Department of Microbiology, Ume University, Ume
S-90187, Sweden1
Author for correspondence: Xin-He Lai. Tel: +46 90 785 6735. Fax: +46 90 772630. e-mail: Lai.X-H{at}micro.umu.se
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ABSTRACT |
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Keywords: cytotoxicity, haemolysin, enteroaggregative Escherichia coli, enteroaggregative heat-stable enterotoxin, ECOR
Abbreviations: AA, aggregative adherence; DAEC, diffusely adherent E. coli; ECOR, E. coli collection of reference strains; EAEC, enteroaggregative E. coli; EAST1, EAEC heat-stable enterotoxin 1; EHEC, enterohaemorrhagic E. coli; EIEC, enteroinvasive E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli; LDH, lactate dehydrogenase; MLEE, multilocus enzyme electrophoresis; RBC, horse erythrocyte
The GenBank accession numbers for the sequences reported in this paper are AF159702 and AF160993161002.
a Permanent address: Priority Laboratory of Molecular Medical Bacteriology of Ministry of Public Health, Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Beijing, China.
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INTRODUCTION |
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The E. coli standard collection of reference (ECOR) strains is a set of 72 wild-type E. coli isolates (Ochman & Selander, 1984b ) from humans and 16 other mammalian species from a large collection of approximately 2600 isolates (Milkman, 1973
). The collection is thought to broadly represent genotypic variation in E. coli (Ochman & Selander, 1984a
). Using results from multilocus enzyme electrophoresis (MLEE) the major lineages of the ECOR collection are divided into five groups: A, B1, B2, D and E (Herzer et al., 1990
). Although it has been stated that none of the ECOR strains is pathogenic (Milkman & McKane, 1995
), it is evident that different pathogenic E. coli may be grouped among the ECOR strains on the basis of MLEE (Pupo et al., 1997
). Furthermore, phylogenetic studies demonstrate the existence of genes encoding typical pathogenicity determinants for uropathogens, such as pap, hly, kps and sfa, among some of the ECOR strains (Bingen et al., 1998
; Boyd & Hartl, 1998
; Marklund et al., 1992
), though it is unclear whether or not these genes are active. Moreover, none of the ECOR strains gives a hybridization signal with an ehxA probe from the EHEC hly sequence (Boyd & Hartl, 1998
).
In this work, we examined the hlyA genotype, phenotype and expression of some ECOR strains, assessed these strains for their cellular toxicity towards different host cells, determined the existence of EAEC in the ECOR collection and conducted a sequence comparison of the EAST1 (astA) genes from these enteropathogenic strains.
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METHODS |
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Detection of haemolysis by liquid- and solid-phase assays.
Bacteria were routinely diluted 1:100 from overnight cultures and grown with shaking. Supernatants (50 µl) of exponential-phase (about 100 Klett units) or overnight bacterial cultures were incubated with 50 µl 2% (final concentration) suspension of horse erythrocytes (RBCs) at 37 °C for 3 h (Bauer & Welch, 1996 ). The amount of lysis was determined by measuring released haemoglobin spectrophotometrically at A540. RBCs were incubated in distilled H2O to measure total lysis (100%), and background lysis was determined with RBCs incubated in saline (Bauer & Welch, 1996
). Percentage lysis was calculated from A540 measurements as follows: 100x[(A540 of sample - A540 of background)/(A540 of total - A540 of background)]. Blood agar plates with washed or unwashed RBCs were also used to check the haemolytic activity of both the supernatants and colonies of some of the ECOR strains carrying or not carrying haemolysin gene(s). A clear haemolytic zone of at least 1 mm around an isolated colony was scored as positive.
Western blot analysis of haemolysin.
Exponential-phase or overnight bacterial cultures were centrifuged and TCA added to 20 ml of the supernatants at 10% final concentration. Samples were kept on ice for at least 60 min. Precipitated proteins were pelleted by centrifugation at 4 °C (Bauer & Welch, 1996 ) and resuspended in SDS-PAGE loading buffer. Proteins were separated on 10% SDS-PAGE gels and transferred to a 0·2 µm pore size PVDF membrane with a semi-dry transfer cell Trans-Blot system (Bio-Rad). The membrane was blocked in PBS (80 mM Na2HPO3, 20 mM NaH2PO3, 100 mM NaCl, pH 7·5)/0·1% (v/v) Tween 20/5% milk powder overnight at room temperature. The primary antibody was rabbit polyclonal antiserum raised against HlyA (a kind gift from Dr A. Juarez, Universidad de Barcelona, Spain) and used at 1:100 dilution as described by Balsalobre et al. (1996)
. All the remaining procedures were according to the instructions from the ECL kit (Amersham Pharmacia Biotech).
Macrophage cytotoxicity by lactate dehydrogenase (LDH) assay and cell detachment.
The murine macrophage cell line J774 was routinely grown, infected, and assessed for cytotoxicity and detachment as described by Lai et al. (1999) , Vanmaele et al. (1995)
and Oscarsson et al. (1999)
. These experiments were repeated twice and representative results are shown. Briefly, 1 d before infection with bacteria, about 2x104 J774 cells were seeded into each well of flat-bottom 96-well plates for cytotoxicity assays and about 1·25x105 cells into each well of 24-well tissue culture plates for detachment experiments. J774 cells were infected with bacteria at a multiplicity of infection (MOI) of 100. At 2 h post-infection, cells were washed and incubated with medium containing 100 µg gentamicin ml-1. Supernatants of the infected macrophages from designated time points were sampled and assayed for the activity of the released intracellular enzyme LDH (Korzeniewski & Callewaert, 1983
) using the Cytotox 96 kit (Promega) according to the manufacturers instructions. The percentage cytotoxicity was calculated as described previously (Lai et al.,1999
) and total release (100%) was taken as the activity in macrophage lysates after treating with Triton X-100 provided with the kit. HB101 and ECOR strains do not have endogenous LDH activity when grown aerobically.
For detachment assay at 24 h post-infection, the monolayers were washed three times with PBS to remove nonadherent cells. J774 cells remaining in each well were fixed for 10 min with 70% methanol and then stained with Giemsa stain for 30 min. The monolayers were then washed three times with water to remove excess Giemsa stain, and the stained cells were lysed with 2% SDS. A portion of the lysates were transferred to 96-well microtitre plates and the absorbance of the contents of each well was recorded using a multiscan plate reader at 620 nm. The percentage of monolayer detachment was calculated as follows: 100-[(A620 of inoculated well/A620 of uninoculated well)x100]. At least four independent experiments were performed for each strain. Values of wells containing uninfected J774 cells were taken as zero detachment (100% attachment).
Clump formation assay.
Bacterial clump formation at the surface of liquid culture was assayed as described by Albert et al. (1993) . Each of the 72 ECOR strains was inoculated into 5 ml Luria broth in glass test tubes and incubated at 37 °C in a shaker incubator at 140 r.p.m. for 1620 h. EAEC strain JM221, which forms clumps visible as a thick scum in liquid overnight cultures, was used as positive control (Albert et al., 1993
).
PCR.
Primers for the whole hlyA gene were from Boyd & Hartl (1998) and can amplify a product of 2930 bp size using a protocol (1 cycle of 94 °C for 30 s; 30 cycles of 94 °C for 30 s, 55 °C for 1 min and 68 °C for 6 min; 1 cycle of 72 °C for 10 min). Primers for the AA probe were from Schmidt et al. (1995b)
; this primer set generated a 630 bp fragment using the original procedure. PCR primers for astA were as described by Itoh et al. (1997)
and Savarino et al. (1993)
. astA was amplified by 35 cycles of 94 °C for 30 s, 55 °C for 1 min and 72 °C for 1 min followed by 1 cycle of 72 °C for 7 min, generating a 124 bp product. PCR analysis was performed with a programmable thermal controller (MiniCycler, M J Research). Amplified products were analysed by electrophoresis in agarose gels of appropriate concentrations. DNA ladder markers of 100 bp and/or 1 kb (Bio-Rad) were used as molecular size standards.
Nucleotide sequencing and sequence analysis.
To sequence the AA probe and the astA gene, the PCR products using bacterial templates were purified with GeneClean III kit (Bio101). A total of 50150 ng purified PCR product was subjected to Taq cycle-sequencing reactions with the dye terminator cycle sequencing Ready Reaction kit (PE Applied Biosystems) according to the manufacturers instructions. Both 3' and 5' primers were used to crosscheck the sequencing results. Electrophoresis of sequencing products was performed on 4% polyacrylamide gels with an automated sequencer (model 377XL; Applied Biosystems). The nucleotide sequences were analysed with the ANALYSIS program version 3.0 (Applied Biosystems).
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RESULTS |
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Macrophage cytotoxicity and detachment
All the 72 ECOR strains were tested for expression of cytotoxicity towards J774 cells as monitored by LDH release and cell detachment (see Methods). Results from cytotoxicity and cell detachment experiments were closely related to haemolytic activity for those strains of strong haemolytic phenotype (Table 1, Fig. 1c
). We found that ECOR strains with strong haemolytic activity (ECOR5154, 56 and 60 in Table 1
and Fig. 1c
) were extremely cytotoxic to J774 cells and caused substantial cell detachment. The weakly haemolytic strain ECOR48 displayed higher cytotoxicity than ECOR24 for unknown reason(s). In spite of their equally weak positive haemolysis on blood agar plates, ECOR48 gave a higher haemolytic value than ECOR24 in the quantitative liquid assay (Fig. 1c
). It remained unclear what factors contribute to the marginal cytotoxicity of ECOR43 and ECOR57. ECOR43 happened to harbour astA (Fig. 2
, lane 9), but it was nonhaemolytic (Fig. 1
and Table 1
). However, cells infected with other ECOR strains without the haemolysin gene (ECOR8 and 65 in Fig. 1
and Table 1
) or even with this gene but without haemolytic activity at all (ECOR63 in Fig. 1
and Table 1
) were still alive and did not display a significant cytotoxic or detaching activity after being cultured for 24 h.
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Nucleotide sequences of the AA probe and the astA from ECOR
Comparing the original EAEC AA probe sequence (Schmidt et al., 1995b ) with that of ECOR8 from this work (data not shown), we found only 2 nucleotides changed at positions 387 (T
C) and 484 (G
A). It is not yet known if that region is within the translated part and whether or not these two changes lead to predicted amino acid changes.
All the previous astA sequences were derived from diarrhoeagenic E. coli (Savarino et al., 1993 ; Yamamoto & Echeverria, 1996
; Yamamoto & Nakazawa, 1997
; Yamamoto et al., 1997
). We sequenced the astA gene from strains in the ECOR collection. Comparison of the published astA sequences and our data from this study is shown in Fig. 3
. The astA sequences of strains ECOR5, ECOR43 and ECOR68 were identical to the published common astA sequences of EAEC strain 042 (Yamamoto et al., 1997
), DAEC (Yamamoto et al., 1997
), EPEC (Yamamoto et al., 1997
) and ETEC (Yamamoto & Echeverria, 1996
; Yamamoto & Nakazawa, 1997
). The astA sequences from strains ECOR10 and ECOR44 were nearly identical to the common sequences, differing by only one base change at position 23 (GCG
GCC) with no amino acid substitution. The astA sequence from ECOR33 differed from EAEC strain 042 by three bases at the eighth, ninth and eleventh codon positions (CGG
CGA, AGA
AGG and ACA
GCA, respectively), resulting in a change in the deduced amino acid residue (Ala
Thr); this was also the case for ECOR32 and ECOR35 except that these two strains had two more base changes at position 23 (GCG
GTC), leading to an amino acid change (Ala
Val). Besides these three bases, they also differed from EAEC strain 17-2 (Savarino et al., 1993
) by one more base at codon position 21 (GCA
ACA), resulting in a total of two deduced amino acid changes at positions 11 (Ala
Thr) and 21 (Thr
Ala). ECOR strains 30 and 71 also differed in their astA sequences from the consensus sequence in the third bases at either positions 8, 10 or 23 (see Fig. 3
for details).
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Distribution of astA among ECOR strains
astA was sparsely distributed among four groups but absent from group B2, with two strains in group A, five strains in group B1, two strains in group D and one strain in group E. Preliminary studies of the location of the astA sequences suggested that the gene may be plasmid-located. We obtained amplified fragments of the same size using plasmid DNA preparations as template from all astA-positive strains (data not shown). One interesting finding was that two isolates from the same giraffe in Washington Zoo (Ochman & Selander, 1984b ), ECOR32 (O7:H21) and ECOR68 (ON:NM), were both found in this study to harbour astA, but their nucleotide sequences differed from each other by five bases, causing two deduced amino acid changes (Fig. 3
). This result may indicate that astA has been subjected to some genetic exchanges, such as intragenic recombination, rather than horizontal transfer. Most of the astA-positive strains (8/10) were from North America (Canada, 1; USA, 7), and the remaining two from Europe (2 out of the 22 strains from Sweden) whose sequences were identical to the common astA sequence.
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DISCUSSION |
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We found that obvious cell detachment and toxicity correlated well with the expression of highly active haemolysin, but not with the existence of the astA gene or the silent hlyA gene under the conditions used. Possible explanations for the differential expression of HlyA and haemolytic activity of these hlyA-positive strains are that some strains (ECOR24, 48, 63) have an incomplete hlyCABD operon (Stanley et al., 1998
) or that the potential amino acid substitutions deduced from their partially sequenced hlyA genes (Boyd & Hartl, 1998
) lead to a decrease (or loss) of haemolytic activity. ECOR51 has a strong haemolytic phenotype but seems to express less of the mature 110 kDa HlyA. It is feasible that the HlyA from ECOR51 either has higher specific activity than those from other ECOR strains or that its partially degraded HlyA (i.e. 60 kDa) product confers this phenotype. Earlier studies have shown that this breakdown product of HlyA recognized by HlyA antibodies may have haemolytic activity (Noegel et al., 1979
; Goebel & Hedgpeth, 1982
; Nicaud et al., 1985
).
ECOR is a complex collection and evidently some of the strains expressed active haemolysin and are potential pathogens. The hly operon is commonly present in multiple copies in the chromosome of extraintestinal E. coli isolates (Blum et al., 1995 ). The linkage between genes for haemolysin and P fimbriae is estimated to be 415 kbp for several O6 and O4 isolates (Low et al., 1984
), which are also the serotypes of ECOR strains 53, 56 and 60 (Table 1
). Elliott et al. (1998)
demonstrated that
haemolysin confers virulence to a nonpathogen in the RITARD animal model, causing inflammation. Island et al. (1998)
reported that haemolytic and CNF1-negative E. coli show cytotoxicity in T24 human bladder cells. Results from this study further prove the relatedness between target cell toxicity and detachment and the expression of highly active haemolysin. The hallmark of infections due to EPEC and EHEC is the attaching-and-effacing (A/E) histopathology (Law & Chart, 1998
; Nataro & Kaper, 1998
). A recombinant plasmid clone containing the entire locus for enterocyte effacement (LEE) region is sufficient to confer the A/E phenotype when cloned into K-12 or E. coli from the normal flora (McDaniel & Kaper, 1997
). Using a PCR-based assay, it was found that ECOR37, a healthy animal isolate of untypable serotype, contains a LEE island at the selC locus (Bergthorsson & Ochman, 1998
), which is consistent with MLEE results repeatedly showing this strain most closely linked to EHEC O157:H7 strains (Perna et al., 1998
; Pupo et al., 1997
) and with a newly proposed evolutionary model where an EPEC-like strain with LEE is the ancestor of O157:H7 (Feng et al., 1998
). One study shows that the chromosomes of some ECOR strains are several hundred kilobases larger than others; thus they could possibly harbour additional pathogenicity islands (Bergthorsson & Ochman, 1998
).
It was reported that the PCR assay with primers derived from the AA probe sequence shows similar sensitivity (89%) and specificity to those of the AA probe (Schmidt et al., 1995b ), which is used as an alternative standard to define EAEC (Law & Chart, 1998
; Nataro & Kaper, 1998
). ECOR8 (O86:NM, one of the EAEC typical serotypes) was positive with this PCR assay and we further confirmed by sequence analysis that it contained the AA probe sequence. Previous work showed that ECOR8 carries genes for PapI and Kps, which are absent in E. coli K-12 (Boyd & Hartl, 1998
), and harbours a single copy of IS200 (Bisercic & Ochman, 1993
). The pathogenicity potential of ECOR8 remains to be determined.
Since the first astA sequence from EAEC strain 17-2 was published (Savarino et al., 1993 ), Yamamoto & Echeverria (1996)
and Yamamoto et al. (1997)
subsequently sequenced its homologues from other diarrhoeagenic E. coli and found that all of their astA genes were nearly identical to each other but differed from the original one from 17-2. Comparison of the astA sequences from EAEC (Yamamoto et al., 1997
), ETEC (Yamamoto & Echeverria, 1996
; Yamamoto & Nakazawa, 1997
), EPEC (Yamamoto et al., 1997
), EPEC-related E. coli (Yamamoto et al., 1997
) and ECOR strains (this report) made it possible to propose the common nucleotide sequence shared by EAEC, ETEC, EPEC and ECOR strains as the astA consensus sequence, whilst those which differed in deduced amino acid sequence were variants (including the astA sequence from strains 17-2, N1 and ECOR strains 32, 33 and 35) (Savarino et al., 1993
; Yamamoto et al., 1997
; and this study). Savarino et al. (1993)
first designed a synthetic peptide spanning the region of EAST1 from residue 8 to 29 with enterotoxic activity. From Fig. 3
one can clearly see that within the functional domain, positions 811, 20, 21, 23 and 33 are hot spots for nucleotide changes. Interestingly, sequence analysis of astA from ECOR strains reveals that all the astA nucleotide variations are located in this functional region. At the eighth and ninth (arginine) residues, both changes in the third base do not cause the deduced amino acid substitution, whilst the alanine residue at positions 11 and 23 leads to substitutions with threonine and valine, respectively. It will be interesting to further investigate the biological significance of the amino acid change from the tiny, hydrophobic, non-polar alanine to the small, less hydrophobic, polar threonine or to the small, aliphatic, less hydrophobic valine. Also interesting would be to check the expression of astA in vivo and in vitro when specific antiserum becomes available, and its activity with the Ussing chamber (Savarino et al., 1993
).
The pathogenesis of EAEC infection is not well understood. The significance of EAST1 in pathogenesis is unknown although E. coli categories other than EAEC, notably the EHEC, ETEC and EPEC strains, have been shown to produce this toxin with high frequency (Savarino et al., 1996 ). According to the three-stage model of EAEC pathogenesis proposed by Nataro & Kaper (1998)
, EAST1 might be involved at the third stage where the elaboration of an EAEC cytotoxin could damage the intestinal cells. It was also suggested recently that EAST1 toxin is closely associated with an adherence factor, CS31A, among pathogenic bovine E. coli (Bertin et al., 1998
). We are more in favour of the notion that astA may confer an ecological advantage to intestinal E. coli isolates simply by contributing to pathogenesis (Savarino et al., 1996
), although this hypothesis remains to be verified.
Due to the heterogeneous status of the ECOR collection for carrying various virulence factors, it seems appropriate to conclude that some of the ECOR strains of both human and animal origin are potential pathogens with features of cell-detaching E. coli. Caution should be taken when handling or disposing of these strains.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Swedish Medical Research Council, the Swedish Natural Science Research Council and the Göran Gustafsson Foundation for Research in Natural Sciences and Medicine.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balsalobre, C., Juárez, A., Madrid, C., Mouriño, M., Prenafeta, A. & Muñoa, F. J. (1996). Complementation of the hha mutation in Escherichia coli by the ymoA gene from Yersinia enterocolitica: dependence on the gene dosage. Microbiology 142, 1841-1846.[Abstract]
Bauer, M. E. & Welch, R. A. (1996). Characterization of an RTX toxin from enterohemorrhagic Escherichia coli O157:H7. Infect Immun 64, 167-175.[Abstract]
Bergthorsson, U. & Ochman, H. (1998). Distribution of chromosome length variation in natural isolates of Escherichia coli. Mol Biol Evol 15, 6-16.[Abstract]
Bertin, Y., Martin, C., Girardeau, J.-P., Pohl, P. & Contrepois, M. (1998). Association of genes encoding P fimbriae, CS31A antigen and EAST 1 toxin among CNF1-producing Escherichia coli strains from cattle with septicemia and diarrhea. FEMS Microbiol Lett 162, 235-239.[Medline]
Bingen, E., Picard, B., Brahimi, N., Mathy, S., Desjardins, P., Elion, J. & Denamur, E. (1998). Phylogenetic analysis of Escherichia coli strains causing neonatal meningitis suggests horizontal gene transfer from a predominant pool of highly virulent B2 group strains. J Infect Dis 177, 642-650.[Medline]
Bisercic, M. & Ochman, H. (1993). Natural populations of Escherichia coli and Salmonella typhimurium harbor the same classes of insertion sequences. Genetics 133, 449-454.
Blum, G., Ott, M., Lischewski, A., Ritter, A., Imrich, H., Taschape, H. & Hacker, J. (1994). Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen. Infect Immun 62, 606-614.[Abstract]
Blum, G., Falbo, V., Caprioli, A. & Hacker, J. (1995). Gene clusters encoding the cytotoxic necrotizing factor type 1, Prs-fimbriae and alpha-hemolysin from the pathogenicity island II of the uropathogenic Escherichia coli strain J96. FEMS Microbiol Lett 126, 189-196.[Medline]
Boyd, E. F. & Hartl, D. L. (1998). Chromosomal regions specific to pathogenic isolates of Escherichia coli have a phylogenetically clustered distribution. J Bacteriol 180, 1159-1165.
Elliott, S. J., Srinivas, S., Albert, M. J., Alam, K., Robins-Browne, R. M., Gunzburg, S. T., Mee, B. J. & Chang, B. J. (1998). Characterization of the roles of hemolysin and other toxins in enteropathy caused by alpha-hemolytic Escherichia coli linked to human diarrhea. Infect Immun 66, 2040-2051.
Felmlee, T., Pellett, S. & Welch, R. A. (1985). The nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol 163, 94-105.[Medline]
Feng, P., Lampel, K. A., Karch, H. & Whittam, T. S. (1998). Genotypic and phenotypic changes in the emergence of Escherichia coli O157:H7. J Infect Dis 177, 1750-1753.[Medline]
Goebel, W. & Hedgpeth, J. (1982). Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli. J Bacteriol 151, 1290-1298.[Medline]
Gunzburg, S. T., Chang, B. J., Elliott, S. J., Burke, V. & Gracey, M. (1993). Diffuse and enteroaggregative patterns of adherence of enteric Escherichia coli isolated from aboriginal children from the Kimberley region of Western Australia. J Infect Dis 167, 755-758.[Medline]
Herzer, P. J., Inouye, S., Inouye, M. & Whittam, T. S. (1990). Phylogenetic distribution of branched RNA-linked multicopy single-stranded DNA among natural isolates of Escherichia coli. J Bacteriol 172, 6175-6181.[Medline]
Island, M. D., Cui, X., Foxman, B., Marrs, C. F., Stamm, W. E., Stapleton, A. E. & Warren, J. W. (1998). Cytotoxicity of hemolytic, cytotoxic necrotizing factor 1-positive and -negative Escherichia coli to human T24 bladder cells. Infect Immun 66, 3384-3389.
Itoh, Y., Nagano, I., Kunishima, M. & Ezaki, T. (1997). Laboratory investigation of enteroaggregative Escherichia coli O untypeable:H10 associated with a massive outbreak of gastrointestinal illness. J Clin Microbiol 35, 2546-2550.[Abstract]
Johnson, J. R. (1991). Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev 4, 80-128.[Medline]
Korzeniewski, C. & Callewaert, D. M. (1983). An enzyme-release assay for natural cytotoxicity. J Immunol Methods 64, 313-320.[Medline]
Lai, X.-H., Xu, J.-G., Melgar, S. & Uhlin, B. E. (1999). An apoptotic response by J774 macrophage cells is common upon infection with diarrheagenic Escherichia coli. FEMS Microbiol Lett 172, 29-34.[Medline]
Law, D. & Chart, H. (1998). Enteroaggregative Escherichia coli. J Appl Microbiol 84, 685-697.[Medline]
Low, D., David, V., Lark, D., Schoolnik, G. & Falkow, S. (1984). Gene clusters governing the production of hemolysin and mannose-resistant hemagglutination are closely linked in Escherichia coli serotype O4 and O6 isolates from urinary tract infections. Infect Immun 43, 353-358.[Medline]
McDaniel, T. K. & Kaper, J. B. (1997). A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. Mol Microbiol 23, 399-407.[Medline]
Marklund, B.-I., Tennent, J. M., Garcia, E., Hamers, A., Bga, M., Lindberg, F., Gaastra, W. & Normark, S. (1992). Horizontal gene transfer of the Escherichia coli pap and prs pili operons as a mechanism for the development of tissue-specific adhesive properties. Mol Microbiol 6, 2225-2242.[Medline]
Marques, L. R. M., Abe, C. M., Griffin, P. M. & Gomes, T. A. T. (1995). Association between alpha-hemolysin production and HeLa cell-detaching activity in fecal isolates of Escherichia coli. J Clin Microbiol 33, 2707-2709.[Abstract]
Milkman, R. (1973). Electrophoretic variation in Escherichia coli from natural sources. Science 182, 1024-1026.[Medline]
Milkman, R. & McKane, M. (1995). DNA sequence variation and recombination in E. coli. In Population Genetics of Bacteria (Society for General Microbiology Symposium 52), pp. 127-142. Edited by S. Baumberg, J. P. W. Young, E. M. H. Wellington & J. R. Saunders. Cambridge: Cambridge University Press.
Nataro, J. P. & Kaper, J. B. (1998). Diarrheagenic Escherichia coli. Clin Microbiol Rev 11, 142-201.
Nicaud, J.-M., Mackman, N., Gray, L. & Holland, I. B. (1985). Regulation of haemolysin synthesis in E. coli determined by HLY genes of human origin. Mol Gen Genet 199, 111-116.[Medline]
Noegel, A., Rdest, U., Springer, W. & Goebel, W. (1979). Plasmid cistrons controlling synthesis and excretion of the exotoxin and haemolysin of Escherichia coli. Mol Gen Genet 175, 343-350.[Medline]
Ochman, H. & Selander, R. K. (1984a). Evidence for clonal population structure in Escherichia coli. Proc Natl Acad Sci USA 81, 198-201.[Abstract]
Ochman, H. & Selander, R. K. (1984b). Standard reference strains of Escherichia coli from natural populations. J Bacteriol 157, 690-693.[Medline]
Oscarsson, J., Mizunoe, Y., Li, L., Lai, X.-H., Wieslander, . & Uhlin, B. E. (1999). Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli. Mol Microbiol 32, 1226-1238.[Medline]
Perna, N. T., Mayhew, G. F., Posfai, G., Elliott, S., Donnenberg, M. S., Kaper, J. B. & Blattner, F. R. (1998). Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7. Infect Immun 66, 3810-3817.
Pupo, G. M., Karaolis, D. K. R., Lan, R. T. & Reeves, P. (1997). Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies. Infect Immun 65, 2685-2692.[Abstract]
Savarino, S. J., Fasano, A., Watson, J., Martin, B. M., Levine, M. M., Guandalini, S. & Guerry, P. (1993). Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin. Proc Natl Acad Sci USA 90, 3093-3097.[Abstract]
Savarino, S. J., McVeigh, A., Watson, J., Cravioto, A., Molina, J., Echeverria, P., Bhan, M. K., Levine, M. M. & Fasano, A. (1996). Enteroaggregative Escherichia coli heat-stable enterotoxin is not restricted to enteroaggregative E. coli. J Infect Dis 173, 1019-1022.[Medline]
Schmidt, H., Beutin, L. & Karch, H. (1995a). Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL933. Infect Immun 63, 1055-1061.[Abstract]
Schmidt, H., Knop, C., Franke, S., Aleksic, S., Heesemann, J. & Karch, H. (1995b). Development of PCR for screening of enteroaggregative Escherichia coli. J Clin Microbiol 33, 701-705.[Abstract]
Stanley, P., Koronakis, V. & Hughes, C. (1998). Acylation of Escherichia coli hemolysin: a unique protein lipidation mechanism underlying toxin function. Microbiol Mol Biol Rev 62, 309-333.
Vanmaele, R. P., Finlayson, M. C. & Armstrong, G. D. (1995). Effect of enteropathogenic Escherichia coli on adherent properties of Chinese hamster ovary cells. Infect Immun 63, 191-198.[Abstract]
Welch, R. A., Dellinger, E. P., Minshew, B. & Falkow, S. (1981). Hemolysin contributes to virulence of extraintestinal Escherichia coli infections. Nature 294, 665-667.[Medline]
Yamamoto, T. & Echeverria, P. (1996). Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene sequences in enterotoxigenic E. coli strains pathogenic to humans. Infect Immun 64, 1141-1145.
Yamamoto, T. & Nakazawa, M. (1997). Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E. coli strains isolated from piglets and calves with diarrhea. J Clin Microbiol 35, 223-227.[Abstract]
Yamamoto, T., Wakisaka, N., Sato, F. & Kato, A. (1997). Comparison of the nucleotide sequence of enteroaggregative Escherichia coli heat-stable enterotoxin 1 genes among diarrhea-associated Escherichia coli. FEMS Microbiol Lett 147, 89-95.[Medline]
Received 28 May 1999;
revised 30 July 1999;
accepted 5 August 1999.