Phenotypic and Molecular Analysis of Tellurite Res
http://www.100md.com
微生物临床杂志 2005年第1期
Institut für Hygiene, Universittsklinikum Münster, Münster, Germany
Division of Pediatric Gastroenterology and Nutrition, Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine and St. Louis Children's Hospital, St. Louis, Missouri
ABSTRACT
A total of 66 (98.5%) of 67 enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains had increased potassium tellurite (Te) MICs (32 to 1,024 μg/ml), grew on Te-containing media, and possessed Te resistance (ter) genes, whereas 83 (96.5%) of 86 sorbitol-fermenting (SF) EHEC O157:NM strains had Te MICs of 4 μg/ml, did not grow on Te-containing media, and lacked ter genes. Optimal detection of SF EHEC O157:NM strains requires Te-independent strategies.
TEXT
Tellurite (Te)-resistant (Ter) non-sorbitol-fermenting enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains cause diarrhea and hemolytic-uremic syndrome (HUS) worldwide (17), but sorbitol-fermenting (SF) EHEC O157:NM (nonmotile) strains have emerged as pathogens only in Europe (1, 6, 8, 12) and Australia (3) so far. SF EHEC O157:NM strains are not distinguishable from commensal E. coli strains on sorbitol MacConkey agar (SMAC), and do not grow (11) on cefixime-Te (CT)-SMAC (22), which is frequently used for selective isolation of EHEC O157:H7 strains from feces, foods, and the environment (2, 4, 5, 9, 13, 20, 21). Ter in EHEC O157:H7 is associated with the ter (terZABCDEF) gene cluster (19), duplicated in strain EDL933 in O islands OI 43 and OI 48 (14). One of these islands was originally identified in strain 86-24 (16) as the Ter and adherence-conferring island (16). Ter in SF EHEC O157:NM strains has not been investigated. Because Te susceptibility (Tes) could thwart the detection of such strains on media containing Te, we investigated Ter and the presence of ter genes in a large collection of SF EHEC O157:NM clinical isolates. We compared these characteristics with those of EHEC O157:H7.
Isolation and characterization of strains. A total of 67 EHEC O157:H7 and 86 SF EHEC O157:NM strains were isolated between 1987 and 2003 from patients with HUS (n = 118) or bloody (n = 11) or watery (n = 19) diarrhea and from asymptomatic carriers (n = 5). To avoid biases from strains selected by their Ter, only EHEC O157 strains isolated on Te-free media by methods described previously (6, 10, 11) were included in this study, and they comprise a subset of the 572 E. coli O157 strains recovered during this interval. The 67 EHEC O157:H7 strains belonged to Shiga toxin (Stx) genotypes stx1 (2 strains), stx2 (28 strains), stx1 + stx2 (6 strains), stx2c (8 strains), stx1 + stx2c (4 strains), and stx2 + stx2c (19 strains). All 86 SF EHEC O157:NM strains contained stx2 only. Te MICs (the lowest Te concentrations which inhibited growth) were determined using a microdilution broth method (15). Each strain was tested in duplicate and in two independent experiments using 4 x 104 to 5 x 104 CFU/well and serial twofold concentrations (1 to 1,024 μg/ml) of potassium Te (Sigma, Taufkirchen, Germany) in 100 μl of Luria-Bertani (LB) broth. The ability of EHEC O157 to grow on solid media containing the Te concentration routinely used for E. coli O157:H7 selective isolation was tested by inoculating 105 CFU from overnight LB broth cultures on CT-SMAC (potassium Te 2.5, μg/ml; cefixime, 0.05 μg/ml [Oxoid, Basingstoke, United Kingdom]) and LB agar plus 2.5 μg of potassium Te per ml (LB-Te agar). The presence of ter genes was determined by PCRs using primer pairs TerZ1 plus TerZ2 (terZ), TerA1 plus TerA2 (terA), TerB1 plus TerB2 (terB), TerC1 plus TerC2 (terC), TerD1 plus TerD2 (terD), TerE1 plus TerE2 (terE), and TerF1 plus TerF2 (terF) (19), with strains EDL933 and C600 as positive and negative controls, respectively. Genomic DNA was digested (BamHI and PstI; New England Biolabs, Frankfurt, Germany), separated in 0.6% agarose, and probed under stringent conditions with digoxigenin-labeled terC (19) (DIG DNA labeling and detection kit; Roche Molecular Biochemicals, Mannheim, Germany).
Ter and ter presence among EHEC O157. Of 67 EHEC O157:H7 strains, 50 (74.6%) and 16 (23.9%) had high (256 to 1,024 μg/ml) or intermediate (32 to 128 μg/ml) Te MICs, respectively (Table 1). All 66 Ter strains grew well on CT-SMAC and LB-Te agar and contained terZ, terA, terB, terC, terD, terE, and terF (Table 1). One EHEC O157:H7 strain (5288/91) had a Te MIC of <1 μg/ml, failed to grow on CT-SMAC and LB-Te agar, and lacked all ter genes (Table 1). In contrast, 83 (96.5%) of 86 SF EHEC O157:NM strains were susceptible to Te (Te MICs of 4 μg/ml) (Table 1). Of these 83 strains, 70 (84.3%) failed to grow on CT-SMAC and LB-Te agar and 13 strains (15.7%) were strongly inhibited on both media (<10 colonies grew after overnight incubation). All 83 Tes SF EHEC O157:NM strains lacked ter genes (Table 1). Two of three Ter SF EHEC O157:NM isolates (3226/98 and 3323/98) had Te MICs of 128 μg/ml, grew well on CT-SMAC and LB-Te agar, and contained all ter genes; the remaining Ter (MIC, 256 μg/ml) SF EHEC O157:NM strain (4180/97) had no ter genes (Table 1).
Southern hybridization. terC was carried on a ca. 9-kb DNA fragment in ter+ SF EHEC O157:NM strains 3226/98 and 3323/98 (Fig. 1, lanes 6 and 7), on a ca. 6.3-kb DNA fragment in strain EDL933 (lane 1), and on no DNA fragments in strains 5288/91, 4180/97, and ter-negative SF EHEC O157:NM strain 493/89 (lanes 3 to 5).
Effect of Ter on detection of EHEC strains. Our study provides for the first time a basis for the inability of SF EHEC O157:NM to grow on CT-SMAC (11), which was until now only speculated to be caused by their Tes (12, 16). In contrast to EHEC O157:H7, almost all SF EHEC O157:NM strains lack ter genes and are Tes. Low Te MICs for SF EHEC O157 strains and the comparable growth inhibition of these isolates on CT-SMAC and LB-Te agar suggest that Te, and not cefixime, is the growth-inhibiting component in CT-SMAC. The Ter and Tes correlated with the presence and absence, respectively, of ter genes in all but one of the 153 EHEC O157 strains investigated. The single Ter, ter-negative SF EHEC O157:NM strain (4180/97) is currently being investigated for other presently known mechanisms of Ter (18). Interestingly, Southern hybridization suggests that the genomic positions of terC differ in the two ter+ SF EHEC O157:NM isolates and EHEC O157:H7 strain EDL933 (Fig. 1). Studies are under way to determine if the ter genes in these SF EHEC O157:NM strains are clustered, similar to those in EDL933 (14), and to determine the genomic location of the ter cluster as well as its copy number. Also, further studies should clarify the reasons for the substantially lower frequency of Tes found among central European EHEC O157:H7 isolates (1.5%) than among North American E. coli O157:H7 (20%) (19). Taken together, our data demonstrate a significant difference between EHEC O157:H7 and SF EHEC O157:NM in the frequency of Ter and ter genes, demonstrate a diversity among SF EHEC O157:NM strains as far as the presence of ter genes is concerned, and suggest that other mechanisms of Ter exist in a minority of such strains. However, most importantly, our data clearly indicate that, because of their Tes, most SF EHEC O157:NM strains are missed by strategies currently used for the isolation of EHEC O157:H7 strains in many clinical laboratories. A similar observation has been reported for a subset of other Tes EHEC strains (5). The selectivity offered by incorporating Te into agar media, while appropriate for isolating E. coli O157:H7 (the most important EHEC serotype worldwide), hinders the assessment of the geographic distribution, medical significance, and epidemiology of SF EHEC O157:NM strains, which in Germany are the second most common cause of HUS (6). Detection opportunities that do not select against SF EHEC O157:NM and, optimally, specifically target these organisms (7) are needed to answer the question about the relative significance of both EHEC O157 pathogens in human diseases.
ACKNOWLEDGMENTS
This study was supported by grant from the Bundesministerium für Bildung und Forschung (BMBF) Project Network of Competence Pathogenomics Alliance "Functional genomic research on enterohaemorrhagic Escherichia coli" (BD no. 119523) and by NIH grant R01 AI47499.
REFERENCES
Alison, L. 2003. HUS due to a sorbitol-fermenting verotoxigenic E. coli O157 in Scotland. Eurosurveillance Wkly. 6:2-3.
Barkocy-Gallagher, G. A., E. D. Berry, M. Rivera-Betancourt, T. M. Arthur, X. Nou, and M. Koohmaraie. 2002. Development of methods for the recovery of Escherichia coli O157:H7 and Salmonella from beef carcass sponge samples and bovine fecal and hide samples. J. Food Prot. 65:1527-1534.
Bettelheim, K. A., M. Whipp, S. P. Djordjevic, and V. Ramachandran. 2002. First isolation outside Europe of sorbitol-fermenting verocytotoxigenic Escherichia coli (VTEC) belonging to O group O157. J.Med. Microbiol. 51:713-714.
Chapman, P. A., A. T. Cerdan Malo, M. Ellin, R. Ashton, and M. A. Harkin. 2001. Escherichia coli O157 in cattle and sheep at slaughter, on beef and lamb carcasses and in raw beef and lamb products in South Yorkshire, UK. Int. J. Food Microbiol. 64:139-150.
De Boer, E., and A. E. Heuvelink. 2000. Methods for the detection and isolation of Shiga toxin-producing Escherichia coli. Symp. Ser. Soc. Appl. Microbiol. 29:133S-143S.
Friedrich, A. W., M. Bielaszewska, W.-L. Zhang, M. Pulz, T. Kuczius, A. Ammon, and H. Karch. 2002. Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J. Infect. Dis. 185:74-84.
Friedrich, A. W., K. V. Nierhoff, M. Bielaszewska, A. Mellmann, and H. Karch. 2004. Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic Escherichia coli O157:H. J. Clin. Microbiol. 42:4697-4701.
Gerber, A., H. Karch, F. Allerberger, H. M. Verweyen, and L. B. Zimmerhackl. 2002. Clinical course and the role of Shiga toxin-producing Escherichia coli infection in the hemolytic-uremic syndrome in pediatric patients, 1997-2000, in Germany and Austria: a prospective study. J. Infect. Dis. 186:493-500.
Hepburn, N. F., M. MacRae, M. Johnston, J. Mooney, and I. D. Ogden. 2002. Optimizing enrichment conditions for the isolation of Escherichia coli O157 in soils by immunomagnetic separation. Lett. Appl. Microbiol. 34:365-369.
Karch, H., and T. Meyer. 1989. Single primer pair for amplifying segments of distinct Shiga-like-toxin genes by polymerase chain reaction. J. Clin. Microbiol. 27: 2751-2757.
Karch, H., C. Janetzki-Mittmann, S. Aleksic, and M. Datz. 1996. Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods and direct culture. J. Clin. Microbiol. 34:516-519.
Karch, H., and M. Bielaszewska. 2001. Sorbitol-fermenting Shiga toxin-producing Escherichia coli O157:H strains: epidemiology, phenotypic and molecular characteristics, and microbiological diagnosis. J. Clin. Microbiol. 39:2043-2049.
Onoue, Y., H. Konuma, H. Nakagawa, Y. Hara-Kudo, T. Fujita, and S. Kumagai. 1999. Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef. Int. J. Food Microbiol. 46:27-36.
Perna, N. T., G. Plunkett, V. Burland, B. Mau, J. D. Glasner, D. J. Rose, G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J. Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis, A. Lim, E. T. Dimalanta, K. D. Potamousis, J. Apodaca, T. S. Anantharaman, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409:529-533.
Sahm, D. F., and J. A. Washington. 1991. Antibacterial susceptibility tests: dilution methods, p. 1105-1116. In A. Balows, W. J. Hausler, Jr., K. L. Hermann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical miocrobiology, 5th ed. American Society for Microbiology, Washington, D.C.
Tarr, P. I., S. S. Bilge, J. C. Vary, S. Jelacic, R. L. Habeeb, T. R. Ward, M. R. Baylor, and T. E. Besser. 2000. Iha: a novel Escherichia coli O157:H7 adherence-conferring molecule encoded on a recently acquired chromosomal island of conserved structure. Infect. Immun. 68:1400-1407.
Tarr, P. I., and M. A. Neill. 2001. Escherichia coli O157:H7. Gastroenterol. Clin. North Am. 30:735-751.
Taylor, D. E. 1999. Bacterial tellurite resistance. Trends Microbiol. 7:111-115.
Taylor, D., M. Rooker, M. Keelen, L.-K. Ng, I. Martin, N. T. Perna, N. T. V. Burland, and F. R. Blattner. 2002. Genome variability of O islands encoding tellurite resistance in enterohemorrhagic Escherichia coli O157:H7 isolates. J. Bacteriol. 184:4690-4698.
Van Duynhoven, Y. T., C. M. de Jager, A. E. Heuvelink, W. K. van der Zwaluw, H. M. Maas, W. van Pelt, and W. J. Wannet. 2002. Enhanced laboratory-based surveillance of Shiga toxin-producing Escherichia coli O157 in the Netherlands. Eur. J. Clin. Microbiol. Infect. Dis. 21:513-522.
Voitoux, E., V. Lafarge, C. Colette, and B. Lombard. 2002. Applicability of the draft standard method for the detection of Escherichia coli O157 in dairy products. Int. J. Food Microbiol. 25:213-221.
Zadik, P. M., P. A. Chapman, and C. A. Siddons. 1993. Use of tellurite for the selection of verocytotoxigenic Escherichia coli O157. J. Med. Microbiol. 39:155-158.(Martina Bielaszewska, Phi)
婵犵數鍎戠徊钘壝洪悩璇茬婵犻潧娲ら閬嶆煕濞戝崬鏋ゆい鈺冨厴閺屾稑鈽夐崡鐐差潾闁哄鏅滃Λ鍐蓟濞戞ǚ鏋庨煫鍥ㄦ尨閸嬫挻绂掔€ｎ亞鍔﹀銈嗗坊閸嬫捇鏌涢悩宕囥€掓俊鍙夊姇閳规垿宕堕埞鐐亙闁诲骸绠嶉崕鍗炍涘☉銏犵劦妞ゆ帒顦悘锔筋殽閻愬樊鍎旀鐐叉喘椤㈡棃宕ㄩ鐐靛搸婵犵數鍋犻幓顏嗗緤閹灐娲箣閻樺吀绗夐梺鎸庣箓閹峰宕甸崼婢棃鏁傜粵瀣妼闂佸摜鍋為幐鎶藉蓟閺囥垹骞㈤柡鍥╁Т婵′粙鏌ｉ姀鈺佺仩缂傚秴锕獮濠囨晸閻樿尙鐤€濡炪倖鎸鹃崑鐔哥閹扮増鈷戦柛锔诲帎閻熸噴娲Χ閸ヮ煈娼熼梺鍐叉惈閹冲氦绻氶梻浣呵归張顒傜矙閹烘鍊垫い鏂垮⒔绾惧ジ鏌￠崘銊モ偓绋挎毄濠电姭鎷冮崟鍨杹閻庢鍠栭悥鐓庣暦濮椻偓婵℃瓕顦抽柛鎾村灦缁绘稓鈧稒岣块惌濠偽旈悩鍙夋喐闁轰緡鍣ｉ、鏇㈡晜閽樺鈧稑鈹戦敍鍕粶濠⒀呮櫕缁瑦绻濋崶銊у幐婵犮垼娉涢敃銈夊汲閺囩喐鍙忛柣鐔煎亰濡偓闂佽桨绀佺粔鎾偩濠靛绀冩い顓熷灣閹寸兘姊绘担绛嬪殐闁哥姵鎹囧畷婵婄疀濞戣鲸鏅ｇ紓鍌欑劍宀ｅ潡鍩㈤弮鍫熺厽闁瑰鍎戞笟娑㈡煕閺傚灝鏆ｉ柡宀嬬節瀹曟帒顫濋鐘靛幀缂傚倷鐒﹂〃鍛此囬柆宥呯劦妞ゆ帒鍠氬ḿ鎰磼椤旇偐绠婚柨婵堝仱閺佸啴宕掑鍗炴憢闂佽崵濞€缂傛艾鈻嶉敐鍥╃煋闁割煈鍠撻埀顒佸笒椤繈顢橀悩顐ｎ潔闂備線娼уú銈吤洪妸鈺佺劦妞ゆ帒鍋嗛弨鐗堢箾婢跺娲寸€规洏鍨芥俊鍫曞炊閵娿儺浼曢柣鐔哥矌婢ф鏁Δ鍜冪稏濠㈣埖鍔栭崑锝夋煕閵夘垰顩☉鎾瑰皺缁辨帗娼忛妸褏鐣奸梺褰掝棑婵炩偓闁诡喗绮撻幐濠冨緞婢跺瞼姊炬繝鐢靛仜椤曨厽鎱ㄦィ鍐ㄦ槬闁哄稁鍘奸崹鍌炴煏婵炵偓娅嗛柛瀣ㄥ妼闇夐柨婵嗘噹閺嗙喐淇婇姘卞ⅵ婵﹥妞介、鏇㈡晲閸℃瑦顓婚梻浣虹帛閹碱偆鎹㈠┑瀣祦閻庯綆鍠栫粻锝嗙節婵犲倸顏柟鏋姂濮婃椽骞愭惔锝傛闂佸搫鐗滈崜鐔风暦閻熸壋鍫柛鏇ㄥ弾濞村嫬顪冮妶鍡楃瑐闁绘帪绠撳鎶筋敂閸喓鍘遍梺鐟版惈缁夋潙鐣甸崱娑欑厓鐟滄粓宕滃顒夋僵闁靛ň鏅滈崑鍌炴煥閻斿搫孝閻熸瑱绠撻獮鏍箹椤撶偟浠紓浣插亾濠㈣泛鈯曡ぐ鎺戠闁稿繗鍋愬▓銈夋⒑缂佹ḿ绠栭柣鈺婂灠閻ｇ兘鏁撻悩鑼槰闂佽偐鈷堥崜姘额敊閹达附鈷戦悹鍥ｂ偓铏亖闂佸憡鏌ㄦ鎼佸煝閹捐绠ｉ柣鎰綑椤庢挸鈹戦悩璇у伐闁哥噥鍨堕獮鍡涘磼濮ｎ厼缍婇幃鈺呭箵閹烘繂濡锋繝鐢靛Л閸嬫捇鏌熷▓鍨灓缁鹃箖绠栭弻鐔衡偓鐢登瑰暩閻熸粎澧楅悡锟犲蓟濞戙垹绠抽柡鍌氱氨閺嬪懎鈹戦悙鍙夊櫣闂佸府绲炬穱濠囧箻椤旇姤娅㈤梺璺ㄥ櫐閹凤拷Division of Pediatric Gastroenterology and Nutrition, Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine and St. Louis Children's Hospital, St. Louis, Missouri
ABSTRACT
A total of 66 (98.5%) of 67 enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains had increased potassium tellurite (Te) MICs (32 to 1,024 μg/ml), grew on Te-containing media, and possessed Te resistance (ter) genes, whereas 83 (96.5%) of 86 sorbitol-fermenting (SF) EHEC O157:NM strains had Te MICs of 4 μg/ml, did not grow on Te-containing media, and lacked ter genes. Optimal detection of SF EHEC O157:NM strains requires Te-independent strategies.
TEXT
Tellurite (Te)-resistant (Ter) non-sorbitol-fermenting enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains cause diarrhea and hemolytic-uremic syndrome (HUS) worldwide (17), but sorbitol-fermenting (SF) EHEC O157:NM (nonmotile) strains have emerged as pathogens only in Europe (1, 6, 8, 12) and Australia (3) so far. SF EHEC O157:NM strains are not distinguishable from commensal E. coli strains on sorbitol MacConkey agar (SMAC), and do not grow (11) on cefixime-Te (CT)-SMAC (22), which is frequently used for selective isolation of EHEC O157:H7 strains from feces, foods, and the environment (2, 4, 5, 9, 13, 20, 21). Ter in EHEC O157:H7 is associated with the ter (terZABCDEF) gene cluster (19), duplicated in strain EDL933 in O islands OI 43 and OI 48 (14). One of these islands was originally identified in strain 86-24 (16) as the Ter and adherence-conferring island (16). Ter in SF EHEC O157:NM strains has not been investigated. Because Te susceptibility (Tes) could thwart the detection of such strains on media containing Te, we investigated Ter and the presence of ter genes in a large collection of SF EHEC O157:NM clinical isolates. We compared these characteristics with those of EHEC O157:H7.
Isolation and characterization of strains. A total of 67 EHEC O157:H7 and 86 SF EHEC O157:NM strains were isolated between 1987 and 2003 from patients with HUS (n = 118) or bloody (n = 11) or watery (n = 19) diarrhea and from asymptomatic carriers (n = 5). To avoid biases from strains selected by their Ter, only EHEC O157 strains isolated on Te-free media by methods described previously (6, 10, 11) were included in this study, and they comprise a subset of the 572 E. coli O157 strains recovered during this interval. The 67 EHEC O157:H7 strains belonged to Shiga toxin (Stx) genotypes stx1 (2 strains), stx2 (28 strains), stx1 + stx2 (6 strains), stx2c (8 strains), stx1 + stx2c (4 strains), and stx2 + stx2c (19 strains). All 86 SF EHEC O157:NM strains contained stx2 only. Te MICs (the lowest Te concentrations which inhibited growth) were determined using a microdilution broth method (15). Each strain was tested in duplicate and in two independent experiments using 4 x 104 to 5 x 104 CFU/well and serial twofold concentrations (1 to 1,024 μg/ml) of potassium Te (Sigma, Taufkirchen, Germany) in 100 μl of Luria-Bertani (LB) broth. The ability of EHEC O157 to grow on solid media containing the Te concentration routinely used for E. coli O157:H7 selective isolation was tested by inoculating 105 CFU from overnight LB broth cultures on CT-SMAC (potassium Te 2.5, μg/ml; cefixime, 0.05 μg/ml [Oxoid, Basingstoke, United Kingdom]) and LB agar plus 2.5 μg of potassium Te per ml (LB-Te agar). The presence of ter genes was determined by PCRs using primer pairs TerZ1 plus TerZ2 (terZ), TerA1 plus TerA2 (terA), TerB1 plus TerB2 (terB), TerC1 plus TerC2 (terC), TerD1 plus TerD2 (terD), TerE1 plus TerE2 (terE), and TerF1 plus TerF2 (terF) (19), with strains EDL933 and C600 as positive and negative controls, respectively. Genomic DNA was digested (BamHI and PstI; New England Biolabs, Frankfurt, Germany), separated in 0.6% agarose, and probed under stringent conditions with digoxigenin-labeled terC (19) (DIG DNA labeling and detection kit; Roche Molecular Biochemicals, Mannheim, Germany).
Ter and ter presence among EHEC O157. Of 67 EHEC O157:H7 strains, 50 (74.6%) and 16 (23.9%) had high (256 to 1,024 μg/ml) or intermediate (32 to 128 μg/ml) Te MICs, respectively (Table 1). All 66 Ter strains grew well on CT-SMAC and LB-Te agar and contained terZ, terA, terB, terC, terD, terE, and terF (Table 1). One EHEC O157:H7 strain (5288/91) had a Te MIC of <1 μg/ml, failed to grow on CT-SMAC and LB-Te agar, and lacked all ter genes (Table 1). In contrast, 83 (96.5%) of 86 SF EHEC O157:NM strains were susceptible to Te (Te MICs of 4 μg/ml) (Table 1). Of these 83 strains, 70 (84.3%) failed to grow on CT-SMAC and LB-Te agar and 13 strains (15.7%) were strongly inhibited on both media (<10 colonies grew after overnight incubation). All 83 Tes SF EHEC O157:NM strains lacked ter genes (Table 1). Two of three Ter SF EHEC O157:NM isolates (3226/98 and 3323/98) had Te MICs of 128 μg/ml, grew well on CT-SMAC and LB-Te agar, and contained all ter genes; the remaining Ter (MIC, 256 μg/ml) SF EHEC O157:NM strain (4180/97) had no ter genes (Table 1).
Southern hybridization. terC was carried on a ca. 9-kb DNA fragment in ter+ SF EHEC O157:NM strains 3226/98 and 3323/98 (Fig. 1, lanes 6 and 7), on a ca. 6.3-kb DNA fragment in strain EDL933 (lane 1), and on no DNA fragments in strains 5288/91, 4180/97, and ter-negative SF EHEC O157:NM strain 493/89 (lanes 3 to 5).
Effect of Ter on detection of EHEC strains. Our study provides for the first time a basis for the inability of SF EHEC O157:NM to grow on CT-SMAC (11), which was until now only speculated to be caused by their Tes (12, 16). In contrast to EHEC O157:H7, almost all SF EHEC O157:NM strains lack ter genes and are Tes. Low Te MICs for SF EHEC O157 strains and the comparable growth inhibition of these isolates on CT-SMAC and LB-Te agar suggest that Te, and not cefixime, is the growth-inhibiting component in CT-SMAC. The Ter and Tes correlated with the presence and absence, respectively, of ter genes in all but one of the 153 EHEC O157 strains investigated. The single Ter, ter-negative SF EHEC O157:NM strain (4180/97) is currently being investigated for other presently known mechanisms of Ter (18). Interestingly, Southern hybridization suggests that the genomic positions of terC differ in the two ter+ SF EHEC O157:NM isolates and EHEC O157:H7 strain EDL933 (Fig. 1). Studies are under way to determine if the ter genes in these SF EHEC O157:NM strains are clustered, similar to those in EDL933 (14), and to determine the genomic location of the ter cluster as well as its copy number. Also, further studies should clarify the reasons for the substantially lower frequency of Tes found among central European EHEC O157:H7 isolates (1.5%) than among North American E. coli O157:H7 (20%) (19). Taken together, our data demonstrate a significant difference between EHEC O157:H7 and SF EHEC O157:NM in the frequency of Ter and ter genes, demonstrate a diversity among SF EHEC O157:NM strains as far as the presence of ter genes is concerned, and suggest that other mechanisms of Ter exist in a minority of such strains. However, most importantly, our data clearly indicate that, because of their Tes, most SF EHEC O157:NM strains are missed by strategies currently used for the isolation of EHEC O157:H7 strains in many clinical laboratories. A similar observation has been reported for a subset of other Tes EHEC strains (5). The selectivity offered by incorporating Te into agar media, while appropriate for isolating E. coli O157:H7 (the most important EHEC serotype worldwide), hinders the assessment of the geographic distribution, medical significance, and epidemiology of SF EHEC O157:NM strains, which in Germany are the second most common cause of HUS (6). Detection opportunities that do not select against SF EHEC O157:NM and, optimally, specifically target these organisms (7) are needed to answer the question about the relative significance of both EHEC O157 pathogens in human diseases.
ACKNOWLEDGMENTS
This study was supported by grant from the Bundesministerium für Bildung und Forschung (BMBF) Project Network of Competence Pathogenomics Alliance "Functional genomic research on enterohaemorrhagic Escherichia coli" (BD no. 119523) and by NIH grant R01 AI47499.
REFERENCES
Alison, L. 2003. HUS due to a sorbitol-fermenting verotoxigenic E. coli O157 in Scotland. Eurosurveillance Wkly. 6:2-3.
Barkocy-Gallagher, G. A., E. D. Berry, M. Rivera-Betancourt, T. M. Arthur, X. Nou, and M. Koohmaraie. 2002. Development of methods for the recovery of Escherichia coli O157:H7 and Salmonella from beef carcass sponge samples and bovine fecal and hide samples. J. Food Prot. 65:1527-1534.
Bettelheim, K. A., M. Whipp, S. P. Djordjevic, and V. Ramachandran. 2002. First isolation outside Europe of sorbitol-fermenting verocytotoxigenic Escherichia coli (VTEC) belonging to O group O157. J.Med. Microbiol. 51:713-714.
Chapman, P. A., A. T. Cerdan Malo, M. Ellin, R. Ashton, and M. A. Harkin. 2001. Escherichia coli O157 in cattle and sheep at slaughter, on beef and lamb carcasses and in raw beef and lamb products in South Yorkshire, UK. Int. J. Food Microbiol. 64:139-150.
De Boer, E., and A. E. Heuvelink. 2000. Methods for the detection and isolation of Shiga toxin-producing Escherichia coli. Symp. Ser. Soc. Appl. Microbiol. 29:133S-143S.
Friedrich, A. W., M. Bielaszewska, W.-L. Zhang, M. Pulz, T. Kuczius, A. Ammon, and H. Karch. 2002. Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J. Infect. Dis. 185:74-84.
Friedrich, A. W., K. V. Nierhoff, M. Bielaszewska, A. Mellmann, and H. Karch. 2004. Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic Escherichia coli O157:H. J. Clin. Microbiol. 42:4697-4701.
Gerber, A., H. Karch, F. Allerberger, H. M. Verweyen, and L. B. Zimmerhackl. 2002. Clinical course and the role of Shiga toxin-producing Escherichia coli infection in the hemolytic-uremic syndrome in pediatric patients, 1997-2000, in Germany and Austria: a prospective study. J. Infect. Dis. 186:493-500.
Hepburn, N. F., M. MacRae, M. Johnston, J. Mooney, and I. D. Ogden. 2002. Optimizing enrichment conditions for the isolation of Escherichia coli O157 in soils by immunomagnetic separation. Lett. Appl. Microbiol. 34:365-369.
Karch, H., and T. Meyer. 1989. Single primer pair for amplifying segments of distinct Shiga-like-toxin genes by polymerase chain reaction. J. Clin. Microbiol. 27: 2751-2757.
Karch, H., C. Janetzki-Mittmann, S. Aleksic, and M. Datz. 1996. Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods and direct culture. J. Clin. Microbiol. 34:516-519.
Karch, H., and M. Bielaszewska. 2001. Sorbitol-fermenting Shiga toxin-producing Escherichia coli O157:H strains: epidemiology, phenotypic and molecular characteristics, and microbiological diagnosis. J. Clin. Microbiol. 39:2043-2049.
Onoue, Y., H. Konuma, H. Nakagawa, Y. Hara-Kudo, T. Fujita, and S. Kumagai. 1999. Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef. Int. J. Food Microbiol. 46:27-36.
Perna, N. T., G. Plunkett, V. Burland, B. Mau, J. D. Glasner, D. J. Rose, G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J. Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis, A. Lim, E. T. Dimalanta, K. D. Potamousis, J. Apodaca, T. S. Anantharaman, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409:529-533.
Sahm, D. F., and J. A. Washington. 1991. Antibacterial susceptibility tests: dilution methods, p. 1105-1116. In A. Balows, W. J. Hausler, Jr., K. L. Hermann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical miocrobiology, 5th ed. American Society for Microbiology, Washington, D.C.
Tarr, P. I., S. S. Bilge, J. C. Vary, S. Jelacic, R. L. Habeeb, T. R. Ward, M. R. Baylor, and T. E. Besser. 2000. Iha: a novel Escherichia coli O157:H7 adherence-conferring molecule encoded on a recently acquired chromosomal island of conserved structure. Infect. Immun. 68:1400-1407.
Tarr, P. I., and M. A. Neill. 2001. Escherichia coli O157:H7. Gastroenterol. Clin. North Am. 30:735-751.
Taylor, D. E. 1999. Bacterial tellurite resistance. Trends Microbiol. 7:111-115.
Taylor, D., M. Rooker, M. Keelen, L.-K. Ng, I. Martin, N. T. Perna, N. T. V. Burland, and F. R. Blattner. 2002. Genome variability of O islands encoding tellurite resistance in enterohemorrhagic Escherichia coli O157:H7 isolates. J. Bacteriol. 184:4690-4698.
Van Duynhoven, Y. T., C. M. de Jager, A. E. Heuvelink, W. K. van der Zwaluw, H. M. Maas, W. van Pelt, and W. J. Wannet. 2002. Enhanced laboratory-based surveillance of Shiga toxin-producing Escherichia coli O157 in the Netherlands. Eur. J. Clin. Microbiol. Infect. Dis. 21:513-522.
Voitoux, E., V. Lafarge, C. Colette, and B. Lombard. 2002. Applicability of the draft standard method for the detection of Escherichia coli O157 in dairy products. Int. J. Food Microbiol. 25:213-221.
Zadik, P. M., P. A. Chapman, and C. A. Siddons. 1993. Use of tellurite for the selection of verocytotoxigenic Escherichia coli O157. J. Med. Microbiol. 39:155-158.(Martina Bielaszewska, Phi)