J Med Microbiol 53 (2004), 1037-1043; DOI: 10.1099/jmm.0.05381-0
© 2004 Society for General Microbiology
ISSN 0022-2615
Phenotypic and genotypic characterization of ß-D-glucuronidase-positive Shiga toxin-producing Escherichia coli O157 : H7 isolates from deer
Hideki Nagano1,
Takashi Hirochi2,
Kozo Fujita2,
Yoshihiro Wakamori1,
Koichi Takeshi1 and
Shoki Yano1
1Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819, Japan 2Sapporo City Institute of Public Health, Kikusui 9-1, Shiroishi-ku, Sapporo 003-8505, Japan
Correspondence Hideki Nagano nagano{at}iph.pref.hokkaido.jp
Received July 7, 2003
Accepted June 7, 2004
ß-Glucuronidase-positive (GUD+) Shiga toxin-producing Escherichia coli (STEC) O157 : H7 was isolated from both an asymptomatic woman and uncooked deer meat in her possession in Hokkaido, Japan. The phenotypic and genotypic characteristics of the two isolates were identical or closely related, indicating probable transmission of the deer isolate to the woman. Moreover, several other GUD+ STEC O157 : H7 strains investigated belonged to the distinct atypical GUD+ STEC O157 : H7 group that has been identified previously. This is the first report that deer can be a reservoir of GUD+ STEC O157 : H7 in Japan.
Abbreviations: EHEC, enterohaemorrhagic Escherichia coli; HUS, haemolytic uraemic syndrome; RPLA, reverse passive latex agglutination; STEC, Shiga toxin-producing Escherichia coli.
 |
INTRODUCTION
|
|---|
Shiga toxin (Stx)-producing Escherichia coli (STEC) O157 is the predominant cause of haemorrhagic colitis and haemolytic uraemic syndrome (HUS) (Tarr, 1995). The ability of STEC strains to cause severe disease in humans is associated with their capacity to secrete Stxs (Stx1, Stx2 or variants) (Melton-Celsa & O'Brien, 1998; O'Brien & Holmes, 1987). Several other virulence factors may contribute to the pathogenicity of these STEC strains. Among them, intimin, encoded by the eae gene, is involved in the intimate attachment of bacteria to enterocytes (Kaper et al., 1998). Some factors which are responsible for the secretion systems are encoded by pas (Kresse et al., 1998) and etp (Schmidt et al., 1997). Other putative virulence factors have been described: enterohaemorrhagic E. coli haemolysin (EHEC-Hly) (Schmidt et al., 1995), a serine protease (EspP) which can cleave human coagulation factor V (Brunder et al., 1997), a bifunctional catalase peroxidase (KatP) (Brunder et al., 1996) and the EHEC type of enteroaggregative E. coli heat-stable enterotoxin (SHEAST) (Yamamoto & Taneike, 2000).
During the last decade, phenotypically atypical strains of STEC O157 have been isolated in Germany (Ammon et al., 1999; Gunzer et al., 1992), the USA (Hayes et al., 1995) and Japan (Nagano et al., 2002). Although they all characteristically produced ß-D-glucuronidase activity (GUD+), those of German origin were non-motile, fermented sorbitol within 24 h (Ammon et al., 1999; Gunzer et al., 1992), and caused not only sporadic cases but also outbreaks, including 28 HUS cases (Ammon et al., 1999). Phenotypic and molecular characterization of the German GUD+ STEC O157 : H– strains revealed that they represented a distinct clone within the E. coli O157 complex (Karch et al., 1993). However, those of Japanese origin isolated in Hokkaido were obtained from asymptomatic subjects or those with mild clinical symptoms. They were shown to represent a distinct clone within the STEC O157 serogroup (Nagano et al., 2002).
Some STEC strains can be found in the faecal flora of a wide variety of animals including cattle, sheep, goats, pigs, cats, dogs, chickens, gulls and pigeons (Beutin et al., 1993; Johnson et al., 1996; Makino et al., 2000; Schmidt et al., 2000; Wallace et al., 1997). However, the great majority of these isolates are of serotypes other than O157 : H7 and are of questionable pathogenicity. Among these animals, cattle are regarded as the most important animal species in terms of human infection. It has been reported that cattle can also be the reservoirs of atypical GUD+ STEC O157 : H– isolates in the Czech Republic (Bielaszewska et al., 2000). In contrast to the cases in central Europe, there are no reports on natural reservoirs and the routes of transmission of STEC GUD+ O157 : H7 strains in Japan.
In this study, we isolated GUD+ STEC O157 : H7 strains from an asymptomatic woman and uncooked deer meat in her possession following a routine health inspection for food-handlers. We analysed the phenotypic and genotypic characteristics of both the deer and human isolates, and compared them with those of GUD+ STEC O157 : H7 isolates that we identified previously (Nagano et al., 2002).
|
METHODS
|
|---|
Bacterial isolates.
The isolates used in this study are listed in Table 1, including one GUD+ STEC O157 : H7 isolate (EC00125) from a deer and another (EC00124) from a 40-year-old woman. An additional four isolates of GUD+ STEC O157 : H7 were obtained from men with no clinical symptoms (EC99070, SREC9912 and SREC0116) or with mild diarrhoea (EC00045). Four isolates of GUD+ STEC O157 : H7 (EC97119, EC96112, EC97144 and EC96073) obtained between 1996 and 1997 (Nagano et al., 2002) were used as controls for PFGE patterns as they gave representative PFGE patterns of GUD+ STEC O157 : H7 isolates in Hokkaido. GUD+ STEC O157 : H7 strains were isolated with a standard method using sorbitol-MacConkey agar (SMAC) (Difco) and SMAC supplemented with cefixime (0.05 mg l–1; Dynal) and potassium tellurite (2.5 mg l–1; Dynal) (CT-SMAC) as previously described (Nagano et al., 2002). In addition, one isolate of typical GUD– STEC O157 : H7 and two isolates of Stx non-producing E. coli O157 : H12 and O157 : H16 were used as controls for phenotypic and genetic characterization.
View this table:
[in this window]
[in a new window]
|
Table 1. Epidemiological data, and plasmid and chromosomal genes encoding accessory virulence factors of atypical and typical isolates of E. coli O157 : H7 and Stx non-producing E. coli O157 isolates from individuals in Hokkaido, Japan
|
|
Phenotypic characterization of E. coli strains.
The biochemical properties of these strains were determined with the API 20E system (bioMérieux). The GUD activity of the isolates was examined on media containing 4-methylumbelliferyl ß-D-glucuronide as previously described (Nagano et al., 2002). An overnight culture at 37 °C which fluoresced under UV light was judged to be positive. Production of Stx was examined by the reverse passive latex agglutination (RPLA) test according to the manufacturer's instructions (Denkaseiken). The haemolytic activity of the isolates was detected after culture on blood agar plates containing 5 % defibrinated and washed sheep red blood cells and 10 mM CaCl2 (Beutin et al., 1989; Schmidt et al., 1996, 1999).
Antimicrobial susceptibility test.
The susceptibility of the isolates to antimicrobial agents was examined by the ‘Etest’ (AB Biodisk). The 10 antimicrobial agents used were: ampicillin, cefotaxime, gentamicin, ciprofloxacin, norfloxacin, tetracycline, sulfamethoxazole-trimethoprim, trimethoprim, fosfomycin and chloramphenicol.
PCR.
PCR for detecting the specific genes in Table 2 was performed with the GeneAmp PCR System 9600 (Perkin-Elmer Applied Biosystems) in a volume of 20 µl containing 1 µl purified bacterial DNA (approx. 200 ng), 200 mM deoxynucleotide triphosphates (dATP, dCTP, dGTP and dTTP), 30 pmol of each primer, 2 µl of a 10-fold-concentrated buffer mixture and 2.5 U Taq polymerase (Takara). The PCR conditions, primer designations and sequences are given in Table 2.
Southern blot hybridization.
E. coli DNA was isolated from 1 ml of an overnight culture using a Wizard Genomic DNA Purification kit (Promega) according to the manufacturer's instructions. The DNA concentration was calculated from its A260 value. For Southern blot hybridization, DNA was digested with BamHI (Toyobo) and electrophoresed through 0.8 % agarose gels in half-strength Tris/borate/EDTA (TBE) buffer, pH 8.3 (Sambrook et al., 1989). The separated DNA fragments were transferred to MAGNA nylon membranes (Micron Separation) by standard methods (Sambrook et al., 1989). Non-radioactive direct DNA labelling and Southern blot hybridization were performed with a Gene Images AlkPhos Direct Labelling and Detection kit (Amersham Pharmacia) following the supplier's procedures. The probes used in this study were prepared by PCR amplification of katP (primers wkat-B/wkat-F; Brunder et al., 1996), hlyA (primers hlyA1/hlyA4; Schmidt et al., 1995), etpD (primers D1/D13R; Schmidt et al., 1997) and espP (primers esp-A/esp-B; Brunder et al., 1997).
DNA-fingerprint analysis by PFGE.
PFGE analysis was conducted according to the methods described previously (Nagano et al., 2002) with a minor modification of the electrophoresis conditions. Briefly, the genomic DNA in agarose plugs after proteinase K treatment (Roche) was digested with 70 U XbaI (Roche) or 50 U NotI (Roche) following the conditions indicated by the manufacturer. The resulting DNA fragments were electrophoresed through 1 % agarose gels with the CHEF-DRIII apparatus (Bio-Rad) at 6 V cm–1 and 14 °C for 9 h with switch times ranging from 4 to 8 s followed by 13 h with the 8–50 s switch times. 
DNA concatemers (Bio-Rad) were used as DNA size markers. After staining with ethidium bromide, the agarose gel was photographed under UV light and the band image was digitized for computer analysis. The GelCompar software package (Applied Maths) was used for clustering analysis. Cluster analysis of the PFGE patterns was performed with the DICE similarity coefficient by using position tolerance at 1.2 %. Dendrograms were calculated by the unweighted pair-group method by using average linkage (UPGMA).
|
RESULTS AND DISCUSSION
|
|---|
Isolation of GUD+ STEC O157 : H7 isolates from a woman and her uncooked deer meat
A GUD+ STEC O157 : H7 strain (EC00124) was isolated from a 40-year-old female who was working in a food-processing company, following a routine health inspection in 2000. She had no symptoms at the time. She volunteered that she had cooked and eaten meat from a wild deer captured in Hokkaido. Another GUD+ STEC O157 : H7 isolate (EC00125) was obtained from the remaining uncooked deer meat. Colonies of non-sorbitol fermenters (SOR–) that agglutinated anti-O157 serum (Denkaseiken) were biochemically confirmed as E. coli and checked for the presence of stx genes by nucleic acid amplification. These isolates were further confirmed as STEC from production of Stx1 and Stx2 using a commercially available RPLA test (Verotox-F; Denkaseiken), and they were serotyped with rabbit anti-O157 and anti-H7 sera (Denkaseiken).
Phenotypic characteristics of GUD+ STEC O157 : H7
Phenotypic characteristics of all isolates are shown in Table 1. All GUD+ STEC O157 : H7 isolates produced Stx1 and Stx2 as demonstrated by the RPLA test. These strains also produced EHEC-Hly on blood agar and GUD after incubation for 16–24 h, but were unable to ferment sorbitol on SMAC agar plates within 24 h. No GUD+ STEC O157 : H7 isolates fermented rhamnose in API 20E strips, while most GUD– STEC O157 : H7 isolates did so. All isolates were susceptible to all 10 antimicrobial agents.
Genetic characterization of GUD+ STEC O157 : H7 isolates
PCR and Southern hybridization techniques were employed to detect virulence genes of the E. coli O157 isolates. As shown in Table 1, five isolates of GUD+ STEC O157 : H7 obtained in Hokkaido between 1999 and 2001, including those from the deer meat and the 40-year-old female, possessed the same virulence factors as those observed in the isolates of GUD+ STEC O157 : H7 reported previously (Nagano et al., 2002). In all of the GUD+ STEC O157 : H7 isolates, EHEC-hlyA, etpD, stx1, stx2, pas and SHEAST genes were detected by PCR; however, no amplicon was observed for katP or espP genes, which are usually detected in typical GUD– STEC O157 : H7 isolates. Southern hybridization with katP or espP DNA fragments showed no signals on blots of BamHI-restricted DNA of GUD+ STEC O157 : H7 isolates. For identification and determination of eaeA type, PCR-RFLP was employed. All STEC O157 : H7 isolates showed a RFLP pattern corresponding to the 
1 type as reported by Oswald et al. (2000). In addition, both GUD+ and GUD– STEC O157 : H7 isolates harboured virulence genes encoding SHEAST type 1 and they were indistinguishable from typical STEC O157 strains. In contrast, stx-negative E. coli isolates of O157 : H12 had no virulence genes and O157 : H16 contained just the 
type of intimin protein.
Although GUD+ STEC O157 : H7 isolates contained chromosomal stx1, stx2, pas and eae genes and plasmid EHEC-hlyA and etpD genes (which are the virulence characteristics shared in common with the typical STEC O157 : H7 strains obtained from patients), these atypical isolates appear to be less pathogenic. This is supported by the finding that no individuals shedding these atypical isolates have developed serious illness such as HUS. In contrast, atypical STEC O157 : H– strains with SOR+ and GUD+ phenotypes caused HUS in Germany and the Czech Republic (Ammon et al., 1999; Bielaszewska et al., 1998, 2000; Gunzer et al., 1992). Some European atypical STEC O157 : H– strains harboured EHEC-hlyA and etpD genes on a large plasmid but lacked espP and katP genes (Bielaszewska et al., 1998, 2000; Brunder et al., 1999) as with the isolates that we report here, but other atypical STEC O157 : H– strains lacked even these two plasmid-carried virulence-associated genes (Bielaszewska et al., 2000). In their report, Bielaszewska et al. (2000) stated that these plasmid-encoded virulence factors may not be essential for pathogenesis or they might have been lost during infection or storage. In contrast, the data obtained here, as well as in the previous study (Nagano et al., 2002), indicate that the composition of virulence genes on the large plasmid in GUD+ STEC O157 : H7 isolates in Hokkaido has been constant for 5 years between 1996 and 2001. Thus the set of putative virulence factors of STEC strains may not be directly linked to the severity of the illness, leading to the implication that the severity of illness is more related to the immunological status of the host.
Fingerprint analysis of GUD+ STEC O157 : H7
Fingerprint analysis of GUD+ STEC O157 : H7 isolates was performed using PFGE. As shown in Fig. 1(a), the XbaI-digested PFGE pattern of the isolate from the deer (lane 3) was very similar to that of the isolate from the female carrier (lane 2) with two differences in bands at positions around 388 and 291 kb. Very similar patterns were seen for another seven GUD+ STEC O157 : H7 isolates: four representatives (lanes 4–7) from the previous study (Nagano et al., 2002), two from 1999 (lanes 8 and 9) and one from 2001 (lane 10). In contrast, all of the PFGE patterns of GUD+ STEC O157 : H7 isolates (lanes 2–10) were different from those of control E. coli O157 isolates, including typical GUD– STEC O157 : H7 (lane 11) and two GUD+ Stx-negative strains of serotypes O157 : H12 (lane 12) and O157 : H16 (lane 13). These findings were confirmed using NotI for PFGE analysis (Fig. 2a). In addition, no differences in NotI-digested PFGE patterns were observed between the deer and female carrier isolates. Analysis of XbaI and NotI PFGE patterns by the GelCompar software (Figs 1b and 2b, respectively) demonstrated that all nine GUD+ STEC O157 : H7 strains, including the deer and human isolates, belong to a distinct cluster, only distantly related to both the GUD– STEC O157 : H7 isolate and the two GUD+ Stx-negative E. coli O157 : H12 and O157 : H16 isolates.
View larger version (44K):
[in this window]
[in a new window]
|
Fig. 1. PFGE patterns of XbaI-digested genomic DNA from GUD+ STEC O157 : H7 isolates and control E. coli O157 isolates (a) and dendrogram of the similarities of PFGE patterns of XbaI-digested DNA derived using the GelCompar software package and calculated by UPGMA (b). Lanes 1 and 14 show molecular mass marker of lambda DNA concatemers (sizes are shown to the left). In lanes 2–10, the following GUD+ STEC O157 : H7 isolates are shown: 2, EC00124; 3, EC00125; 4, EC97119; 5, EC96112; 6, EC97144; 7, EC96073; 8, EC99070; 9, SREC9912; 10, SREC0116. In lanes 11–13, control E. coli O157 isolates are shown as follows: 11, EC00045 (GUD– STEC O157 : H7); 12, EC01068 (GUD+, Stx-negative E. coli O157 : H12); 13, EC01028 (GUD+, Stx-negative E. coli O157 : H16). The strains in this study are indicated by an asterisk.
|
|
View larger version (46K):
[in this window]
[in a new window]
|
Fig. 2. PFGE patterns of NotI-digested genomic DNA from GUD+ STEC O157 : H7 isolates and control E. coli O157 isolates (a) and dendrogram of the similarities of PFGE patterns of NotI-digested DNA derived using the GelCompar software package and calculated by UPGMA (b). Lane numbers are the same as shown in Fig. 1. The strains in this study are indicated by an asterisk.
|
|
The minor difference in the XbaI PFGE patterns between the deer and the human isolates seems to be due to genetic drift of these isolates. According to the criteria of Tenover et al. (1995), these two isolates are closely related. Based on the phenotypic and genetic characteristics of both isolates, and epidemiological data, it is most probable that the woman was infected with the STEC GUD+ O157 : H7 from the deer meat.
We reported previously the presence of a stable and novel distinct cluster of atypical GUD+ STEC O157 : H7 isolates which were initially obtained between 1996 and 1998 in Hokkaido (Nagano et al., 2002). Now we have shown that the GUD+ STEC O157 : H7 isolates obtained between 1999 and 2001 belong to this cluster, as judged from the results of PFGE analyses, the composition of virulence genes, and their phenotypic characteristics. In particular, the computed cluster analysis for those PFGE results clearly revealed that the GUD+ STEC O157 : H7 strains belong to a distinguishable cluster among the STEC O157 complex (about 300 isolates) that has been spread in Hokkaido (data not shown). In total, 17 GUD+ STEC O157 : H7 isolates were obtained in Hokkaido in the 5 years from 1996 to 2001. The major type of XbaI-digested PFGE pattern [lane 4 (EC97119) of Fig. 1a, b] was evident in eight isolates of GUD+ STEC O157 : H7 (Nagano et al., 2002). Although dendrogram analyses of the PFGE patterns of atypical STEC isolates obtained in 1999 (Fig. 1b, lane 9) and 2000 (Fig. 1b, lanes 2 and 3) revealed some tendency for genetic drift, which might lead to disappearance of this group, another isolate obtained in 2001 (Fig. 1b, lane 10) belonged to this group as checked by PFGE analyses. This implies that the GUD+ STEC O157 : H7 strains have slowly spread in Hokkaido with less genetic drift.
STEC can inhabit the intestine of wild animals as well as that of the domestic animals. There was a sporadic case of GUD– STEC O157 : H7 infection due to contaminated wild deer meat of Hokkaido origin in 1997 (Report by Ministry of Health and Welfare, Japan). Asakura et al. (1998) also isolated seven STEC strains from 43 faecal samples of wild deer in Hokkaido. These STEC strains were O111 : H45, Out : H45 and O96 : H– but not O157. It seems likely that wild deer have become a reservoir of STEC in Hokkaido. The rapid increase in wild deer numbers in Hokkaido is becoming a problem, as they have easier access to farms and more frequent contact with cattle. The sporadic case of STEC O157 infection from deer to human reported here is a timely warning about infections from not only domestic animals but also wild deer.
|
REFERENCES
|
|---|
Ammon, A., Peterson, L. R. & Karch, H. (1999). A large outbreak of hemolytic uremic syndrome caused by an unusual sorbitol-fermenting strain of E.coli O157 : H–. J Infect Dis 179, 1274–1277.[CrossRef][Medline]
Asakura, H., Makino, S., Shirahata, T., Tsukamoto, T., Kurazono, H., Ikeda, T. & Takeshi, K. (1998). Detection and genetical characterization of shiga toxin-producing Escherichia coli from wild deer. Microbiol Immunol 42, 815–822.[Medline]
Beutin, L., Monteneglo, M. A., Ørskov, I., Ørskov, F., Prada, J., Zimmermann, S. & Stephan, R. (1989). Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli. J Clin Microbiol 27, 2559–2564.[Abstract/Free Full Text]
Beutin, L., Geier, D., Steinrück, H., Zimmermann, S. & Scheutz, F. (1993). Prevalence and some properties of verotoxin (Shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals. J Clin Microbiol 31, 2483–2488.[Abstract/Free Full Text]
Bielaszewska, M., Schmidt, H., Karmali, M. A., Khakhria, R., Janda, J., Bláhová, K. & Karch, H. (1998). Isolation and characterization of sorbitol-fermenting shiga toxin (verocytotoxin)-producing Escherichia coli O157 : H– strains in the Czech Republic. J Clin Microbiol 36, 2135–2137.[Abstract/Free Full Text]
Bielaszewska, M., Schmidt, H., Liesegang, A. & 7 other authors (2000). Cattle can be a reservoir of sorbitol-fermenting shiga toxin-producing Escherichia coli O157 : H– strains and a source of human diseases. J Clin Microbiol 38, 3470–3473.[Abstract/Free Full Text]
Brunder, W., Schmidt, H. & Karch, H. (1996). KatP, a novel catalase-peroxidase encoded by the large plasmid of enterohaemorrhagic Escherichia coli O157 : H7. Microbiology 142, 3305–3315.[Abstract]
Brunder, W., Schmidt, H. & Karch, H. (1997). EspP, a novel extracellular serine protease of enterohaemorrhagic Escherichia coli O157 : H7 cleaves human coagulation factor V. Mol Microbiol 24, 767–778.[CrossRef][Medline]
Brunder, W., Schmidt, H., Frosch, M. & Karch, H. (1999). The large plasmids of shiga-toxin-producing Escherichia coli (STEC) are highly variable genetic elements. Microbiology 145, 1005–1014.[Abstract]
Cebula, T. A., Payne, W. L. & Feng, P. (1995). Simultaneous identification of strains of Escherichia coli serotype O157 : H7 and their shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. J Clin Microbiol 33, 248–250.[Abstract]
Gunzer, F., Böhm, H., Rüsmann, M., Bitzan, S., Aleksic, S. & Karch, H. (1992). Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome. J Clin Microbiol 30, 1807–1810.[Abstract/Free Full Text]
Hayes, P. S., Blom, K., Feng, P., Lewis, J., Strokbine, N. A. & Swaminathan, B. S. (1995). Isolation and characterization of a ß-D-glucuronidase-producing strain of Escherichia coli serotype O157 : H7 in the United States. J Clin Microbiol 33, 3347–3348.[Abstract]
Johnson, R. P., Clarke, R. C., Wilson, J. B. & 10 other authors (1996). Growing concerns and recent outbreaks involving non-O157 : H7 serotypes of verotoxigenic Escherichia coli. J Food Prot 59, 1112–1122.
Kaper, J. B., Elliotto, S., Sperandio, V., Perna, N. T., Mayhew, G. F. & Blattner, F. R. (1998). Attaching-and-effacing intestinal histopathology and the locus of enterocyte effacement. In Escherichia coli O157 : H7 and other Shiga Toxin-Producing E. coli Strains, pp. 163–182. Edited by J. B. Kaper & A. D. O'Brien. Washington, DC: American Society for Microbiology.
Karch, H., Böhm, H., Schmidt, H., Gunzer, F., Aleksic, S. & Heesemann, J. (1993). Clonal structure and pathogenicity of shiga-like toxin-producing sorbitol-fermenting Escherichia coli O157 : H–. J Clin Microbiol 31, 1200–1205.[Abstract/Free Full Text]
Kresse, A. U., Schulze, K., Deibel, C., Ebel, F., Rohde, M., Chakraborty, T. & Guzmán, C. A. (1998). Pas, a novel protein required for protein secretion and attaching and effacing activities of enterohemorrhagic Escherichia coli. J Bacteriol 180, 4370–4379.[Abstract/Free Full Text]
Makino, S., Kobori, H., Asakura, H., Watarai, M., Shirahata, T., Ikeda, T., Takeshi, K. & Tsukamoto, T. (2000). Detection and characterization of shiga toxin-producing Escherichia coli from seagulls. Epidemiol Infect 125, 55–61.[CrossRef][Medline]
Melton-Celsa, A. R. & O'Brien, A. (1998). Structure, biology, and relative toxicity of Shiga toxin family members for cells and animals. In Escherichia coli O157 : H7 and other Shiga Toxin-Producing E. coli Strains, pp. 121–128. Edited by J. B. Kaper & A. D. O'Brien. Washington, DC: American Society for Microbiology.
Nagano, H., Okui, T., Fujiwara, O., Uchiyama, Y., Tamate, N., Kumada, H., Morimoto, Y. & Yano, S. (2002). Clonal structure of shiga-toxin (Stx)-producing and ß-D-glucuronidase positive Escherichia coli O157 : H7 strains isolated from outbreaks and sporadic cases in Hokkaido, Japan. J Med Microbiol 51, 405–416.[Abstract/Free Full Text]
O'Brien, A. D. & Holmes, R. K. (1987). Shiga and Shiga-like toxins. Microbiol Rev 51, 206–220.[Free Full Text]
Oswald, E., Schmidt, H., Morabito, S., Karch, H., Marchès, O. & Caprioli, A. (2000). Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli: characterization of a new intimin variant. Infect Immun 68, 64–71.[Abstract/Free Full Text]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schmidt, H., Beutin, L. & Karch, H. (1995). Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157 : H7 strain EDL 933. Infect Immun 63, 1055–1061.[Abstract]
Schmidt, H., Maier, E., Karch, H. & Benz, R. (1996). Pore-forming properties of the plasmid-encoded hemolysin of enterohemorrhagic Escherichia coli O157 : H7. Eur J Biochem 241, 594–601.[Medline]
Schmidt, H., Henkel, B. & Karch, H. (1997). A gene cluster closely related to type II secretion pathway operons of gram-negative bacteria is located on the large plasmid of enterohemorrhagic Escherichia coli O157 : H7 strains. FEMS Microbiol Lett 148, 265–271.[CrossRef][Medline]
Schmidt, H., Geitz, C., Tarr, P. I., Frosch, M. & Karch, H. (1999). Non-O157 : H7 pathogenic shiga toxin-producing Escherichia coli: phenotypic and genetic profiling of virulence traits and evidence for clonality. J Infect Dis 179, 115–123.[CrossRef][Medline]
Schmidt, H., Scheef, J., Morabito, S., Caprioli, A., Wieler, L. H. & Karch, H. (2000). A new shiga toxin 2 variant (Stx2f) from Escherichia coli isolated from pigeons. Appl Environ Microbiol 66, 1205–1208.[Abstract/Free Full Text]
Tarr, P. I. (1995). Escherichia coli O157 : H7: clinical, diagnostic, and epidemiological aspects of human infection. Clin Infect Dis 20, 1–10.[Medline]
Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelson, P. A., Murray, B. E., Persing, D. H. & Swaminathan, B. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33, 2233–2239.[Medline]
Wallace, J. S., Cheasty, T. & Jones, K. (1997). Isolation of vero cytotoxin-producing Escherichia coli O157 from wild birds. J Appl Microbiol 82, 399–404.[CrossRef][Medline]
Yamamoto, T. & Taneike, I. (2000). The sequences of enterohemorrhagic Escherichia coli and Yersinia pestis that are homologous to the enteroaggregative E.coli heat-stable enterotoxin gene: cross-species transfer in evolution. FEBS Lett 472, 22–26.[CrossRef][Medline]
TOP
INTRODUCTION
METHODS
