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Clonal distributionof septicaemic escherichia coli 078 isolates, evidenced by enzyme electrophoretic polymorphism
Paris
A~m. hTst. Pasteur/MicrohioL
!~gg
1988, 139~ 23%242
BRiL!: ,,iOTE CI_t'.)NAL DISTRIBUTION OF SEPTICAEM!C E S C H E R I C H I A C O L I 078 ISOLATES,
EVIDENCED BY ENZYME ELECTROPHORETIC POLYMORPHISM B. Picard (i), p. Goullet (t) and M. Contrepois (~) (i) Laboratoire de Microbiologie, Facultd X. Bichat, Universitd Paris VII, 75018 Pari,~ and (2) Institut National de la Recherche Agronomique, Laboratoire de Microbiologie, Centre de Recherches de Clermont-Ferrand- Theix, 63122 Ceyrat (Ft cmce)
INTRODUCTION
Escherichia coli isolates with serogroup 078 have been identified in septicaemia in various animal species [8]. They have also been isolated in human extraintestinal infections [71.
A previous study indicated that the animal septicaemic 078 E. coli ColV producers are very closely related in their outer membrane protein (OMP) patterns and could constitute a clone [2]. Since meaningful ctonal groupings are best recog~ized by the combination of OMP and electrophoretic enzyme patterns [1, 9J, we have compared these two characters in 24 E. coli 078 isolates from diverse animal aud human origins. For this purpose, electropl-:aretic variability of esterases A, B, C and I [3] and of acid phosphatase (AP), glutamate (GDH), lactate (LDH) and L-malate (MDH) dehydrogenases, was analysed by polyacrylamide agarose gel electrophoresis (PAGE). MATERIALS AND METHODS Bacterial strains. - - The 24 tested strains are listed in table 1. Culture conditions. - - All isolates were cultured in Fernbach flasks containing 500 ml L broth [3] without glucose. The flasks were shaken vigourousty for 18 h at 37°C in a recip, ocating water bath shaker set at about 70 o s c i l l a t i o n s / m i n
Submitted Novembcr 30, 1987, accepted January 2, 1988.
B. PICARD, P. G O U L L E T A N D M. CON7 REPOIS
240
TABtL- I. - -
Laboratory code 1-6 7-9 10-12 13-15 16-18 19 20 2t 22 23 24
Strain designation 3, 4, 31, 34, 914, V2019 Ri5, X54, X429 R2, R3, R4 MT99, $61, MLM R 9 8 1 , R789, R1528 M 10 1404 JL 9 G 2820 RVC 330 JL 10
Strains examined. Source
Country of origin
OMP pattern
Calf
France
t
Calf Lamb Chicken Human
England dngland France Hungary
1 1 1 1
Piglet Calf Calf Calf Calf Calf
England France Canada France England Canada
i 2a 2a 2b 3 4
C~MP patterns previously analysed for strains 1-15 and 19-24 by Dassouli-Mrani-Belkebir et al. [2]. O M P pattern of human isolates 16-18 have been added for the present study.
E l e c t r o p h o r e l i c analysis. - - The preparation of extracts, inhibition by di-isopropyl fluorophosphate (DFP), heat inactivation, horizontal slab PAGE (7 % w/v, acrylamide, Tris/glycine buffer pH 8.6), estimation of electrophoretic mobility (Em, or Mf) and esterase staining were described by Goullet [3]. Glutamate dehydrogenase, lactate dehydrogenase, L-malade dehydrogenase and acid phosphatase were stained as previously described [4].
RESULTS
AP, GDH, LDH, MDH and esterase~ B and C were m o n o m o r p h i c and detected in all strains, whereas esterase A was evidenced in only 2 strains and esterase I showed two type~ of mobility. The mean of genetic diversity coefficient for the four esterases was 0.12. In addition, one faint band hydrolysing ~-naphthyl acetate and migrating at Em -- 74 was detected in strains 1 to 22. The 24 E. coli isolates were distributed into 4 electrophoretic types (Et) (distinct combination of electrophoretic variants for all enzymes) (table I I). Et I was the most frequent (18 strains) and included the 3 isolates of human origin.
AP DFP Et GDH LDH
= = = = =
acid phosphatase. di-isopropyl fluorophosphate. electrophoretype. glutamate dehydrogenase. lactate dehydrogenase.
MDH Em OMP PAGE
= = = =
L-malate dehydrogenase. electrophoretic mobiliD t vlf). outer m e m b r a n e protein. polyacrylamide agarose gel electrophoresis.
ENZYME ELECTROPHORETYPES
O F E. COLI
241
TABLE II. -- Electrophoretypes distinguished in |he 24 strains of E. coli 078. Electrophoretypes (Em or Mf values) Electrophoretic mobilities of AP GDH LDH MDH Estcrase Esterase Esterase Esterase
A B C I
1 (strains ~ 18)
2 (strains 19 to 21)
3 (strain 22)
4 (strains 23 and 24)
38 12 23 35 -70 57 60
38 12 23 35 -70 57 68
38 12 23 35 -70 57 --
~8 12 23 35 81 70 57 68
DISCUSSION The m o n o m o r p h i s m of A P , G D H , L D H , M D H and esterases B and C and the low degree of variability of esterases A and I suggest the clonal structure of the 24 E. coli 0 7 8 strains analysed. The four Et were distinct from those of 610 E. co# strains and 182 Shigella strains previously studied [5, 6]. Most of the E. coli 0 7 8 strains belonged to E t l , which included the 3 strains isolated from humans. Our work demonstrated the strong association between O M P patterns and Et : 18 of the 19 strains exhibiting O M P pattern 1 showed Et 1. Consequently, this homogeneous group could be considered as an E. coli clone isolated from septicaemia in animals and also from h u man infections. Strain M 10 differed only in esterase I mobility and can be denoted as a strain variant of the clone [1]. Interestingly, this piglet isolate was also the only haemolytic strain. The five o~aer strains which were of distinct O M P pattern were distributed in the 2 other Et. MOTS-CLES: Escherichia coli, Septic6mie, Enzyme; Distribu,ion clonale, Electrophoretype.
REFERENCES [1] ACHTMAN,M., HEUZENROEDER,M., KUSECEK,B., OCHMAN, Ho, CAUGANT, D., SELANDER,R.K., "vAISANEN-RHEN,V., KORHONEN,T.K., STUART,S., ORSKOV, F. & ORSKOV, I., Clonal analysis of Escherichia coli O2:K1 isolated from diseased humans and animals. Infect. Immun., 1986, 51, 268-276.
242
B. P i C A R D ,
P. G O U L L E T
A N D M. C O N T R E P O L q
[2{] DASSOUH-MRANI-BELKEBIP,,A., CONTREPOIS, M., G1RARDEAU,J.P. & Dee V aRTANIAN~M.~ Outer membrane analysis of Esc;,e:ichia coli 078 isolated from septicemic animals. Vet. Microbiot. (in press). [3] GOULLLT, Ph., An esterase zymogram of Escherichia coll. J. gen. MicrobioL, 1973, 77, 27-35. [4] GOUH_ET, P. & PICARD, B., Etude du polymorphisme 6lectrophordtique des lactate-, malate- et glutamate-deshydrogenases, de la phosphatase acide et des est&ases de Providencia alcalifaciens, P. stuartii et P. rustigianii. Ann. hTst. Pasteur/Microbiol., 1985, 136 A, 347-358. [51 GOULLH, Pb. & PICARD, Bo, Comparative esterase electrophoretic polymorphism of Escherichia coil isolates obtained from animals and human sources. J. gen. MtcrooloJ., 1986, 132, 1843-1831. [6] GOt'LLEt, Ph. & P!CARD, B., Differentiation of Shigella by esterase electrophoretic polymorphism. J. gen. Microbiol., 1987, 133, 1005-1017. [7] MncH, H., NIKOt.NIKOV,S. & CZlROK, E., Escherichia coli CoW plasmids and their role in pathogenicity. Acta Microbiol. Hung., i984, 31, 117-125. [8] OrSKOV, F. & ORSKOV, l., Serotyping of Escherichia coli, in ~Methods in Microbiology ~, (T. Bergan), 14 (pp. 44-112). Academic Press, London, New York, 1984. [9] SEI.ANDER,R.K., CAUGANT,D.A. & WHIFTAM,T.S., Genetic structure and variation in natural populations of Escherichia coli, in ¢ Cellular and molecular biology~> (Neidhardt), 2 (pp. 1625-1648). American Society for Microbiology, Washington, 1987.
A~m. hTst. Pasteur/MicrohioL
!~gg
1988, 139~ 23%242
BRiL!: ,,iOTE CI_t'.)NAL DISTRIBUTION OF SEPTICAEM!C E S C H E R I C H I A C O L I 078 ISOLATES,
EVIDENCED BY ENZYME ELECTROPHORETIC POLYMORPHISM B. Picard (i), p. Goullet (t) and M. Contrepois (~) (i) Laboratoire de Microbiologie, Facultd X. Bichat, Universitd Paris VII, 75018 Pari,~ and (2) Institut National de la Recherche Agronomique, Laboratoire de Microbiologie, Centre de Recherches de Clermont-Ferrand- Theix, 63122 Ceyrat (Ft cmce)
INTRODUCTION
Escherichia coli isolates with serogroup 078 have been identified in septicaemia in various animal species [8]. They have also been isolated in human extraintestinal infections [71.
A previous study indicated that the animal septicaemic 078 E. coli ColV producers are very closely related in their outer membrane protein (OMP) patterns and could constitute a clone [2]. Since meaningful ctonal groupings are best recog~ized by the combination of OMP and electrophoretic enzyme patterns [1, 9J, we have compared these two characters in 24 E. coli 078 isolates from diverse animal aud human origins. For this purpose, electropl-:aretic variability of esterases A, B, C and I [3] and of acid phosphatase (AP), glutamate (GDH), lactate (LDH) and L-malate (MDH) dehydrogenases, was analysed by polyacrylamide agarose gel electrophoresis (PAGE). MATERIALS AND METHODS Bacterial strains. - - The 24 tested strains are listed in table 1. Culture conditions. - - All isolates were cultured in Fernbach flasks containing 500 ml L broth [3] without glucose. The flasks were shaken vigourousty for 18 h at 37°C in a recip, ocating water bath shaker set at about 70 o s c i l l a t i o n s / m i n
Submitted Novembcr 30, 1987, accepted January 2, 1988.
B. PICARD, P. G O U L L E T A N D M. CON7 REPOIS
240
TABtL- I. - -
Laboratory code 1-6 7-9 10-12 13-15 16-18 19 20 2t 22 23 24
Strain designation 3, 4, 31, 34, 914, V2019 Ri5, X54, X429 R2, R3, R4 MT99, $61, MLM R 9 8 1 , R789, R1528 M 10 1404 JL 9 G 2820 RVC 330 JL 10
Strains examined. Source
Country of origin
OMP pattern
Calf
France
t
Calf Lamb Chicken Human
England dngland France Hungary
1 1 1 1
Piglet Calf Calf Calf Calf Calf
England France Canada France England Canada
i 2a 2a 2b 3 4
C~MP patterns previously analysed for strains 1-15 and 19-24 by Dassouli-Mrani-Belkebir et al. [2]. O M P pattern of human isolates 16-18 have been added for the present study.
E l e c t r o p h o r e l i c analysis. - - The preparation of extracts, inhibition by di-isopropyl fluorophosphate (DFP), heat inactivation, horizontal slab PAGE (7 % w/v, acrylamide, Tris/glycine buffer pH 8.6), estimation of electrophoretic mobility (Em, or Mf) and esterase staining were described by Goullet [3]. Glutamate dehydrogenase, lactate dehydrogenase, L-malade dehydrogenase and acid phosphatase were stained as previously described [4].
RESULTS
AP, GDH, LDH, MDH and esterase~ B and C were m o n o m o r p h i c and detected in all strains, whereas esterase A was evidenced in only 2 strains and esterase I showed two type~ of mobility. The mean of genetic diversity coefficient for the four esterases was 0.12. In addition, one faint band hydrolysing ~-naphthyl acetate and migrating at Em -- 74 was detected in strains 1 to 22. The 24 E. coli isolates were distributed into 4 electrophoretic types (Et) (distinct combination of electrophoretic variants for all enzymes) (table I I). Et I was the most frequent (18 strains) and included the 3 isolates of human origin.
AP DFP Et GDH LDH
= = = = =
acid phosphatase. di-isopropyl fluorophosphate. electrophoretype. glutamate dehydrogenase. lactate dehydrogenase.
MDH Em OMP PAGE
= = = =
L-malate dehydrogenase. electrophoretic mobiliD t vlf). outer m e m b r a n e protein. polyacrylamide agarose gel electrophoresis.
ENZYME ELECTROPHORETYPES
O F E. COLI
241
TABLE II. -- Electrophoretypes distinguished in |he 24 strains of E. coli 078. Electrophoretypes (Em or Mf values) Electrophoretic mobilities of AP GDH LDH MDH Estcrase Esterase Esterase Esterase
A B C I
1 (strains ~ 18)
2 (strains 19 to 21)
3 (strain 22)
4 (strains 23 and 24)
38 12 23 35 -70 57 60
38 12 23 35 -70 57 68
38 12 23 35 -70 57 --
~8 12 23 35 81 70 57 68
DISCUSSION The m o n o m o r p h i s m of A P , G D H , L D H , M D H and esterases B and C and the low degree of variability of esterases A and I suggest the clonal structure of the 24 E. coli 0 7 8 strains analysed. The four Et were distinct from those of 610 E. co# strains and 182 Shigella strains previously studied [5, 6]. Most of the E. coli 0 7 8 strains belonged to E t l , which included the 3 strains isolated from humans. Our work demonstrated the strong association between O M P patterns and Et : 18 of the 19 strains exhibiting O M P pattern 1 showed Et 1. Consequently, this homogeneous group could be considered as an E. coli clone isolated from septicaemia in animals and also from h u man infections. Strain M 10 differed only in esterase I mobility and can be denoted as a strain variant of the clone [1]. Interestingly, this piglet isolate was also the only haemolytic strain. The five o~aer strains which were of distinct O M P pattern were distributed in the 2 other Et. MOTS-CLES: Escherichia coli, Septic6mie, Enzyme; Distribu,ion clonale, Electrophoretype.
REFERENCES [1] ACHTMAN,M., HEUZENROEDER,M., KUSECEK,B., OCHMAN, Ho, CAUGANT, D., SELANDER,R.K., "vAISANEN-RHEN,V., KORHONEN,T.K., STUART,S., ORSKOV, F. & ORSKOV, I., Clonal analysis of Escherichia coli O2:K1 isolated from diseased humans and animals. Infect. Immun., 1986, 51, 268-276.
242
B. P i C A R D ,
P. G O U L L E T
A N D M. C O N T R E P O L q
[2{] DASSOUH-MRANI-BELKEBIP,,A., CONTREPOIS, M., G1RARDEAU,J.P. & Dee V aRTANIAN~M.~ Outer membrane analysis of Esc;,e:ichia coli 078 isolated from septicemic animals. Vet. Microbiot. (in press). [3] GOULLLT, Ph., An esterase zymogram of Escherichia coll. J. gen. MicrobioL, 1973, 77, 27-35. [4] GOUH_ET, P. & PICARD, B., Etude du polymorphisme 6lectrophordtique des lactate-, malate- et glutamate-deshydrogenases, de la phosphatase acide et des est&ases de Providencia alcalifaciens, P. stuartii et P. rustigianii. Ann. hTst. Pasteur/Microbiol., 1985, 136 A, 347-358. [51 GOULLH, Pb. & PICARD, Bo, Comparative esterase electrophoretic polymorphism of Escherichia coil isolates obtained from animals and human sources. J. gen. MtcrooloJ., 1986, 132, 1843-1831. [6] GOt'LLEt, Ph. & P!CARD, B., Differentiation of Shigella by esterase electrophoretic polymorphism. J. gen. Microbiol., 1987, 133, 1005-1017. [7] MncH, H., NIKOt.NIKOV,S. & CZlROK, E., Escherichia coli CoW plasmids and their role in pathogenicity. Acta Microbiol. Hung., i984, 31, 117-125. [8] OrSKOV, F. & ORSKOV, l., Serotyping of Escherichia coli, in ~Methods in Microbiology ~, (T. Bergan), 14 (pp. 44-112). Academic Press, London, New York, 1984. [9] SEI.ANDER,R.K., CAUGANT,D.A. & WHIFTAM,T.S., Genetic structure and variation in natural populations of Escherichia coli, in ¢ Cellular and molecular biology~> (Neidhardt), 2 (pp. 1625-1648). American Society for Microbiology, Washington, 1987.