INFECTION AND IMMUNITY, Aug. 1992, p. 3376-3380
Vol. 60, No. 8
0019-9567/92/083376-05$02.00/0
Replicon Typing of Virulence Plasmids of Enterotoxigenic Escherichia coli Isolates from Cattle JACQUES G. MAINIL,1* FRAN(OISE BEX,2 PIERRE DREZE,2 ALBERT KAECKENBEECK,' AND
MARTINE COUTURIER2
Departement de Bacteriologie et de Pathologie des Maladies Bacteriennes, Faculte de Medecine Veterinaire, Universite de Liege, Sart Tilman, Batiment B43, Liege B-4000,1 and Departement de Biologie Moleculaire, Faculte des Sciences, Universite de Bnruelles, Rhodes St-Genese B-1640,2 Belgium Received 24 February 1992/Accepted 9 May 1992
Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle. The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins. The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repll, repHIl, repHI2, repVM, repN, repP, repQ, repT, repU, repW, repX, and repY). The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate. The sizes of most of these virulence plasmids were from 65 to 95 MDa. The F41 probe failed to hybridize with any plasmid band. The virulence plasmids had multireplicon types typical of plasmids of the IncF groups. The most common basic replicon association was the triple RepFIA- RepFIB-RepFIC family association.
The purposes of the study reported here were (i) to identify plasmid basic replicons present in ETEC isolates from calves; (ii) to identify virulence plasmids in the same isolates by using virulence factor gene probes; and (iii) to classify the virulence plasmids by using the replicon typing
Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in newborn farm animals (calves, lambs, and piglets) and in human beings (babies and travellers). Specific virulence factors of ETEC isolates are (i) classical heat-stable (STa, with two genotypes [STaP and STaH], and STh) or heatlabile (LT1) enterotoxins and (ii) fimbrial adhesins that promote adherence to enterocytes (F2 or CFA1, F3 or CFA2, F4 or K88, F5 or K99, F6 or 987P, etc.) (for reviews, see references 14, 20, 23, and 36). In ETEC isolates from cattle, association of an STa enterotoxin (STaP) with the production of F5 fimbrial adhesin or with the corresponding genes is common (17-19, 25). Moreover, many bovine ETEC isolates belonging to serogroups 09 and 0101 also synthesize F41 adhesin or contain the genes that code for it (17, 20). Whereas the genes encoding F41 adhesin are located on the chromosome, production of STaP enterotoxin and F5 adhesin is plasmid mediated. These virulence plasmids can be transferred and can also carry genes for antibiotic resistance and/or colicin production (13, 21; for a review, see reference 32). Some of the virulence plasmids of bovine ETEC have been classified in the IncFI or IncFII incompatibility group, whereas others, though having DNA sequence homology with F (IncFI) and/or Rl (IncFII) reference plasmids, are unclassifiable in any of the IncF incompatibility groups (for a review, see reference 32). Recently, Couturier et al. (7) proposed another plasmid classification scheme based on DNA probes derived from incompatibility loci of plasmids (replication and partition loci). This scheme, replicon typing, allows more precise classification of most of the plasmids studied. The technique has been applied to plasmids of E. coli in general (2), to resistance plasmids of E. coli (6), and to the virulenceassociated plasmids of Salmonella spp. (24, 37), enteroinvasive E. coli, and Shigella spp. (31). *
system. (This report constitutes part of a thesis submitted by J. G. Mainil to the University of Liege [Liege, Belgium] in partial fulfillment of the requirements for the Docteur en Sciences Veterinaires degree [1988]. This work was part of presentations at the 3rd European Congress of Clinical Microbiology [The Hague, The Netherlands, May 1987, abstract 48] and at the 2nd International Meeting on Bacterial Epidemiological Markers [Rhodes, Greece, April 1990, abstracts 23I and
16II].)
TABLE 1. Results of colony hybridization with replicon probes of 116 ETEC and 116 non-ETEC isolates from cattle Replicon probe
repFIA repFIB repFIC repFIIA
repHIl repHI2 repll repL/M repN repP repQ repT repU repW repX repY
Hybridizationa by ETEC isolates
Non-ETEC isolates
89.6 96.6 100 100 0 1.7 28.4 0 4.3 31.0 0.9 0 0 0 6.9 0
31.9 58.6 72.4 68.1 3.4 0 20.8 0 4.3 13.8 14.9 0 0 0 1.7 1.7
a % of probe-positive isolates.
Corresponding author. 3376
VOL. 60, 1992
REPLICON TYPING OF PLASMIDS OF ETEC ISOLATES
4 5 6 7 8 91011 121314151617181920
B 1 23 to
-..*
3377
~f
40
40
em
"m m4e
STa P C
1 2 3 4 5 6 7 8 91011 121314151617181920
D 1 2 3 4 5 6 7 8 9101112131415161718192G I....
-.
_
e_ 0."w
FIA/R
"
-W* _sm .to ,
....
FIe^C
FIG. 1. Hybridization of plasmids isolated from ETEC isolates from cattle with virulence and replicon probes. (A) Agarose gel (0.6%) electrophoresis showing the profile of plasmids isolated by the method of Kado and Liu (15). Also shown are radioautographs of this agarose gel after hybridization with the probe for the STaP enterotoxin gene (B), with the repFIA probe (C), and with the repFIC probe (D). Lanes, isolates or plasmids, and pathotypes or replicon types are as follows: 1, isolate 81A, STaP+ F5' F41+; 2, isolate 81B, F5+ F41+; 3, isolate 81D, STaP+ F41+; 4, isolate 122A, STaP+ F5+ F41+; 5, isolate 122B, STaP+ F5+ F41+; 6, isolate 126A, STaP+ F5+; 7, isolate 317A, non-ETEC; 8, isolate 510, STaP+ F5+ F41+; 9, strain B41, STaP+ F5+ F41+; 10, plasmid F'lac,pro, repFIA+ repFIB+ repFIC+ repFIIA+ (7); 11, plasmid Rldrdl9, repFIC+ repFIIA+ (7); 12, isolate 29552, STaP+ F5+ F41+; 13, isolate 29745, STaP+ F5+ F41+; 14, isolate 31109, STaP+ F5+ F41+; 15, isolate 31371, STaP+ F5+ F41+; 16, isolate 34336, STaP+ F41+; 17, isolate 35358, STaP+ F5+ F41+; 18, strain 431, STaP+ F5+ F41+ porcine ETEC (21); 19, plasmid P307, repFIB+ repFIC+ repFIIA+ repI1+ (7); 20, E. coli K12-C600, chromosomal marker.
MATERIALS AND METHODS Gene probes. The gene probes for STaP enterotoxin and F5 and F41 adhesins (virulence probes) were derived from recombinant plasmids pRIT10130 (12), pFK99 (8), and pDGA17 (la), respectively, as described by Mainil et al. (17). The DNA probes for 16 plasmid basic replicons (probes
repFIA, repFIB, repFIC, repFIlA, repIl, repHIl, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY) were derived according to the method of Couturier et al. (7). The repFIC probe cross hybridizes with other basic replicons (RepFIIA, Rep9, RepIl, RepK, and RepB/0). These basic replicons are carried by plasmids belonging to different incompatibility groups that are put into only one group by the replicon typing: the RepFIC family. E. coli isolates and strains. One hundred sixteen STaP+ F5+ or STaP+ F5+ F41+ ETEC isolates, including bovine
ETEC reference strain B41 (0101:H-) (33), and 116 nonETEC bovine isolates (17) were studied by colony hybridization with the different replicon probes. They were isolated at the Veterinary School during the years 1967 to 1970 (28) and 1979 to 1986 (17) from calves (one to four isolates per calf) from different herds, less than 3 months old, and with enteric or systemic diseases thought to be caused by E. coli. Two of the STaP+ F5+ isolates from the years 1967 to 1970 and 15 of the STaP+ F5+ F41+ isolates (eight from the years 1967 to 1970, six from the years 1979 to 1986, and strain B41) were chosen for study with both the virulence probes and the replicon probes so as to identify the virulence plasmids and the basic replicons present on them. The 2 STaP+ F5+ isolates belonged to serogroup 08; 10 of the STaP+ F5+ F41+ isolates belonged to serogroup 0101; 1 belonged to serogroup 09; and 5 belonged to serogroups other than 08, 09, 020, and 0101. Several controls for virulence probe
3378
MAINIL ET AL.
INFECT. IMMUN.
TABLE 2. Localization of genes encoding virulence factors in bovine ETEC isolates Isolate
Isolate pathotypea
81Ab.c 122Ab,d
STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ STaP+ F5+ STaP+ F41+ STaP+ F41+ F5+ F41+
122Bb,d 122Cb d
122F/b d 156Kb 385Ab
510b 28587e 29552e 29745e 31109e 31371e
35358e B41 AHI-308
AIHI-455
126AW 349AW
81Db,c 34336e 81Bb,c
Plasmidsizesprofile (plasmid [MDaJ) 85, 75, 60, 45, 17 105, 75, 45 85, 70, 65, 45 120, 80, 75, 70, 4 95, 75 80, 65, 30 95, 80 65, 25 80 75, 35 75, 65 75, 35 75, 65 70 65, 50 100, 70, 50, 45 85 65, 40, 14 80, 65, 45, 25 80, 75, 65 95, 40 85, 45
Virulence plasmid size (MDa)
85, 75 105 85 120 95 80 95 65 80 75 75 75 75 70 65 100 85 65 80 80 95 85
Plasmid pathotype
STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ F5+
F5+, STaP+ F5+ F5+ F5+ F5+ F5+ F5+ F5+
F5+ F5+ F5+ F5+ F5+ F5+ F5+ F5+ F5+ F5+ F5+
a By colony hybridization (17).
b Isolates from 1967 to 1970. c d
e
Isolates 81A, 81B, and 81D come from the same animal. Isolates 122A, 122B, 122C, and 122F come from the same animal. Isolates from 1979 to 1986.
hybridization were included: two STaP+ F5+ F41+ bovine ETEC strains (16a), AHI-308 (serogroup 0101) and AHI-455 (serogroup 0101) (29), two STaP+ F41+ isolates (serogroup 0101), one F5+ F41+ isolate (serogroup 0101), three nonETEC isolates (17), and E. coli K12-C600. Four STaP+ F5+ F41+ isolates came from the same calf as did another STaP+ F5+ F41+ isolate, one STaP+ F41+ isolate, and the F5+ F41+ isolate. Colony DNA hybridization with replicon probes. Filters with replicas of the isolates to be tested were treated and allowed to hybridize with the replicon probes as described by Couturier et al. (7). Hybridization conditions allowed 15 to 20% mismatch. Plasmid DNA hybridization. Plasmid DNA was extracted from the E. coli isolates according to the method of Kado and Liu (15), with some modifications (4). The plasmids were separated by agarose gel electrophoresis and hybridized with the virulence and replicon probes as described by Maas et al. (16), with modifications according to the method of Broes et al. (4). These hybridization conditions also allowed 15 to 20% mismatch. The replicon probe controls were the plasmids from which the probes were derived (7). Plasmid size markers were the six plasmids from Erwinia uredovora 391 and had molecular masses of 173.3, 120, 70, 43.3, 18.7, and 5.2 MDa (38). RESULTS Colony hybridization with replicon probes. Most of the 116 ETEC isolates (>90%) hybridized with the repFIA, repFIB, repFIC, and repFIIA probes; a third of them hybridized with the repll and repP probes. None or very few hybridized with the other replicon probes (Table 1). Fewer non-ETEC isolates were replicon probe positive, but again, hybridization
occurred most frequently with the repFIA, repFIB, repFIC, and repFIIA probes (Table 1). Virulence plasmid identification. After extraction, the different plasmids were separated by agarose gel electrophoresis. The 25 bovine E. coli isolates included in the study contained one to five plasmids (Fig. 1 and Table 2). In all 22 virulence probe-positive E. coli isolates, the STaP and the F5 probes hybridized with plasmid DNA but the F41 probe did not (Fig. 1). In the two STaP+ F5+ and in the 17 STaP+ F5+ F41+ bovine ETEC isolates, the STaP and F5 probes hybridized with a same plasmid band (STaP+ F5+ plasmids). The size range for these virulence plasmid bands was from 65 to 120 MDa. The STaP+ F5+ plasmids from the four ETEC isolates from the same animal had different molecular masses (Table 2). In one ETEC isolate, a second plasmid band hybridized with the STaP probe but not with the F5 probe (STaP+ plasmid) (Table 2). Each of the two STaP+ F41+ isolates had a single STaP probe-positive plasmid band (STaP+ plasmids), whereas the F5+ F41+ isolate yielded a single F5 probe-positive plasmid band (F5+ plasmid) (Table 2). The three non-ETEC isolates were negative with the three virulence probes. Identification of basic replicons present on STaP+ F5+, STaP+, and F5+ virulence plasmids. The following replicon probes were chosen according to the colony hybridization results of the 22 virulence probe-positive E. coli isolates: repFIA, repFIB, repFIC, repFIIA, repIl, repHI2, repN, repP, repQ, and repX. The only probes found to hybridize with the virulence plasmids were repFIA, repFIB, repFIC, repFIIA, and repll (Fig. 1). All virulence plasmids (STaP+ F5+, STaP+, and F5+) were positive with the repFIC and repFIIA probes, and
REPLICON TYPING OF PLASMIDS OF ETEC ISOLATES
VOL. 60, 1992
3379
TABLE 3. Replicon typing of the 23 virulence plasmids present in 22 ETEC isolatesa Replicon probes hybridized
Plasmid pathotype
STaP+ F5+
STaP+ F5+
repFIB, repFIB, repFIC, repFIC, repFIA, repFIB, repFIB, repFIC, repFIA, repFIB,
repFIA, repFIA, repFIA, repFIB,
repFIC, repFIIA repFIC, repFIIA, repll repFIIA repFIIA repFIC, repFIIA repFIIA repFIC, repFIIA
Replicon typesb
RepFIA, RepFIB, RepFIA, RepFIB, RepFIA, RepFIC RepFIB, RepFIC RepFIA, RepFIB, RepFIB, RepFIC RepFIA, RepFIB,
No. of isolates
RepFIC RepFIC
15c 1 2 1
RepFIC
2e
RepFIC
if
1
a One isolate contained one STaP+ F5+ plasmid and one STaP+ plasmid (Table 2). RepFIC = one basic replicon of the RepFIC family (see Materials and Methods for replicon probe specificity). c Including the STaP+ F5+ plasmids from isolates 122A, 122B, 122C, and 81A. d Including the STaP+ F5+ plasmid from isolate 122F. e STaP+ plasmids from isolates 81A and 81D. f F5+ plasmid from isolate 81B.
most were positive with the repFIA and repFIB probes (Table 3). In most isolates, the repFIC and repFIIA probes
hybridized with more than one plasmid band and the repFIA and repFIB probes hybridized with the virulence plasmids only. In only one isolate did the repll probe hybridize with the STaP+ F5+ plasmid band, whereas in the other positive isolates another plasmid band was revealed. The repHI2, repN, repP, repQ, and repX probes hybridized with other plasmid bands obtained from the isolates. Not all virulence plasmids from E. coli isolates from the same animal had identical replicon types (Table 3). DISCUSSION The bovine ETEC isolates studied in this work harbor one to five plasmids. The STaP enterotoxin and F5 fimbrial adhesin genes are located on a plasmid, whereas the F41 adhesin genes are not. Involvement of plasmids in the production of STaP enterotoxin and F5 adhesin has already been reported (13; for a review, see reference 32). In each strain of this study, moreover, the STaP and the F5 probes reveal the same plasmid band. These two virulence factor genes are thus most probably associated on a single plasmid molecule ranging in size from 65 to 95 MDa for most of them. The existence of a second copy of the STaP enterotoxin genes in one isolate can be explained by a transposition event involving the STaP-encoding transposon Tn1681 (35). The presence of two plasmids of identical size, one being STaP+ and the other being F5+, cannot be excluded absolutely (10), but association of the STaP enterotoxin and F5 adhesin genes on the same plasmid molecule seems to be widespread in STa+ F5+ ETEC isolates from cattle and from pigs (5, 13, 30; for a review, see reference 32). Associations of genes coding for other adhesins (F2, F3, and F6) with genes coding for enterotoxins (STa, STh, and LT1) are also common in human ETEC and other porcine ETEC (9, 11, 22, 27; for a review, see reference 32). Several results, sometimes contradictory as to plasmid number, size, and phenotype, have been published for strain B41 (3, 10, 34), and the results for strains AHI-308 and AHI-455 also differ from the original description (29) in plasmid number and type. These differences may arise from differences in the plasmid detection methods used or from genetic rearrangements having occurred during storage of the strains in the different laboratories. Virulence-associated plasmids of members of the family Enterobacteriaceae belong to the IncF groups. These plasmids express incompatibility with IncF plasmids or share DNA sequence similarities with IncF plasmids (for reviews,
see references 7 and 32). Those studied by replicon typing have multireplicon types typical of the IncF group plasmids (7, 24, 31, 37): they carry at least one basic replicon of the RepFIC family (7, 26) and RepFIA, RepFIB, or both (24, 31, 37). The triple RepFIA-RepFIB-RepFIC association was the most common in our work: it was found in 19 of the 23 virulence plasmids that were present in the 22 ETEC isolates
examined (Table 3). The wide distribution of basic replicons of the family RepFIC on plasmids of E. coli is also illustrated by the high rate of hybridization of non-ETEC isolates with the repFIC and repFIIA probes and by the multiple plasmid bands detected by the same two probes in most of the ETEC isolates. IncF group plasmid multireplicon types are also common on non-virulence-associated plasmids of members of the family Enterobacteriaceae (2, 6), suggesting that such plasmids existed in these bacterial species before they were colonized by virulence factor-encoding genes. As no specific basic replicon is present in virulent strains of E. coli (enterotoxigenic or otherwise) or of other enterobacterial species, replicon typing in this case cannot provide an epidemiological marker. However, such large-scale colony hybridization replicon typing does provide an overview of the types of plasmids present in such bacterial species and is useful for analyzing the extent of plasmid transfer between related and taxonomically distant bacterial species (1). Furthermore, recognition of replicon types on plasmids involved in virulence of members of the family Enterobacteriaceae could considerably facilitate future typing and would help to rapidly sight the emergence of virulence plasmids belonging to a new replicon type. ACKNOWLEDGMENTS We thank Pierre Pohl (National Institute for Veterinary Research) for helpful discussion and constant encouragement and Etienne Jacquemin for technical help. This work was supported by grants from IRSIA (Institut pour l'encouragement de la Recherche dans l'Industrie et l'Agriculture) (convention CEMA Bacteriologie 4897), from the FNRS (Fonds National de la Recherche Scientifique) (credits aux chercheurs), and from the Banque Nationale de Belgique. REFERENCES 1. American Society for Microbiology. 1991. Far-flung gene transfers in nature. ASM News 57:450.
la.Anderson, D. G., and S. L. Moseley. 1988. Escherichia coli F41 adhesin: genetic organization, nucleotide sequence, and homology with the K88 determinant. J. Bacteriol. 170:4890-4896. 2. Bergquist, P. L., S. Saadi, and W. K. Maas. 1986. Distribution of basic replicons having homology with RepFIA, RepFIB and RepFIC among incF group plasmids. Plasmid 15:19-34.
3380
MAINIL ET AL.
3. Bertin, A. 1985. F41 antigen as a virulence factor in the infant mouse model of Escherichia coli. J. Gen. Microbiol. 131:30373045. 4. Broes, A., J. M. Fairbrother, J. Mainil, J. Harel, and S. Lariviere. 1988. Phenotypic and genotypic characterization of enterotoxigenic Escherichia coli serotype 08:KX105 and 08: K"2829" strains isolated from piglets with diarrhea. J. Clin. Microbiol. 26:2402-2409. 5. Casey, T. A., and H. W. Moon. 1990. Genetic characterization and virulence of enterotoxigenic Escherichia coli mutants which have lost virulence genes in vivo. Infect. Immun. 58:4156-4158. 6. Chaslus-Dancla, E., P. Pohl, M. Meurisse, M. Marin, and J. P. Lafont. 1991. High genetic homology between plasmids of human and animal origins conferring resistance to the aminoglycosides gentamicin and apramycin. Antimicrob. Agents Chemother. 35:590-593. 7. Couturier, M., F. Bex, P. L. Bergquist, and W. K. Maas. 1988. Identification and classification of bacterial plasmids. Microbiol. Rev. 52:305-317. 8. de Graaf, F. K., B. E. Krenn, and P. Klaasen. 1984. Organization and expression of genes involved in the biosynthesis of K99 fimbriae. Infect. Immun. 43:508-514. 9. Echeverria, P., J. Seriwatana, D. N. Taylor, S. Changchawalit, C. J. Smyth, J. Twohig, and B. Rowe. 1986. Plasmids coding for colonization antigens I and II, heat-labile enterotoxin, and heat-stable enterotoxin A2 in Eschenchia coli. Infect. Immun. 51:626-630. 10. Falkow, S., L. P. Williams, Jr., S. L. Seaman, and L. D. Rollins. 1976. Increased survival in calves of Escherichia coli K-12 carrying an Ent plasmid. Infect. Immun. 13:1005-1007. 11. Gonzalez, E. A., J. Blanco, I. Garabal, and M. Blanco. 1991. Biotypes, antibiotic resistance and plasmids coding for CFA/I and STa in enterotoxigenic Escherichia coli strains of serotype 0153:H45 isolated in Spain. J. Med. Microbiol. 34:89-95. 12. Harford, N., M. De Wilde, and T. Cabezon. 1981. In S. B. Levy, R. C. Clowes, and E. L. Koenig (ed.), Molecular biology, pathogenicity and ecology of bacterial plasmids, p. 611. Plenum Publishing Corp., New York. 13. Harnett, N. M., and C. L. Gyles. 1985. Linkage of genes for heat-stable enterotoxin, drug resistance, K99 antigen, and colicin production in bovine and porcine strains of enterotoxigenic Escherichia coli. Am. J. Vet. Res. 46:428-433. 14. Holland, R. E. 1990. Some infectious causes of diarrhea in young farm animals. Clin. Microbiol. Rev. 3:345-375. 15. Kado, C. I., and S. T. Liu. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145:13651373. 16. Maas, R., R. M. Silva, T. A. T. Gomes, L. R. Trabulsi, and W. K. Maas. 1985. Detection of genes for heat-stable enterotoxin I in Escherichia coli strains isolated in Brazil. Infect. Immun. 49:46-51. 16a.Mainil, J. G. Unpublished data. 17. Mainil, J. G., F. Bex, E. Jacquemin, P. Pohl, M. Couturier, and A. Kaeckenbeeck. 1990. Prevalence of four enterotoxin (STaP, STaH, STh, and LT) and four adhesin subunit (K99, K88, 987P, and F41) genes among Escherichia coli isolates from cattle. Am. J. Vet. Res. 51:187-190. 18. Mainil, J. G., S. L. Moseley, R. A. Schneider, K. Sutch, T. A. Casey, and H. W. Moon. 1986. Hybridization of bovine Escherichia coli isolates with gene probes for four enterotoxins (STaP, STaH, STh, LT) and one adhesion factor (K99). Am. J. Vet. Res. 47:1145-1148. 19. Moon, H. W., S. C. Whipp, and S. M. Skarvedt. 1976. Etiologic diagnosis of diarrheal diseases of calves: frequency and methods for detecting enterotoxin and K99 antigen production by Escherichia coli. Am. J. Vet. Res. 37:1025-1029. 20. Morrs, J. A., and W. J. Sojka. 1985. Eschenchia coli as a
INFECT. IMMUN. pathogen in animals, p. 47-77. In M. Sussman (ed.), The virulence of Escherichia coli: reviews and methods. Academic Press, London. 21. Moseley, S. L., G. Dougan, R. A. Schneider, and H. W. Moon. 1986. Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88. J. Bacteriol. 167:799-804. 22. Murray, B. E., and J. Tsao. 1987. Comparison of CFAII:ST plasmids of enterotoxigenic Escherichia coli. J. Infect. Dis. 156:398-401. 23. Orskov, F., and I. Orskov. 1984. Serotyping of Escherichia coli. Methods Microbiol. 14:43-112. 24. Pohl, P., P. Lintermans, F. Bex, A. Desmyter, P. Dreze, P. A. Fonteyne, and M. Couturier. 1987. Proprietes phenotypiques et genotypiques de quatre plasmides de virulence de Salmonella typhimurium. Ann. Inst. Pasteur (Paris) 138:523-528. 25. Pohl, P., P. Lintermans, A. Kaeckenbeeck, K. Van Muylem, and C. Schlicker. 1984. Correlation entre la production de l'antigene K99 et celle de l'enterotoxine STI chez les colibacilles du veau. Ann. Med. Vet. 128:119-124. 26. Saadi, S., W. K. Maas, D. F. Hill, and P. L. Bergquist. 1987. Nucleotide sequence of RepFIC, a basic replicon present in IncFI plasmids P307 and F, and its relation to the RepA replicon of IncFII plasmids. J. Bacteriol. 169:1836-1846. 27. Schifferli, D. M., E. H. Beachey, and R. K. Taylor. 1990. The 987P fimbrial gene cluster of enterotoxigenic Escherichia coli is plasmid encoded. Infect. Immun. 58:149-156. 28. Schoenaers, F., M. Josse, and A. Kaeckenbeeck. 1978. Detection des Escherichia coli ent6rotoxiques, d'origine bovine, par inoculation au souriceau. Ann. Med. Vet. 122:435-439. 29. Sekizaki, T., N. Terakado, H. Ueda, and K. Hashimoto. 1982. Isolation and characterization of plasmids encoding heat-stable enterotoxin from Escherichia coli of cattle origin. Jpn. J. Vet. Sci. 44:619-627. 30. Shimizu, M., T. Sakano, and S. Satoh. 1988. Linkage of K99 production and STa activity in a plasmid of an Escherichia coli porcine isolate. Microbiol. Immunol. 32:635-639. 31. Silva, R. M., S. Saadi, and W. K. Maas. 1988. A basic replicon of virulence-associated plasmids of Shigella spp. and enteroinvasive Escherichia coli is homologous with a basic replicon in plasmids of IncF groups. Infect. Immun. 56:836-842. 32. Smith, H. R., S. M. Scotland, and B. Rowe. 1985. Genetics of Escherichia coli virulence, p. 227-269. In M. Sussman (ed.), The virulence of Escherichia coli: reviews and methods. Academic Press, London. 33. Smith, H. W., and S. Halls. 1967. Observation by the ligated intestinal segment and oral inoculation methods on Escherichia coli infections in pigs, calves, lambs and rabbits. J. Pathol. Bacteriol. 93:499-529. 34. So, M., H. W. Boyer, M. Betlach, and S. Falkow. 1976. Molecular cloning of an Escherichia coli plasmid determinant that encodes for the production of heat-stable enterotoxin. J. Bacteriol. 128:463-472. 35. So, M., F. Heifron, and B. J. McCarthy. 1979. The Escherichia coli gene encoding heat-stable toxin is a bacterial transposon flanked by inverted repeats of IS1. Nature (London) 277:453456. 36. Sussman, M. 1985. Escherichia coli in human and animal disease, p. 7-45. In M. Sussman (ed.), The virulence of Escherichia coli: reviews and methods. Academic Press, London. 37. Tinge, S. A., and R. Curtiss III. 1990. Isolation of the replication and partitioning regions of the Salmonella typhimunum virulence plasmid and stabilization of heterologous replicons. J. Bacteriol. 172:5266-5277. 38. Thiry, G. 1984. Plasmids of the epiphytic bacterium Ervinia uredovora. J. Gen. Microbiol. 130:1623-1631.
Vol. 60, No. 8
0019-9567/92/083376-05$02.00/0
Replicon Typing of Virulence Plasmids of Enterotoxigenic Escherichia coli Isolates from Cattle JACQUES G. MAINIL,1* FRAN(OISE BEX,2 PIERRE DREZE,2 ALBERT KAECKENBEECK,' AND
MARTINE COUTURIER2
Departement de Bacteriologie et de Pathologie des Maladies Bacteriennes, Faculte de Medecine Veterinaire, Universite de Liege, Sart Tilman, Batiment B43, Liege B-4000,1 and Departement de Biologie Moleculaire, Faculte des Sciences, Universite de Bnruelles, Rhodes St-Genese B-1640,2 Belgium Received 24 February 1992/Accepted 9 May 1992
Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle. The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins. The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repll, repHIl, repHI2, repVM, repN, repP, repQ, repT, repU, repW, repX, and repY). The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate. The sizes of most of these virulence plasmids were from 65 to 95 MDa. The F41 probe failed to hybridize with any plasmid band. The virulence plasmids had multireplicon types typical of plasmids of the IncF groups. The most common basic replicon association was the triple RepFIA- RepFIB-RepFIC family association.
The purposes of the study reported here were (i) to identify plasmid basic replicons present in ETEC isolates from calves; (ii) to identify virulence plasmids in the same isolates by using virulence factor gene probes; and (iii) to classify the virulence plasmids by using the replicon typing
Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in newborn farm animals (calves, lambs, and piglets) and in human beings (babies and travellers). Specific virulence factors of ETEC isolates are (i) classical heat-stable (STa, with two genotypes [STaP and STaH], and STh) or heatlabile (LT1) enterotoxins and (ii) fimbrial adhesins that promote adherence to enterocytes (F2 or CFA1, F3 or CFA2, F4 or K88, F5 or K99, F6 or 987P, etc.) (for reviews, see references 14, 20, 23, and 36). In ETEC isolates from cattle, association of an STa enterotoxin (STaP) with the production of F5 fimbrial adhesin or with the corresponding genes is common (17-19, 25). Moreover, many bovine ETEC isolates belonging to serogroups 09 and 0101 also synthesize F41 adhesin or contain the genes that code for it (17, 20). Whereas the genes encoding F41 adhesin are located on the chromosome, production of STaP enterotoxin and F5 adhesin is plasmid mediated. These virulence plasmids can be transferred and can also carry genes for antibiotic resistance and/or colicin production (13, 21; for a review, see reference 32). Some of the virulence plasmids of bovine ETEC have been classified in the IncFI or IncFII incompatibility group, whereas others, though having DNA sequence homology with F (IncFI) and/or Rl (IncFII) reference plasmids, are unclassifiable in any of the IncF incompatibility groups (for a review, see reference 32). Recently, Couturier et al. (7) proposed another plasmid classification scheme based on DNA probes derived from incompatibility loci of plasmids (replication and partition loci). This scheme, replicon typing, allows more precise classification of most of the plasmids studied. The technique has been applied to plasmids of E. coli in general (2), to resistance plasmids of E. coli (6), and to the virulenceassociated plasmids of Salmonella spp. (24, 37), enteroinvasive E. coli, and Shigella spp. (31). *
system. (This report constitutes part of a thesis submitted by J. G. Mainil to the University of Liege [Liege, Belgium] in partial fulfillment of the requirements for the Docteur en Sciences Veterinaires degree [1988]. This work was part of presentations at the 3rd European Congress of Clinical Microbiology [The Hague, The Netherlands, May 1987, abstract 48] and at the 2nd International Meeting on Bacterial Epidemiological Markers [Rhodes, Greece, April 1990, abstracts 23I and
16II].)
TABLE 1. Results of colony hybridization with replicon probes of 116 ETEC and 116 non-ETEC isolates from cattle Replicon probe
repFIA repFIB repFIC repFIIA
repHIl repHI2 repll repL/M repN repP repQ repT repU repW repX repY
Hybridizationa by ETEC isolates
Non-ETEC isolates
89.6 96.6 100 100 0 1.7 28.4 0 4.3 31.0 0.9 0 0 0 6.9 0
31.9 58.6 72.4 68.1 3.4 0 20.8 0 4.3 13.8 14.9 0 0 0 1.7 1.7
a % of probe-positive isolates.
Corresponding author. 3376
VOL. 60, 1992
REPLICON TYPING OF PLASMIDS OF ETEC ISOLATES
4 5 6 7 8 91011 121314151617181920
B 1 23 to
-..*
3377
~f
40
40
em
"m m4e
STa P C
1 2 3 4 5 6 7 8 91011 121314151617181920
D 1 2 3 4 5 6 7 8 9101112131415161718192G I....
-.
_
e_ 0."w
FIA/R
"
-W* _sm .to ,
....
FIe^C
FIG. 1. Hybridization of plasmids isolated from ETEC isolates from cattle with virulence and replicon probes. (A) Agarose gel (0.6%) electrophoresis showing the profile of plasmids isolated by the method of Kado and Liu (15). Also shown are radioautographs of this agarose gel after hybridization with the probe for the STaP enterotoxin gene (B), with the repFIA probe (C), and with the repFIC probe (D). Lanes, isolates or plasmids, and pathotypes or replicon types are as follows: 1, isolate 81A, STaP+ F5' F41+; 2, isolate 81B, F5+ F41+; 3, isolate 81D, STaP+ F41+; 4, isolate 122A, STaP+ F5+ F41+; 5, isolate 122B, STaP+ F5+ F41+; 6, isolate 126A, STaP+ F5+; 7, isolate 317A, non-ETEC; 8, isolate 510, STaP+ F5+ F41+; 9, strain B41, STaP+ F5+ F41+; 10, plasmid F'lac,pro, repFIA+ repFIB+ repFIC+ repFIIA+ (7); 11, plasmid Rldrdl9, repFIC+ repFIIA+ (7); 12, isolate 29552, STaP+ F5+ F41+; 13, isolate 29745, STaP+ F5+ F41+; 14, isolate 31109, STaP+ F5+ F41+; 15, isolate 31371, STaP+ F5+ F41+; 16, isolate 34336, STaP+ F41+; 17, isolate 35358, STaP+ F5+ F41+; 18, strain 431, STaP+ F5+ F41+ porcine ETEC (21); 19, plasmid P307, repFIB+ repFIC+ repFIIA+ repI1+ (7); 20, E. coli K12-C600, chromosomal marker.
MATERIALS AND METHODS Gene probes. The gene probes for STaP enterotoxin and F5 and F41 adhesins (virulence probes) were derived from recombinant plasmids pRIT10130 (12), pFK99 (8), and pDGA17 (la), respectively, as described by Mainil et al. (17). The DNA probes for 16 plasmid basic replicons (probes
repFIA, repFIB, repFIC, repFIlA, repIl, repHIl, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY) were derived according to the method of Couturier et al. (7). The repFIC probe cross hybridizes with other basic replicons (RepFIIA, Rep9, RepIl, RepK, and RepB/0). These basic replicons are carried by plasmids belonging to different incompatibility groups that are put into only one group by the replicon typing: the RepFIC family. E. coli isolates and strains. One hundred sixteen STaP+ F5+ or STaP+ F5+ F41+ ETEC isolates, including bovine
ETEC reference strain B41 (0101:H-) (33), and 116 nonETEC bovine isolates (17) were studied by colony hybridization with the different replicon probes. They were isolated at the Veterinary School during the years 1967 to 1970 (28) and 1979 to 1986 (17) from calves (one to four isolates per calf) from different herds, less than 3 months old, and with enteric or systemic diseases thought to be caused by E. coli. Two of the STaP+ F5+ isolates from the years 1967 to 1970 and 15 of the STaP+ F5+ F41+ isolates (eight from the years 1967 to 1970, six from the years 1979 to 1986, and strain B41) were chosen for study with both the virulence probes and the replicon probes so as to identify the virulence plasmids and the basic replicons present on them. The 2 STaP+ F5+ isolates belonged to serogroup 08; 10 of the STaP+ F5+ F41+ isolates belonged to serogroup 0101; 1 belonged to serogroup 09; and 5 belonged to serogroups other than 08, 09, 020, and 0101. Several controls for virulence probe
3378
MAINIL ET AL.
INFECT. IMMUN.
TABLE 2. Localization of genes encoding virulence factors in bovine ETEC isolates Isolate
Isolate pathotypea
81Ab.c 122Ab,d
STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ F41+ STaP+ F5+ STaP+ F5+ STaP+ F41+ STaP+ F41+ F5+ F41+
122Bb,d 122Cb d
122F/b d 156Kb 385Ab
510b 28587e 29552e 29745e 31109e 31371e
35358e B41 AHI-308
AIHI-455
126AW 349AW
81Db,c 34336e 81Bb,c
Plasmidsizesprofile (plasmid [MDaJ) 85, 75, 60, 45, 17 105, 75, 45 85, 70, 65, 45 120, 80, 75, 70, 4 95, 75 80, 65, 30 95, 80 65, 25 80 75, 35 75, 65 75, 35 75, 65 70 65, 50 100, 70, 50, 45 85 65, 40, 14 80, 65, 45, 25 80, 75, 65 95, 40 85, 45
Virulence plasmid size (MDa)
85, 75 105 85 120 95 80 95 65 80 75 75 75 75 70 65 100 85 65 80 80 95 85
Plasmid pathotype
STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ STaP+ F5+
F5+, STaP+ F5+ F5+ F5+ F5+ F5+ F5+ F5+
F5+ F5+ F5+ F5+ F5+ F5+ F5+ F5+ F5+ F5+ F5+
a By colony hybridization (17).
b Isolates from 1967 to 1970. c d
e
Isolates 81A, 81B, and 81D come from the same animal. Isolates 122A, 122B, 122C, and 122F come from the same animal. Isolates from 1979 to 1986.
hybridization were included: two STaP+ F5+ F41+ bovine ETEC strains (16a), AHI-308 (serogroup 0101) and AHI-455 (serogroup 0101) (29), two STaP+ F41+ isolates (serogroup 0101), one F5+ F41+ isolate (serogroup 0101), three nonETEC isolates (17), and E. coli K12-C600. Four STaP+ F5+ F41+ isolates came from the same calf as did another STaP+ F5+ F41+ isolate, one STaP+ F41+ isolate, and the F5+ F41+ isolate. Colony DNA hybridization with replicon probes. Filters with replicas of the isolates to be tested were treated and allowed to hybridize with the replicon probes as described by Couturier et al. (7). Hybridization conditions allowed 15 to 20% mismatch. Plasmid DNA hybridization. Plasmid DNA was extracted from the E. coli isolates according to the method of Kado and Liu (15), with some modifications (4). The plasmids were separated by agarose gel electrophoresis and hybridized with the virulence and replicon probes as described by Maas et al. (16), with modifications according to the method of Broes et al. (4). These hybridization conditions also allowed 15 to 20% mismatch. The replicon probe controls were the plasmids from which the probes were derived (7). Plasmid size markers were the six plasmids from Erwinia uredovora 391 and had molecular masses of 173.3, 120, 70, 43.3, 18.7, and 5.2 MDa (38). RESULTS Colony hybridization with replicon probes. Most of the 116 ETEC isolates (>90%) hybridized with the repFIA, repFIB, repFIC, and repFIIA probes; a third of them hybridized with the repll and repP probes. None or very few hybridized with the other replicon probes (Table 1). Fewer non-ETEC isolates were replicon probe positive, but again, hybridization
occurred most frequently with the repFIA, repFIB, repFIC, and repFIIA probes (Table 1). Virulence plasmid identification. After extraction, the different plasmids were separated by agarose gel electrophoresis. The 25 bovine E. coli isolates included in the study contained one to five plasmids (Fig. 1 and Table 2). In all 22 virulence probe-positive E. coli isolates, the STaP and the F5 probes hybridized with plasmid DNA but the F41 probe did not (Fig. 1). In the two STaP+ F5+ and in the 17 STaP+ F5+ F41+ bovine ETEC isolates, the STaP and F5 probes hybridized with a same plasmid band (STaP+ F5+ plasmids). The size range for these virulence plasmid bands was from 65 to 120 MDa. The STaP+ F5+ plasmids from the four ETEC isolates from the same animal had different molecular masses (Table 2). In one ETEC isolate, a second plasmid band hybridized with the STaP probe but not with the F5 probe (STaP+ plasmid) (Table 2). Each of the two STaP+ F41+ isolates had a single STaP probe-positive plasmid band (STaP+ plasmids), whereas the F5+ F41+ isolate yielded a single F5 probe-positive plasmid band (F5+ plasmid) (Table 2). The three non-ETEC isolates were negative with the three virulence probes. Identification of basic replicons present on STaP+ F5+, STaP+, and F5+ virulence plasmids. The following replicon probes were chosen according to the colony hybridization results of the 22 virulence probe-positive E. coli isolates: repFIA, repFIB, repFIC, repFIIA, repIl, repHI2, repN, repP, repQ, and repX. The only probes found to hybridize with the virulence plasmids were repFIA, repFIB, repFIC, repFIIA, and repll (Fig. 1). All virulence plasmids (STaP+ F5+, STaP+, and F5+) were positive with the repFIC and repFIIA probes, and
REPLICON TYPING OF PLASMIDS OF ETEC ISOLATES
VOL. 60, 1992
3379
TABLE 3. Replicon typing of the 23 virulence plasmids present in 22 ETEC isolatesa Replicon probes hybridized
Plasmid pathotype
STaP+ F5+
STaP+ F5+
repFIB, repFIB, repFIC, repFIC, repFIA, repFIB, repFIB, repFIC, repFIA, repFIB,
repFIA, repFIA, repFIA, repFIB,
repFIC, repFIIA repFIC, repFIIA, repll repFIIA repFIIA repFIC, repFIIA repFIIA repFIC, repFIIA
Replicon typesb
RepFIA, RepFIB, RepFIA, RepFIB, RepFIA, RepFIC RepFIB, RepFIC RepFIA, RepFIB, RepFIB, RepFIC RepFIA, RepFIB,
No. of isolates
RepFIC RepFIC
15c 1 2 1
RepFIC
2e
RepFIC
if
1
a One isolate contained one STaP+ F5+ plasmid and one STaP+ plasmid (Table 2). RepFIC = one basic replicon of the RepFIC family (see Materials and Methods for replicon probe specificity). c Including the STaP+ F5+ plasmids from isolates 122A, 122B, 122C, and 81A. d Including the STaP+ F5+ plasmid from isolate 122F. e STaP+ plasmids from isolates 81A and 81D. f F5+ plasmid from isolate 81B.
most were positive with the repFIA and repFIB probes (Table 3). In most isolates, the repFIC and repFIIA probes
hybridized with more than one plasmid band and the repFIA and repFIB probes hybridized with the virulence plasmids only. In only one isolate did the repll probe hybridize with the STaP+ F5+ plasmid band, whereas in the other positive isolates another plasmid band was revealed. The repHI2, repN, repP, repQ, and repX probes hybridized with other plasmid bands obtained from the isolates. Not all virulence plasmids from E. coli isolates from the same animal had identical replicon types (Table 3). DISCUSSION The bovine ETEC isolates studied in this work harbor one to five plasmids. The STaP enterotoxin and F5 fimbrial adhesin genes are located on a plasmid, whereas the F41 adhesin genes are not. Involvement of plasmids in the production of STaP enterotoxin and F5 adhesin has already been reported (13; for a review, see reference 32). In each strain of this study, moreover, the STaP and the F5 probes reveal the same plasmid band. These two virulence factor genes are thus most probably associated on a single plasmid molecule ranging in size from 65 to 95 MDa for most of them. The existence of a second copy of the STaP enterotoxin genes in one isolate can be explained by a transposition event involving the STaP-encoding transposon Tn1681 (35). The presence of two plasmids of identical size, one being STaP+ and the other being F5+, cannot be excluded absolutely (10), but association of the STaP enterotoxin and F5 adhesin genes on the same plasmid molecule seems to be widespread in STa+ F5+ ETEC isolates from cattle and from pigs (5, 13, 30; for a review, see reference 32). Associations of genes coding for other adhesins (F2, F3, and F6) with genes coding for enterotoxins (STa, STh, and LT1) are also common in human ETEC and other porcine ETEC (9, 11, 22, 27; for a review, see reference 32). Several results, sometimes contradictory as to plasmid number, size, and phenotype, have been published for strain B41 (3, 10, 34), and the results for strains AHI-308 and AHI-455 also differ from the original description (29) in plasmid number and type. These differences may arise from differences in the plasmid detection methods used or from genetic rearrangements having occurred during storage of the strains in the different laboratories. Virulence-associated plasmids of members of the family Enterobacteriaceae belong to the IncF groups. These plasmids express incompatibility with IncF plasmids or share DNA sequence similarities with IncF plasmids (for reviews,
see references 7 and 32). Those studied by replicon typing have multireplicon types typical of the IncF group plasmids (7, 24, 31, 37): they carry at least one basic replicon of the RepFIC family (7, 26) and RepFIA, RepFIB, or both (24, 31, 37). The triple RepFIA-RepFIB-RepFIC association was the most common in our work: it was found in 19 of the 23 virulence plasmids that were present in the 22 ETEC isolates
examined (Table 3). The wide distribution of basic replicons of the family RepFIC on plasmids of E. coli is also illustrated by the high rate of hybridization of non-ETEC isolates with the repFIC and repFIIA probes and by the multiple plasmid bands detected by the same two probes in most of the ETEC isolates. IncF group plasmid multireplicon types are also common on non-virulence-associated plasmids of members of the family Enterobacteriaceae (2, 6), suggesting that such plasmids existed in these bacterial species before they were colonized by virulence factor-encoding genes. As no specific basic replicon is present in virulent strains of E. coli (enterotoxigenic or otherwise) or of other enterobacterial species, replicon typing in this case cannot provide an epidemiological marker. However, such large-scale colony hybridization replicon typing does provide an overview of the types of plasmids present in such bacterial species and is useful for analyzing the extent of plasmid transfer between related and taxonomically distant bacterial species (1). Furthermore, recognition of replicon types on plasmids involved in virulence of members of the family Enterobacteriaceae could considerably facilitate future typing and would help to rapidly sight the emergence of virulence plasmids belonging to a new replicon type. ACKNOWLEDGMENTS We thank Pierre Pohl (National Institute for Veterinary Research) for helpful discussion and constant encouragement and Etienne Jacquemin for technical help. This work was supported by grants from IRSIA (Institut pour l'encouragement de la Recherche dans l'Industrie et l'Agriculture) (convention CEMA Bacteriologie 4897), from the FNRS (Fonds National de la Recherche Scientifique) (credits aux chercheurs), and from the Banque Nationale de Belgique. REFERENCES 1. American Society for Microbiology. 1991. Far-flung gene transfers in nature. ASM News 57:450.
la.Anderson, D. G., and S. L. Moseley. 1988. Escherichia coli F41 adhesin: genetic organization, nucleotide sequence, and homology with the K88 determinant. J. Bacteriol. 170:4890-4896. 2. Bergquist, P. L., S. Saadi, and W. K. Maas. 1986. Distribution of basic replicons having homology with RepFIA, RepFIB and RepFIC among incF group plasmids. Plasmid 15:19-34.
3380
MAINIL ET AL.
3. Bertin, A. 1985. F41 antigen as a virulence factor in the infant mouse model of Escherichia coli. J. Gen. Microbiol. 131:30373045. 4. Broes, A., J. M. Fairbrother, J. Mainil, J. Harel, and S. Lariviere. 1988. Phenotypic and genotypic characterization of enterotoxigenic Escherichia coli serotype 08:KX105 and 08: K"2829" strains isolated from piglets with diarrhea. J. Clin. Microbiol. 26:2402-2409. 5. Casey, T. A., and H. W. Moon. 1990. Genetic characterization and virulence of enterotoxigenic Escherichia coli mutants which have lost virulence genes in vivo. Infect. Immun. 58:4156-4158. 6. Chaslus-Dancla, E., P. Pohl, M. Meurisse, M. Marin, and J. P. Lafont. 1991. High genetic homology between plasmids of human and animal origins conferring resistance to the aminoglycosides gentamicin and apramycin. Antimicrob. Agents Chemother. 35:590-593. 7. Couturier, M., F. Bex, P. L. Bergquist, and W. K. Maas. 1988. Identification and classification of bacterial plasmids. Microbiol. Rev. 52:305-317. 8. de Graaf, F. K., B. E. Krenn, and P. Klaasen. 1984. Organization and expression of genes involved in the biosynthesis of K99 fimbriae. Infect. Immun. 43:508-514. 9. Echeverria, P., J. Seriwatana, D. N. Taylor, S. Changchawalit, C. J. Smyth, J. Twohig, and B. Rowe. 1986. Plasmids coding for colonization antigens I and II, heat-labile enterotoxin, and heat-stable enterotoxin A2 in Eschenchia coli. Infect. Immun. 51:626-630. 10. Falkow, S., L. P. Williams, Jr., S. L. Seaman, and L. D. Rollins. 1976. Increased survival in calves of Escherichia coli K-12 carrying an Ent plasmid. Infect. Immun. 13:1005-1007. 11. Gonzalez, E. A., J. Blanco, I. Garabal, and M. Blanco. 1991. Biotypes, antibiotic resistance and plasmids coding for CFA/I and STa in enterotoxigenic Escherichia coli strains of serotype 0153:H45 isolated in Spain. J. Med. Microbiol. 34:89-95. 12. Harford, N., M. De Wilde, and T. Cabezon. 1981. In S. B. Levy, R. C. Clowes, and E. L. Koenig (ed.), Molecular biology, pathogenicity and ecology of bacterial plasmids, p. 611. Plenum Publishing Corp., New York. 13. Harnett, N. M., and C. L. Gyles. 1985. Linkage of genes for heat-stable enterotoxin, drug resistance, K99 antigen, and colicin production in bovine and porcine strains of enterotoxigenic Escherichia coli. Am. J. Vet. Res. 46:428-433. 14. Holland, R. E. 1990. Some infectious causes of diarrhea in young farm animals. Clin. Microbiol. Rev. 3:345-375. 15. Kado, C. I., and S. T. Liu. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145:13651373. 16. Maas, R., R. M. Silva, T. A. T. Gomes, L. R. Trabulsi, and W. K. Maas. 1985. Detection of genes for heat-stable enterotoxin I in Escherichia coli strains isolated in Brazil. Infect. Immun. 49:46-51. 16a.Mainil, J. G. Unpublished data. 17. Mainil, J. G., F. Bex, E. Jacquemin, P. Pohl, M. Couturier, and A. Kaeckenbeeck. 1990. Prevalence of four enterotoxin (STaP, STaH, STh, and LT) and four adhesin subunit (K99, K88, 987P, and F41) genes among Escherichia coli isolates from cattle. Am. J. Vet. Res. 51:187-190. 18. Mainil, J. G., S. L. Moseley, R. A. Schneider, K. Sutch, T. A. Casey, and H. W. Moon. 1986. Hybridization of bovine Escherichia coli isolates with gene probes for four enterotoxins (STaP, STaH, STh, LT) and one adhesion factor (K99). Am. J. Vet. Res. 47:1145-1148. 19. Moon, H. W., S. C. Whipp, and S. M. Skarvedt. 1976. Etiologic diagnosis of diarrheal diseases of calves: frequency and methods for detecting enterotoxin and K99 antigen production by Escherichia coli. Am. J. Vet. Res. 37:1025-1029. 20. Morrs, J. A., and W. J. Sojka. 1985. Eschenchia coli as a
INFECT. IMMUN. pathogen in animals, p. 47-77. In M. Sussman (ed.), The virulence of Escherichia coli: reviews and methods. Academic Press, London. 21. Moseley, S. L., G. Dougan, R. A. Schneider, and H. W. Moon. 1986. Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88. J. Bacteriol. 167:799-804. 22. Murray, B. E., and J. Tsao. 1987. Comparison of CFAII:ST plasmids of enterotoxigenic Escherichia coli. J. Infect. Dis. 156:398-401. 23. Orskov, F., and I. Orskov. 1984. Serotyping of Escherichia coli. Methods Microbiol. 14:43-112. 24. Pohl, P., P. Lintermans, F. Bex, A. Desmyter, P. Dreze, P. A. Fonteyne, and M. Couturier. 1987. Proprietes phenotypiques et genotypiques de quatre plasmides de virulence de Salmonella typhimurium. Ann. Inst. Pasteur (Paris) 138:523-528. 25. Pohl, P., P. Lintermans, A. Kaeckenbeeck, K. Van Muylem, and C. Schlicker. 1984. Correlation entre la production de l'antigene K99 et celle de l'enterotoxine STI chez les colibacilles du veau. Ann. Med. Vet. 128:119-124. 26. Saadi, S., W. K. Maas, D. F. Hill, and P. L. Bergquist. 1987. Nucleotide sequence of RepFIC, a basic replicon present in IncFI plasmids P307 and F, and its relation to the RepA replicon of IncFII plasmids. J. Bacteriol. 169:1836-1846. 27. Schifferli, D. M., E. H. Beachey, and R. K. Taylor. 1990. The 987P fimbrial gene cluster of enterotoxigenic Escherichia coli is plasmid encoded. Infect. Immun. 58:149-156. 28. Schoenaers, F., M. Josse, and A. Kaeckenbeeck. 1978. Detection des Escherichia coli ent6rotoxiques, d'origine bovine, par inoculation au souriceau. Ann. Med. Vet. 122:435-439. 29. Sekizaki, T., N. Terakado, H. Ueda, and K. Hashimoto. 1982. Isolation and characterization of plasmids encoding heat-stable enterotoxin from Escherichia coli of cattle origin. Jpn. J. Vet. Sci. 44:619-627. 30. Shimizu, M., T. Sakano, and S. Satoh. 1988. Linkage of K99 production and STa activity in a plasmid of an Escherichia coli porcine isolate. Microbiol. Immunol. 32:635-639. 31. Silva, R. M., S. Saadi, and W. K. Maas. 1988. A basic replicon of virulence-associated plasmids of Shigella spp. and enteroinvasive Escherichia coli is homologous with a basic replicon in plasmids of IncF groups. Infect. Immun. 56:836-842. 32. Smith, H. R., S. M. Scotland, and B. Rowe. 1985. Genetics of Escherichia coli virulence, p. 227-269. In M. Sussman (ed.), The virulence of Escherichia coli: reviews and methods. Academic Press, London. 33. Smith, H. W., and S. Halls. 1967. Observation by the ligated intestinal segment and oral inoculation methods on Escherichia coli infections in pigs, calves, lambs and rabbits. J. Pathol. Bacteriol. 93:499-529. 34. So, M., H. W. Boyer, M. Betlach, and S. Falkow. 1976. Molecular cloning of an Escherichia coli plasmid determinant that encodes for the production of heat-stable enterotoxin. J. Bacteriol. 128:463-472. 35. So, M., F. Heifron, and B. J. McCarthy. 1979. The Escherichia coli gene encoding heat-stable toxin is a bacterial transposon flanked by inverted repeats of IS1. Nature (London) 277:453456. 36. Sussman, M. 1985. Escherichia coli in human and animal disease, p. 7-45. In M. Sussman (ed.), The virulence of Escherichia coli: reviews and methods. Academic Press, London. 37. Tinge, S. A., and R. Curtiss III. 1990. Isolation of the replication and partitioning regions of the Salmonella typhimunum virulence plasmid and stabilization of heterologous replicons. J. Bacteriol. 172:5266-5277. 38. Thiry, G. 1984. Plasmids of the epiphytic bacterium Ervinia uredovora. J. Gen. Microbiol. 130:1623-1631.
