This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ito, K.
Right arrow Articles by Ratchtrachenchai, O.-A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ito, K.
Right arrow Articles by Ratchtrachenchai, O.-A.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, March 2007, p. 1038-1041, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01103-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Intimin Types Determined by Heteroduplex Mobility Assay of Intimin Gene (eae)-Positive Escherichia coli Strains{triangledown}

Kenitiro Ito,1* Mariko Iida,1 Mitsugu Yamazaki,2 Kazuo Moriya,3 Sanae Moroishi,3 Jun Yatsuyanagi,4 Takayuki Kurazono,5 Noriaki Hiruta,6 and Orn-Anong Ratchtrachenchai7

Infectious Diseases Surveillance Center, National Institute of Infectious Diseases, Tokyo,1 Aichi Prefectural Institute of Public Health, Nagoya,2 Saga Prefectural Center of Public Health and Sanitary Pharmaceutical Research, Saga,3 Akita Prefectural Institute of Public Health, Akita,4 Saitama Institute of Public Health, Saitama,5 Yokosuka Institute of Public Health, Yokosuka, Japan,6 Department of Medical Sciences, National Institute of Health, Nonthaburi, Thailand7

Received 30 May 2006/ Returned for modification 3 July 2006/ Accepted 3 January 2007


arrow
ABSTRACT
 
We developed a quick genetic approach to screen variants of the intimin gene (eae) by using a heteroduplex mobility assay (HMA) that targets the 5' conserved region of eae. The eae variants were categorized into 4 major HMA types and 10 minor subtypes.


arrow
TEXT
 
Enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) produce characteristic attaching and effacing lesions using intimin encoded by eae (24). The eae genes of several strains have been cloned and sequenced and have a highly conserved 5'-terminal region but are variable in the 3'-terminal region (13). Allele-specific PCRs targeting eae genes in the variable 3' region have been employed to determine eae types (1, 5, 10, 27, 30, 31, 33) and subtypes in combination with restriction fragment length polymorphism (RFLP) (4, 27). Ramachandran et al. (29) designed universal PCR primers to amplify the Int280-encoding region and identified types by RFLP. Recently, methods using real-time PCR (25) and oligonucleotide microarray (14) have been developed. Eighteen types and nine subtypes of intimin, namely, {alpha}, {alpha}2, ß1 to -3, {gamma}1, {gamma}2, {delta}, {varepsilon}, {varepsilon}2 to -4, {zeta}, {eta}, {eta}2, {theta}, {iota}, {iota}2, {kappa}, {lambda}, µ, {nu}, {xi}, o, {pi}, {rho}, and {sigma}, have been deposited in the Entrez nucleotide database.

As an alternative method to DNA sequencing, heteroduplex mobility analysis (HMA) has been developed to determine variations among amplified fragments (12). HMA is a simple, rapid, and inexpensive method (11, 18). In this study we demonstrated the variability of the 5' region of eae genes by HMA.

A total of 211 eae-positive strains of E. coli isolated in Thailand and Japan were used in this study. In addition, eae types E2348/69 (O127:H6, eae-{alpha}) (20), H19 (O26:H11, eae-ß) (2), PMK5 (O103:H2, eae-{varepsilon}) (21), and EDL933 (O157:H7, eae-{gamma}) (26) were used as a control.

To categorize the strains into EPEC or STEC strains, we checked for stx genes by PCR (17) or RPLA (Denka-Seiken, Tokyo, Japan). For HMA typing, an E. coli culture on an agar plate was suspended in 100 µl of distilled water, boiled for 10 min, and used as a PCR template. The 5'-terminal region of the eae genes was amplified by PCR as described by Nakazawa and Itoh (23).

Amplicons were then subjected to HMA (12). Briefly, an appropriate amount of amplicon was mixed with 2 µl of the amplicon obtained from strains taken as a reference, and distilled water containing 10 mM EDTA (pH 8.0) was added to 10 µl. The amplicons were denatured at 94°C for 5 min and reannealed at 72°C for 3 min and at 50°C for 1 h. Heteroduplexes were separated by homemade polyacrylamide gel electrophoresis on a 7.5% gel with a stacking gel without sodium dodecyl sulfate. To test the discriminatory power of commercially available precast gels, PAG mini-"Daiichi" (Daiichi Pure Chemicals. Tokyo, Japan) and e-PAGEL (ATTO Corporation, Tokyo, Japan) were also used. The resolution of commercially available precast gels was as good as the resolution of the homemade gel.

The combination of the amplicons and strain KI1218 or KI1223 gave the most distinctive HMA patterns; therefore, they were used as reference patterns.

By HMA typing, eae genes were categorized into four major groups, groups a to d, which can be subtyped into 10 minor types (Fig. 1). O and H serotypes were determined with antiserum kits (Denka-Seiken) as described by the manufacturer. Of the 211 eae-positive E. coli strains, 124 were distributed among 29 typeable O:H serotypes (Table 1). The same serotypes generated identical HMA types; this was not observed in the case of O26:H11, whose HMA type is associated with stx genes. All stx-positive strains are b1, whereas stx-negative strains are b1 or b2 (Table 1).


Figure 1
View larger version (68K):
[in this window]
[in a new window]

  		FIG. 1. HMA profiles of all HMA types with HMA reference strains. Each of the 10 HMA types, a1 to d2, and 4 eae reference strains, {alpha}, ß, {gamma}, and {varepsilon}, formed heteroduplexes with (A) KI1218 strain (O153:H21, type d1) or (B) KI1223 strain (O157:H45, type a1). Heteroduplexes were separated on 7.5% homemade polyacrylamide gels and stained with ethidium bromide. Electrophoretograms are shown as negative images. Lanes: M, molecular size markers (100-bp ladder); c1, KI1231 (O55:H7); {gamma}, EDL933 (O157:H7); c2, KI1699 (O63:H6); c3, KI1492 (O124:H40); a1, KI1223 (O157:H45); {alpha}, E2348/69 (O127a:H6); a2, KI1458 (O55:H51); a3, KI1434 (OUT [O but untypeable]:HNM [H but nonmotile]); d1, KI1218 (O153:H21); d2, KI1440 (OUT:H7); b1, KI1220 (O128:H2); ß, H19 (O26:H11, stx+); b2, KI1221 (O26:H11, stx mutant); {varepsilon}, PMK5 (O103:H2).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Comparison of serotypes, HMA types, and stx genes among the eae-positive Escherichia coli strains from Thailand and Japana

The reference strains for eae types, eae-{alpha}, -ß, -{gamma}, and -{varepsilon}, were typed into HMA types, a1, b1, c1, and b2, respectively (Fig. 1). Regarding the common serotype strains in published studies (1, 3-5, 7, 19, 27, 29, 30, 32) and this study, we compare HMA types to the allele-specific eae types reported in the references. With a few exceptions, the serotype strains of a1, O55:H6, O127a:H6, O142:H6, O142:H34, and O157:H45 were eae-{alpha}/{alpha}1, except O119:H6 which was typed to eae-ß/ß2. The serotype strains of b1, O15:H2, O26:H11, O119:H2, O128:H2, O153:H7, and O167:H9 were eae-ß/ß1. The serotype strains of c1, O55:H7, and O157:H7 were eae-{gamma}/{gamma}1, and the serotype strains of b2, O103:H2, was eae-{varepsilon}/{varepsilon}1.

The PCR products of 15 strains including each HMA type appearing in Fig. 2 were sequenced (Greiner, Tokyo, Japan). PHYLIP-style tree files were produced using CLUSTALW, DDBJ version available at http://www.ddbj.nig.ac.jp/search/clustalw-j.html, and phylogenies were displayed using TREEVIEW (28). The HMA-a1 sequences of KI1318 and O119:H6 (DDBJ accession no. AB185253) from positions 95 to 528 exactly matched the eae-ß2 sequence of O119:H6 (EMBL accession no. AJ715407). According to phylogenetic analysis among eae-{alpha} (GenBank accession no. M58154), eae-ß (GenBank accession no. AF453441), and eae-ß2 types, eae-ß2 was homologous to eae-{alpha} on the basis of its 5'-terminal region (at positions 1 to 1600) but was homologous to eae-ß on the basis of its 3'-terminal region (position 1600 to the end). The eae sequence of O119:H6 (EMBL accession no. AJ715407) has a mosaic structure as suggested by McGraw et al. (22).


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 2. Phylogram based on 5'-terminal nucleotide sequences of different intimin subtypes (nucleotides 95 to 528) and HMA types. The HMA type, name, and serotype of the strains used in this study are shown in boldface type. The eae type and serotype of the strains deposited in the Entrez nucleotide database are shown in plain text. The horizontal axis shows evolutionary change, and the vertical axis is arbitrary. The scale bar indicates the number of nucleotide replacements per site. DNA sequences with different eae genes were retrieved from the Entrez database, including eae-{alpha} (accession no. M58154), eae-{alpha}2 (AF530555), eae-ß (AF453441), eae-ß2 (AJ715407), eae-ß3 (AJ876651), eae-{gamma} (Z11541), eae-{gamma}2 (AF025311), eae-{delta} (AJ875027), eae-{varepsilon} (AF116899), eae-{varepsilon}2 (AF530554), eae-{varepsilon}3 (AJ876650), eae-{varepsilon}4 (AJ876651), eae-{zeta} (AF449417), eae-{eta} (AJ308550), eae-{eta}2 (AJ876652), eae-{theta} (AF449419), eae-{iota} (AJ308551), eae-{iota}2 (AF530553), eae-{kappa} (U66102), eae-{lambda} (AF530557), eae-µ (AJ705049), eae-{nu} (AJ705050), eae-{xi} (AJ705051), eae-o (AJ584841, partial), eae-{pi} (AJ705052), eae-{rho} (AJ748082), and eae-{sigma} (AJ781125).

Figure 2 shows a phylogram based on the 5'-terminal nucleotide sequences (95-528) of the eae gene in the databases and this study. The sequences could be clustered into five groups. The sequences of b1 differ by 1 to 4 nucleotides from one another. The DNA sequences of eae-ß3, eae-{varepsilon}4, and eae types in cluster x differed from HMA types by more than 5 nucleotides; the HMA types of these eae types will be analyzed in future studies. The phylogram showed (Fig. 2) good correlation with electrophoretic mobility (Fig. 1B).

The HMA method applied to the amplicon from the variable 3' region may have significance in terms of typing, but it is difficult to select universal primers. In a preliminary experiment with universal PCR primers by Ramachandran et al. (29), several HMA types were poorly amplified by HMA.

The 5'-terminal regions of eae genes are highly conserved (13); however, the eae genes contain variations that are sufficient to yield high resolution by the HMA typing method. This facilitates the subtyping of the intimin family in E. coli. To compare the discriminatory ability of typing systems, Simpson's index of diversity described by Hunter and Gaston (15) was applied. Simpson's indices by Blanco et al. (5-9) range from 0.62 (9) to 0.87 (6), with an overall value of 0.79. Simpson's indices by other authors (1, 4, 16, 27, 33) range from 0.66 to 0.82, and the Simpson's index in this study was 0.81, indicating that HMA has a high discriminatory ability which is almost equal to that of the allele-specific PCR system developed by Blanco.

Nucleotide sequence accession numbers. The sequences reported here appear in DDBJ under accession numbers AB118854 to AB118869 and AB185253.


arrow
ACKNOWLEDGMENTS
 
This work was supported by a grant for Food and Chemical Safety from the Ministry of Health, Labor, and Welfare of Japan.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Infectious Diseases Surveillance Center, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama-shi, Tokyo 208-0011, Japan. Phone: 81-42561-0771. Fax: 81-42590-8586. E-mail: kito{at}nih.go.jp. Back

{triangledown} Published ahead of print on 17 January 2007. Back


arrow
REFERENCES
 
    1
  1. Adu-Bobie, J., G. Frankel, C. Bain, A. G. Goncalves, L. Trabulsi, G. Douce, S. Knutton, and G. Dougan. 1998. Detection of intimins {alpha}, ß, {gamma}, and {delta}, four intimin derivatives expressed by attaching and effacing microbial pathogens. J. Clin. Microbiol. 36:662-668.[Abstract/Free Full Text]
    2. 2
  2. Agin, T. S., and M. K. Wolf. 1997. Identification of a family of intimins common to Escherichia coli causing attaching-effacing lesions in rabbits, humans, and swine. Infect. Immun. 65:320-326.[Abstract]
    3. 3
  3. Batchelor, M., S. Knutton, A. Caprioli, V. Huter, M. Zanial, G. Dougan, and G. Frankel. 1999. Development of a universal intimin antiserum and PCR primers. J. Clin. Microbiol. 37:3822-3827.[Abstract/Free Full Text]
    4. 4
  4. Beutin, L., G. Krause, O. Marchés, S. Zimmermann, S. Kaulfuss, and K. Gleier. 2004. Characterization of Shiga toxin-producing Escherichia coli strains isolated from human patients in Germany over a 3-year period. J. Clin. Microbiol. 42:1099-1108.[Abstract/Free Full Text]
    5. 5
  5. Blanco, J. E., M. Blanco, M. P. Alonso, A. Mora, G. Dahbi, M. A. Coira, and J. Blanco. 2004. Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from human patients: prevalence in Lugo, Spain, from 1992 through 1999. J. Clin. Microbiol. 42:311-319.[Abstract/Free Full Text]
    6. 6
  6. Blanco, M., J. E. Blanco, J. Blanco, V. M. de Carvalho, D. L. Onuma, and A. F. Pestana de Castro. 2004. Typing of intimin (eae) genes in attaching and effacing Escherichia coli strains from monkeys. J. Clin. Microbiol. 42:1382-1383.[Free Full Text]
    7. 7
  7. Blanco, M., J. E. Blanco, G. Dahbi, M. P. Alonso, A. Mora, M. A. Coira, C. Madrid, A. Juarez, M. I. Bernardez, E. A. Gonzalez, and J. Blanco. 2006. Identification of two new intimin types in atypical enteropathogenic Escherichia coli. Int. Microbiol. 9:103-110.[Medline]
    8. 8
  8. Blanco, M., J. E. Blanco, G. Dahbi, A. Mora, M. P. Alonso, G. Varela, M. P. Gadea, F. Schelotto, E. A. Gonzalez, and J. Blanco. 2006. Typing of intimin (eae) genes from enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea in Montevideo, Uruguay: identification of two novel intimin variants (µB and {xi}R/ß2B). J. Med. Microbiol. 55:1165-1174.[Abstract/Free Full Text]
    9. 9
  9. Blanco, M., J. E. Blanco, A. Mora, G. Dahbi, M. P. Alonso, E. A. Gonzalez, M. I. Bernardez, and J. Blanco. 2004. Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain and identification of a new intimin variant gene (eae-{xi}). J. Clin. Microbiol. 42:645-651.[Abstract/Free Full Text]
    10. 10
  10. China, B., E. Jacques, A.-C. Devrin, V. Pirson, and J. Mainil. 1999. Heterogeneity of the eae genes in attaching/effacing Escherichia coli from cattle: comparison with human strains. Res. Microbiol. 150:323-332.[Medline]
    11. 11
  11. Delwart, E. L., H. W. Sheppard, B. D. Walker, J. Goudsmit, and J. Mullins. 1994. Human immunodeficiency virus type 1 evolution in vivo tracked by DNA heteroduplex mobility assays. J. Virol. 68:6672-6683.[Abstract/Free Full Text]
    12. 12
  12. Delwart, E. L., G. E. Shpaer, and J. I. Mullins. 1995. Heteroduplex mobility assays for phylogenetic analysis, p. 154-160. In M. A. Innis, D. H. Gelfand, and J. J. Sninsky (ed.), PCR strategies. Academic Press, San Diego, CA.
    13. 13
  13. Frankel, D., D. C. Candy, P. Everest, and G. Dougan. 1994. Characterization of the C-terminal domains of intimin-like proteins of enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, and Hafnia alvei. Infect. Immun. 62:1835-1842.[Abstract/Free Full Text]
    14. 14
  14. Garrido, P., M. Blanco, M. Moreno-Paz, C. Briones, G. Dahbi, J. Blanco, J. Blanco, and V. Parro. 2006. STEC-EPEC oligonucleotide microarray: a new tool for typing genetic variants of the LEE pathogenicity island of human and animal Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains. Clin. Chem. 52:192-201.[Abstract/Free Full Text]
    15. 15
  15. Hunter, P. R., and M. A. Gaston. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 26:2465-2466.[Abstract/Free Full Text]
    16. 16
  16. Jenkins, C., H. R. Smith, A. J. Lawson, G. A. Willshaw, T. Cheasty, J. G. Wheeler, and D. S. Tompkins. 2006. Serotypes, intimin subtypes, and antimicrobial resistance patterns of atypical enteropathogenic Escherichia coli isolated in England from 1993 to 1996. Eur. J. Clin. Microbiol. Infect. Dis. 25:19-24.[CrossRef][Medline]
    17. 17
  17. 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.[Abstract/Free Full Text]
    18. 18
  18. Kumeda, Y., and T. Asao. 2001. Heteroduplex panel analysis, a novel method for genetic identification of Aspergillus section Flavi strains. Appl. Environ. Microbiol. 37:4084-4090.
    19. 19
  19. Lacher, D. W., H. Steinsland, and T. S. Whittam. 2006. Allelic subtyping of the intimin locus (eae) of pathogenic Escherichia coli by fluorescent RFLP. FEMS Microbiol. Lett. 261:80-87.[CrossRef][Medline]
    20. 20
  20. Levine, M. M., J. P. Nataro, H. Karch, M. M. Baldini, J. B. Kaper, R. E. Black, M. L. Clements, and A. D. O'Brien. 1985. The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. J. Infect. Dis. 152:550-559.[Medline]
    21. 21
  21. Mariani-Kurkdjian, P., E. Denamur, A. Milon, B. Picard, H. Cave, N. Lambert-Zechovsky, C. Loirat, P. Goullet, P. J. Sansonetti, and J. Elion. 1993. Identification of a clone of Escherichia coli O103:H2 as a potential agent of hemolytic-uremic syndrome in France. J. Clin. Microbiol. 31:296-301.[Abstract/Free Full Text]
    22. 22
  22. McGraw, E. A., J. Li, R. K. Selander, and T. S. Whittam. 1999. Molecular evolution and mosaic structure of {alpha}, ß, and {gamma} intimins of pathogenic Escherichia coli. Mol. Biol. Evol. 16:12-22.[Medline]
    23. 23
  23. Nakazawa, M., and K. Itoh. 1995. Experimental infection of infant rabbits with verotoxin-producing Escherichia coli of bovine origin. J. Jpn. Assoc. Infect. Dis. 69:772-776.
    24. 24
  24. Nataro, J., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-210.[Abstract/Free Full Text]
    25. 25
  25. Nielsen, E. M., and M. T. Andersen. 2003. Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5' nuclease PCR assay. J. Clin. Microbiol. 41:2884-2893.[Abstract/Free Full Text]
    26. 26
  26. O'Brien, A. D., J. W. Newland, S. F. Miller, R. K. Holmes, H. W. Smith, and S. B. Formal. 1984. Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea. Science 226:694-696.[Abstract/Free Full Text]
    27. 27
  27. Oswald, E., H. Schmidt, S. Morabito, H. Karch, O. Marchès, and A. Caprioli. 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]
    28. 28
  28. Page, R. D. M. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 2:357-358.
    29. 29
  29. Ramachandran, V., K. Brett, M. A. Hornitzky, M. Dowton, K. A. Bettelheim, M. J. Walker, and S. P. Djordjevic. 2003. Distribution of intimin subtypes among Escherichia coli isolates from ruminant and human sources. J. Clin. Microbiol. 41:5022-5032.[Abstract/Free Full Text]
    30. 30
  30. Reid, S. D., D. J. Betting, and T. S. Whittam. 1999. Molecular detection and identification of intimin alleles in pathogenic Escherichia coli by multiplex PCR. J. Clin. Microbiol. 37:2719-2722.[Abstract/Free Full Text]
    31. 31
  31. Schmidt, H., H. Russmann, and H. Karch. 1993. Virulence determinants in nontoxinogenic Escherichia coli O157 strains that cause infantile diarrhea. Infect. Immun. 61:4894-4898.[Abstract/Free Full Text]
    32. 32
  32. Vieira, M. A. M., J. R. C. Andrade, L. R. Trabulsi, A. C. P. Rosa, A. M. G. Dias, S. R. T. S. Ramos, G. Frankel, and T. T. Gomes. 2001. Phenotypic and genotypic characteristics of Escherichia coli strains of non-enteropathogenic E. coli (EPEC) serogroups that carry eae and lack the EPEC adherence factor and Shiga toxin probe sequences. J. Infect. Dis. 183:762-772.[CrossRef][Medline]
    33. 33
  33. Zhang, W. L., B. Köhler, E. Oswald, L. Beutin, H. Karch, S. Morabito, A. Caprioli, S. Suerbaum, and H. Schmidt. 2002. Genetic diversity of intimin genes of attaching and effacing Escherichia coli strains. J. Clin. Microbiol. 40:4486-4492.[Abstract/Free Full Text]

Journal of Clinical Microbiology, March 2007, p. 1038-1041, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01103-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ito, K.
Right arrow Articles by Ratchtrachenchai, O.-A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ito, K.
Right arrow Articles by Ratchtrachenchai, O.-A.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»