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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kwara, A.
Right arrow Articles by Crawford, J. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kwara, A.
Right arrow Articles by Crawford, J. T.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, June 2003, p. 2683-2685, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2683-2685.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Evaluation of the Epidemiologic Utility of Secondary Typing Methods for Differentiation of Mycobacterium tuberculosis Isolates

Awewura Kwara,1* Ronald Schiro,2 Lauren S. Cowan,3 Newton E. Hyslop,1 Mark F. Wiser,4 Stephanie Roahen Harrison,5 Patricia Kissinger,5 Lois Diem,3 and Jack T. Crawford3

Department of Tropical Medicine,4 Department of Epidemiology, Tulane University School of Public HealthTropical Medicine,5 Section of Adult Infectious Diseases, Tulane University Health Sciences Center,1 Office of Public Health, Louisiana Department of Health and Hospitals, New Orleans, Louisiana 70112,2 Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 303333

Received 13 December 2002/ Returned for modification 13 February 2003/ Accepted 24 March 2003


arrow
ABSTRACT
 
Spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis (MIRU-VNTR) were evaluated for the ability to differentiate 64 Mycobacterium tuberculosis isolates from 10 IS6110-defined clusters. MIRU-VNTR performed slightly better than spoligotyping in reducing the number of clustered isolates and the sizes of the clusters. All epidemiologically related isolates remained clustered by MIRU-VNTR but not by spoligotyping.


arrow
TEXT
 
DNA fingerprinting of Mycobacterium tuberculosis isolates is a powerful tool for studying the molecular epidemiology of tuberculosis (TB). IS6110 fingerprinting has proven useful for detecting outbreaks (5) and for conducting population-based studies of recent transmission (1, 9). However, for low-copy-number strains (fewer than six copies of IS6110), it is a less reliable indicator of clonality and therefore a poor predictor of epidemiologic relationships (6, 12, 13). Secondary typing is often recommended for these isolates to decrease the number of falsely clustered isolates. Spoligotyping is the most widely used method for secondary typing; however, recent reports indicate that it does not appreciably improve strain differentiation and suggest that more discriminating methods should be used (3). Mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis (MIRU-VNTR) is a relatively new typing method that determines the number of repeated mycobacterial interspersed repetitive units at 12 independent loci (7). In an initial study, 12 sets of epidemiologically related isolates remained clustered by MIRU-VNTR (7). In a study of low-copy-number isolates, MIRU-VNTR analysis was shown to further resolve some IS6110 clusters, but the epidemiological significance was not investigated (2). In this study, we compared spoligotyping and MIRU-VNTR for the secondary typing of isolates that were previously clustered by IS6110 and correlated the genotyping results with the epidemiologic data.

The study included 64 isolates from patients with TB diagnosed between 1999 and 2001. These isolates represent 74% of 87 IS6110-clustered isolates identified during an investigation of TB transmission in metropolitan New Orleans, La. The institutional review boards of Tulane University and Louisiana State University approved the study, and the consent form waiver was granted in accordance with federal regulatory code (45 CFR 46.116(d) [available online at http://ohrp .osophs.dhhs.gov/humansubjects/guidance/45cfr46.htm#46-116]). IS6110 analysis was performed by the standard method (11), with some modifications. The PCR DIG probe synthesis kit and the DIG nucleic acid detection kit (Roche Diagnostics) were used to detect IS6110 fragments. Images were analyzed with Molecular Analyst v. 1.6 (Bio-Rad, Hercules, Calif.). Spoligotyping (Isogen Bioscience BV, Maarssen, The Netherlands) was performed in accordance with previously published methods (8). Results were recorded as an octal code (4). MIRU loci (10) were amplified with primers specific for sequences flanking each locus (Table 1). The reaction mixture (20 µl) contained a 1-µl DNA sample, 1x Taq PCR buffer, 1x Q solution, 3 mM magnesium chloride, deoxynucleoside triphosphates (each at 0.2 mM), 1 U of AmpliTaq DNA polymerase (Qiagen, Valencia, Calif.), and the primer pair at 0.6 µM (each primer at 0.3 µM). The amplification profile consisted of 1 min at 94°C, followed by 40 cycles of 30 s at 94°C, 10 s at 60°C, and 1 min at 72°C in a GeneAmp 9700 PCR System (Perkin-Elmer Applied Biosystems, Foster City, Calif.). The size of each amplicon was determined with a CEQ2000 capillary sequencer (Beckman Coulter, Fullerton, Calif.). The number of MIRUs was determined in accordance with the convention described in Table 1. The results were recorded as a 12-digit number as previously described (2). Patient data were collected retrospectively from hospital and TB clinic records and from contact-tracing data obtained by TB control disease intervention specialists or by inpatient coordinators. A transmission link was said to exist if two patients lived or worked in the same facility at a time when one of the individuals was likely to have been infectious or if two patients independently identified contact with a prior common case.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Sequences of oligonucleotides used in MIRU-VNTR

Sixty-four isolates from 10 IS6110 clusters were available for secondary typing; 30 were low-copy-number isolates, and 34 were high-copy-number isolates. The IS6110 patterns are shown in Fig. 1. Spoligotyping identified eight types that defined seven clusters (63 isolates) and one unique isolate. Only one IS6110-defined cluster was further spilt by spoligotyping (Table 2). Of the 13 isolates in this cluster, 1 had a spoligotype that differed from that of the others by a single spacer. MIRU-VNTR identified 14 types that defined 11 clusters (61 isolates) and three unique isolates (Table 2). Three of the four largest IS6110 clusters were subdivided into four subclusters and four unique isolates. Two of these clusters contained low-copy-number isolates, and the third contained high-copy-number isolates. The combination of all three methods identified 16 genotypes that defined 12 clusters (60 isolates) and four unique isolates (Table 2). The isolate that was unique by spoligotyping remained clustered by MIRU-VNTR.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1. Representative patterns of clustered isolates identified by IS6110 RFLP. Numbers indicate pattern designations (LTBx) and numbers of IS6110 bands. Clusters of the LTB55, LTB44, LTB32, LTB60, LTB36, LTB35, and LTB30 genotypes were excluded from this study because the isolates were not available for MIRU-VNTR typing.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Genotyping results

Epidemiologic data were available for 61 patients; 16 (25%) patients had definite transmission links with another patient. With one exception, the genotypes of the isolates from these patients were identical (Table 3). Isolates from three family members were identical by IS6110 and MIRU-VNTR, but one isolate had a slightly different spoligotype in that it was missing spacer 40. Such minor variations have been previously reported among outbreak isolates (6). Two of the patients in this study were diagnosed with TB as a result of laboratory cross-contamination. The genotypes of these three isolates were identical.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Genotypes of isolates with documented transmission links

Genotyping of M. tuberculosis isolates from a given community is useful to the TB controller by identifying unsuspected cases of recently transmitted disease and areas in which traditional contact tracing has failed. However, large clusters of unrelated strains can confound these efforts, and therefore, a combination of genotyping techniques that provides the greatest level of discrimination is necessary for molecular typing to be useful to TB control programs. In this study, spoligotyping of IS6110-defined clusters did not improve strain differentiation. The only IS6110-clustered isolates that were subdivided by spoligotyping were epidemiologically related. In contrast, MIRU-VNTR had greater resolving power than spoligotyping and defined four additional genotypes; all epidemiologically related isolates remained clustered. We found that MIRU-VNTR is useful for secondary typing of IS6110-clustered isolates with both low and high numbers of IS6110 copies. MIRU-VNTR further resolved two low-copy-number IS6110 clusters and a high-copy-number cluster containing isolates with the Beijing spoligotype. The reduction in the number of falsely clustered isolates should allow more effective focused contact investigations. The ability of MIRU-VNTR to differentiate some IS6110-spoligotype-defined clusters suggests that the widely used genotyping combination of IS6110 and spoligotyping may result in overestimation of recently transmitted disease.


arrow
ACKNOWLEDGMENTS
 
We thank the nurses and staff at the Wetmore TB Clinic, James Rigol and Richard Adams of the Louisiana Department of Health and Hospitals Office of Public Health Region 1, and Michelle Fernandez and Charlee Jones of the Medical Center of Louisiana in New Orleans for collecting the patient data. We thank Kerri Gerage and Arlene Lefebre of the Louisiana Department of Health and Hospitals Office of Public Health Central Laboratory for assistance with validation of the IS6110 RFLP procedure and with computer analysis of the patterns. We also thank Steven Styron for assistance with graphics and illustrations.

A. Kwara was supported by the Wetmore Foundation.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Infectious Diseases Division, Miriam Hospital, 164 Summit Ave., Providence, RI 02906-0609. Phone: (401) 793-2463. Fax: (401) 793-4704. E-mail: Awewura_Kwara{at}brown.edu. Back


arrow
REFERENCES
 
    1
  1. Alland, D., G. E. Kalkut, A. R. Moss, R. A. McAdam, J. A. Hahn, W. Bosworth, E. Drucker, and B. R. Bloom. 1994. Transmission of tuberculosis in New York City. An analysis by DNA fingerprinting and conventional epidemiologic methods. N. Engl. J. Med. 330:1710-1716.[Abstract/Free Full Text]
    2. 2
  2. Cowan, L. S., L. Mosher, L. Diem, J. P. Massey, and J. T. Crawford. 2002. Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units. J. Clin. Microbiol. 40:1592-1602.[Abstract/Free Full Text]
    3. 3
  3. Cronin, W. A., J. E. Golub, L. S. Magder, N. G. Baruch, M. J. Lathan, L. N. Mukasa, N. Hooper, J. H. Razeq, D. Mulcahy, W. H. Benjamin, and W. R. Bishai. 2001. Epidemiologic usefulness of spoligotyping for secondary typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110. J. Clin. Microbiol. 39:3709-3711.[Abstract/Free Full Text]
    4. 4
  4. Dale, J. W., D. Brittain, A. A. Cataldi, D. Cousins, J. T. Crawford, J. Driscoll, H. Heersma, T. Lillebaek, T. Quitugua, N. Rastogi, R. A. Skuce, C. Sola, D. van Soolingen, and V. Vincent. 2001. Spacer oligonucleotide typing of bacteria of the Mycobacterium tuberculosis complex: recommendations for standard nomenclature. Int. J. Tuber. Lung Dis. 5:216-219.
    5. 5
  5. Edlin, B. R., J. I. Tokars, M. H. Grieco, J. T. Crawford, J. Williams, E. M. Sordillo, K. R. Ong, J. O. Kilburn, S. W. Dooley, K. G. Castro, W. R. Jarvis, and S. D. Holmberg. 1992. An outbreak of multidrug-resistant tuberculosis among hospitalized patients with the acquired immunodeficiency syndrome. N. Engl. J. Med. 326:1514-1521.[Abstract]
    6. 6
  6. Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, S. Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, and J. van Embden. 1997. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35:907-914.[Abstract]
    7. 7
  7. Mazars, E., S. Lesjean, A. L. Banuls, M. Gilbert, V. Vincent, B. Gicquel, M. Tibayrenc, C. Locht, and P. Supply. 2001. High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proc. Natl. Acad. Sci. USA 98:1901-1906.[Abstract/Free Full Text]
    8. 8
  8. Molhuizen, H. O., A. E. Bunschoten, L. M. Schouls, and J. D. van Embden. 1998. Rapid detection and simultaneous strain differentiation of Mycobacterium tuberculosis complex bacteria by spoligotyping. Methods Mol. Biol. 101:381-394.[Medline]
    9. 9
  9. Small, P. M., P. C. Hopewell, S. P. Singh, A. Paz, J. Parsonnet, D. C. Ruston, G. F. Schecter, C. L. Daley, and G. K. Schoolnik. 1994. The epidemiology of tuberculosis in San Francisco: a population-based study using conventional and molecular methods. N. Engl. J. Med. 330:1703-1709.[Abstract/Free Full Text]
    10. 10
  10. Supply, P., E. Mazars, S. Lesjean, V. Vincent, B. Gicquel, and C. Locht. 2000. Variable human mini-satellite-like regions in the Mycobacterium tuberculosis genome. Mol. Microbiol. 36:762-771.[CrossRef][Medline]
    11. 11
  11. van Embden, J. D. A., M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach, B. Gicquel, P. Hermans, C. Martin, R. McAdam, T. M. Shinnick, and P. M. Small. 1993. Strains identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J. Clin. Microbiol. 31:406-409.[Abstract/Free Full Text]
    12. 12
  12. van Soolingen, D., P. E. W. De Haas, P. W. M. Hermans, P. M. A. Groenen, and J. D. A. van Embden. 1993. Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis. J. Clin. Microbiol. 31:1987-1995.[Abstract/Free Full Text]
    13. 13
  13. Warren, R., M. Richardson, S. Sampson, J. H. Hauman, N. Beyers, P. R. Donald, and P. D. van Helden. 1996. Genotyping of Mycobacterium tuberculosis with additional markers enhances accuracy in epidemiological studies. J. Clin. Microbiol. 34:2219-2224.[Abstract]

Journal of Clinical Microbiology, June 2003, p. 2683-2685, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2683-2685.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sun, Z., Chao, Y., Zhang, X., Zhang, J., Li, Y., Qiu, Y., Liu, Y., Nie, L., Guo, A., Li, C. (2008). Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Clinical Isolates in China. J. Clin. Microbiol. 46: 4075-4077 [Abstract] [Full Text]  
  • Han, H., Wang, F., Xiao, Y., Ren, Y., Chao, Y., Guo, A., Ye, L. (2007). Utility of mycobacterial interspersed repetitive unit typing for differentiating Mycobacterium tuberculosis isolates in Wuhan, China. J Med Microbiol 56: 1219-1223 [Abstract] [Full Text]  
  • Shen, X., Shen, G.-m., Wu, J., Gui, X.-h., Li, X., Mei, J., DeRiemer, K., Gao, Q. (2007). Association between embB Codon 306 Mutations and Drug Resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 51: 2618-2620 [Abstract] [Full Text]  
  • Oelemann, M. C., Diel, R., Vatin, V., Haas, W., Rusch-Gerdes, S., Locht, C., Niemann, S., Supply, P. (2007). Assessment of an Optimized Mycobacterial Interspersed Repetitive- Unit-Variable-Number Tandem-Repeat Typing System Combined with Spoligotyping for Population-Based Molecular Epidemiology Studies of Tuberculosis. J. Clin. Microbiol. 45: 691-697 [Abstract] [Full Text]  
  • Godreuil, S., Torrea, G., Terru, D., Chevenet, F., Diagbouga, S., Supply, P., Van de Perre, P., Carriere, C., Banuls, A. L. (2007). First Molecular Epidemiology Study of Mycobacterium tuberculosis in Burkina Faso. J. Clin. Microbiol. 45: 921-927 [Abstract] [Full Text]  
  • Supply, P., Allix, C., Lesjean, S., Cardoso-Oelemann, M., Rusch-Gerdes, S., Willery, E., Savine, E., de Haas, P., van Deutekom, H., Roring, S., Bifani, P., Kurepina, N., Kreiswirth, B., Sola, C., Rastogi, N., Vatin, V., Gutierrez, M. C., Fauville, M., Niemann, S., Skuce, R., Kremer, K., Locht, C., van Soolingen, D. (2006). Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis. J. Clin. Microbiol. 44: 4498-4510 [Abstract] [Full Text]  
  • Gopaul, K. K., Brown, T. J., Gibson, A. L., Yates, M. D., Drobniewski, F. A. (2006). Progression Toward an Improved DNA Amplification-Based Typing Technique in the Study of Mycobacterium tuberculosis Epidemiology.. J. Clin. Microbiol. 44: 2492-2498 [Abstract] [Full Text]  
  • Allix, C., Walravens, K., Saegerman, C., Godfroid, J., Supply, P., Fauville-Dufaux, M. (2006). Evaluation of the Epidemiological Relevance of Variable-Number Tandem-Repeat Genotyping of Mycobacterium bovis and Comparison of the Method with IS6110 Restriction Fragment Length Polymorphism Analysis and Spoligotyping.. J. Clin. Microbiol. 44: 1951-1962 [Abstract] [Full Text]  
  • Burman, W., Benator, D., Vernon, A., Khan, A., Jones, B., Silva, C., Lahart, C., Weis, S., King, B., Mangura, B., Weiner, M., El-Sadr, W., the Tuberculosis Trials Consortium, (2006). Acquired Rifamycin Resistance with Twice-Weekly Treatment of HIV-related Tuberculosis. Am. J. Respir. Crit. Care Med. 173: 350-356 [Abstract] [Full Text]  
  • Gibson, A., Brown, T., Baker, L., Drobniewski, F. (2005). Can 15-Locus Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Analysis Provide Insight into the Evolution of Mycobacterium tuberculosis?. Appl. Environ. Microbiol. 71: 8207-8213 [Abstract] [Full Text]  
  • Kremer, K., Arnold, C., Cataldi, A., Gutierrez, M. C., Haas, W. H., Panaiotov, S., Skuce, R. A., Supply, P., van der Zanden, A. G. M., van Soolingen, D. (2005). Discriminatory Power and Reproducibility of Novel DNA Typing Methods for Mycobacterium tuberculosis Complex Strains. J. Clin. Microbiol. 43: 5628-5638 [Abstract] [Full Text]  
  • Prodinger, W. M., Brandstatter, A., Naumann, L., Pacciarini, M., Kubica, T., Boschiroli, M. L., Aranaz, A., Nagy, G., Cvetnic, Z., Ocepek, M., Skrypnyk, A., Erler, W., Niemann, S., Pavlik, I., Moser, I. (2005). Characterization of Mycobacterium caprae Isolates from Europe by Mycobacterial Interspersed Repetitive Unit Genotyping. J. Clin. Microbiol. 43: 4984-4992 [Abstract] [Full Text]  
  • van Deutekom, H., Supply, P., de Haas, P. E. W., Willery, E., Hoijng, S. P., Locht, C., Coutinho, R. A., van Soolingen, D. (2005). Molecular Typing of Mycobacterium tuberculosis by Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Analysis, a More Accurate Method for Identifying Epidemiological Links between Patients with Tuberculosis. J. Clin. Microbiol. 43: 4473-4479 [Abstract] [Full Text]  
  • Cowan, L. S., Diem, L., Monson, T., Wand, P., Temporado, D., Oemig, T. V., Crawford, J. T. (2005). Evaluation of a Two-Step Approach for Large-Scale, Prospective Genotyping of Mycobacterium tuberculosis Isolates in the United States. J. Clin. Microbiol. 43: 688-695 [Abstract] [Full Text]  
  • Scott, A. N., Menzies, D., Tannenbaum, T.-N., Thibert, L., Kozak, R., Joseph, L., Schwartzman, K., Behr, M. A. (2005). Sensitivities and Specificities of Spoligotyping and Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing Methods for Studying Molecular Epidemiology of Tuberculosis. J. Clin. Microbiol. 43: 89-94 [Abstract] [Full Text]  
  • Kremer, K., Au, B. K. Y., Yip, P. C. W., Skuce, R., Supply, P., Kam, K. M., van Soolingen, D. (2005). Use of Variable-Number Tandem-Repeat Typing To Differentiate Mycobacterium tuberculosis Beijing Family Isolates from Hong Kong and Comparison with IS6110 Restriction Fragment Length Polymorphism Typing and Spoligotyping. J. Clin. Microbiol. 43: 314-320 [Abstract] [Full Text]  
  • Warren, R. M., Victor, T. C., Streicher, E. M., Richardson, M., van der Spuy, G. D., Johnson, R., Chihota, V. N., Locht, C., Supply, P., van Helden, P. D. (2004). Clonal Expansion of a Globally Disseminated Lineage of Mycobacterium tuberculosis with Low IS6110 Copy Numbers. J. Clin. Microbiol. 42: 5774-5782 [Abstract] [Full Text]  
  • Sun, Y.-J., Lee, A. S. G., Ng, S. T., Ravindran, S., Kremer, K., Bellamy, R., Wong, S.-Y., van Soolingen, D., Supply, P., Paton, N. I. (2004). Characterization of Ancestral Mycobacterium tuberculosis by Multiple Genetic Markers and Proposal of Genotyping Strategy. J. Clin. Microbiol. 42: 5058-5064 [Abstract] [Full Text]  
  • Mokrousov, I., Narvskaya, O., Limeschenko, E., Vyazovaya, A., Otten, T., Vyshnevskiy, B. (2004). Analysis of the Allelic Diversity of the Mycobacterial Interspersed Repetitive Units in Mycobacterium tuberculosis Strains of the Beijing Family: Practical Implications and Evolutionary Considerations. J. Clin. Microbiol. 42: 2438-2444 [Abstract] [Full Text]  
  • Cangelosi, G. A., Freeman, R. J., Lewis, K. N., Livingston-Rosanoff, D., Shah, K. S., Milan, S. J., Goldberg, S. V. (2004). Evaluation of a High-Throughput Repetitive-Sequence-Based PCR System for DNA Fingerprinting of Mycobacterium tuberculosis and Mycobacterium avium Complex Strains. J. Clin. Microbiol. 42: 2685-2693 [Abstract] [Full Text]  
  • Sun, Y.-J., Bellamy, R., Lee, A. S. G., Ng, S. T., Ravindran, S., Wong, S.-Y., Locht, C., Supply, P., Paton, N. I. (2004). Use of Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing To Examine Genetic Diversity of Mycobacterium tuberculosis in Singapore. J. Clin. Microbiol. 42: 1986-1993 [Abstract] [Full Text]  
  • Vernon, A. A, McNabb, M S. (2003). Commentary: Can capture-recapture analysis of epidemiological and molecular data help us understand recent tuberculosis transmission?. Int J Epidemiol 32: 770-771 [Full Text]  

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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kwara, A.
Right arrow Articles by Crawford, J. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kwara, A.
Right arrow Articles by Crawford, J. T.

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