5 x 1011bacteria/ml) and one drop was added to one drop of red-cell suspension and one drop of saline in a circular depression, 25 mm in diameter, on a white glazed porcelain tile. The tile was rocked at ambient temperature (c. 20°C) by tilting it to and fro to an angle of 30" below the horizontal 30 times/min. for 10 min. Haemagglutination was read as strongly positive (+ +) or moderately strong (+ +) when coarse clumping of the red cells took place within, respectively, c. 20 s or c. 2 min., and weakly positive (+) when fine clumping became perceptible to the unaided eye within 10 min. Absence of haemagglutination in a parallel test in which a drop of 1.5 or 2.0 % (w/v) D-mannose was substituted for the drop of saline in the mixture showed that the activity was mannose sensitive.
+
MRE-haemagglutination tests Tests for mannose-resistant eluting (MRE) haemagglutination were made with bacteria from cultures grown for 18 h at 37°C on plates of PB agar and with the different species of red blood cells shown in table 1. Rocked-tile method. This was the standard method used with all strains. Growth from the PB agar was made up to a dense suspension in saline (c. 1012 bacteria/ml). One drop of this suspension was mixed with one drop of red blood cells and one drop of 1.5 or 2.0% D-mannose in a depression on a tile, and the tile was rocked for 5 min. at c. 20°C and then for 15 min. in a cold room at 3-5°C before reading. In an alternative method, which gave similar results, a loopful of confluent bacterial growth from the agar was suspended directly in a mixture of one drop each of red cells, mannose and saline on a tile at c. 20°C; the tile was chilled to c. 0°C in the freezing compartment of a refrigerator and the test was read within 30 s of the resumption of mixing by rocking in air at c. 20°C. Elution of the bacteria from the red cells and the resultant disappearance of agglutination were demonstrated when the tile, with continued rocking, was gently warmed to between 20°C and 50°C. Settling method. This method was used for a selection of strains, including those with K88 antigen. We followed the " microhaemagglutination" procedure of Jones and Rutter (1974) in which the bacteria and red cells are allowed to settle for 1 h in the wells of a plastic tray, but modified in that we grew the bacteria on PB agar without added glucose, used methyl-alpha-D-mannoside, which is not fermented by E. coli, instead of D-mannose as the inhibitor of MS activity, tested red-cell suspensions at 1 % and at 3 % (v/v), and held the tests at 3-5°C instead of 0-3°C. Electron microscopy Broth cultures were examined for type-1 fimbriae, and broth and PB agar cultures for typeMRE fimbriae, with a transmission electron microscope at a gun voltage of 60 kV. The bacteria, washed in distilled water, were either shadow-cast with gold-palladium alloy at an angle of 15" or negatively stained with a 2 % (w/v) solution of phosphotungstic acid (pH 7.0). Serological agglutination tests Antifimbrial sera were prepared and agglutination tests done as by Gillies and Duguid ( 1 958).
