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 Hold, G. L.
Right arrow Articles by Flint, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hold, G. L.
Right arrow Articles by Flint, H. J.
Agricola
Right arrow Articles by Hold, G. L.
Right arrow Articles by Flint, H. J.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, July 2003, p. 4320-4324, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.4320-4324.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Oligonucleotide Probes That Detect Quantitatively Significant Groups of Butyrate-Producing Bacteria in Human Feces

Georgina L. Hold,1* Andreas Schwiertz,2,{dagger} Rustam I. Aminov,1 Michael Blaut,2 and Harry J. Flint1

Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, United Kingdom,1 Deutsches Institut für Ernaehrungsforschung, Abteilung Gastrointestinale Mikrobiologie, 14558 Bergholz-Rehbrücke, Germany2

Received 30 December 2002/ Accepted 25 February 2003


arrow
ABSTRACT
 
16S rRNA-targeted oligonucleotide probes were designed for butyrate-producing bacteria from human feces. Three new cluster-specific probes detected bacteria related to Roseburia intestinalis, Faecalibacterium prausnitzii, and Eubacterium hallii at mean populations of 2.3, 3.8, and 0.6%, respectively, in samples from 10 individuals. Additional species-level probes accounted for no more than 1%, with a mean of 7.7%, of the total human fecal microbiota identified as butyrate producers in this study. Bacteria related to E. hallii and the genera Roseburia and Faecalibacterium are therefore among the most abundant known butyrate-producing bacteria in human feces.


arrow
INTRODUCTION
 
The microbiota of the gastrointestinal tract of humans has been studied extensively because of the role played by gut bacteria both in disease and in the maintenance of gut health (7, 17, 27). One important activity of the large intestinal microbiota is to break down substrates, such as resistant starch and plant cell wall polysaccharides. The main fermentation products are the short-chain fatty acids acetate, propionate, and butyrate. Of these, butyrate is known to play an important role in the metabolic welfare of colonocytes (19, 20) and is also implicated in providing protection against cancer and ulcerative colitis (3-5). Despite this prominent role, the taxonomy, population structure, and dynamics of predominant butyrate-producing bacteria in the human intestinal tract are poorly understood.

There is no simple way to selectively isolate butyrate-producing bacteria, and the majority of those recovered from nonselective isolation have proved to be highly oxygen sensitive (2). The purpose of the present study was, therefore, to design 16S rRNA-targeted oligonucleotide probes for butyrate-producing bacteria, including recent isolates from the human gut, many of which represent new species (8, 9, 21). The majority of these butyrate-producing isolates belong to the clusters XIVa and IV of clostridia (6) (Fig. 1 and 2), which account for a significant proportion of total bacterial diversity in the human large intestine on the basis of 16S rRNA sequence analyses (12, 25).



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1. Phylogenetic tree showing the coverage of the newly designed probes within Clostridium cluster XIVa. The tree was constructed by using neighbor-joining analysis of a distance matrix obtained from a multiple-sequence alignment. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branch points; values of 97% or more were considered significant.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. Phylogenetic tree showing the coverage of the newly designed F. prausnitzii probe within Clostridium cluster IV. The tree was constructed by using neighbor-joining analysis of a distance matrix obtained from a multiple-sequence alignment. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branch points; values of 97% or more were considered significant.

Four broad-specificity probes were designed to target the small subunit rRNA of bacteria related to Eubacterium hallii, the recently reclassified Faecalibacterium prausnitzii (formerly Fusobacterium prausnitzii) (9), Coprococcus eutactus, and Roseburia intestinalis clusters (Table 1 and Fig. 1 and 2). The latter probe is predicted to recognize R. intestinalis, Eubacterium rectale, and Eubacterium ramulus as well as the butyrate-producing Eubacterium isolates A2-194 and L1-83 and the Roseburia isolate A2-183 from the study of Barcenilla et al. (2). In addition, six more specific probes were designed to recognize R. intestinalis (8), Anaerostipes caccae (21), the Eubacterium isolates L1-83 and A2-194, Coprococcus isolate L2-50, and E. rectale isolate A1-86. The new probes were designed with the ARB (16) software package, checked against the Ribosomal Database Project (RDP) and EMBL databases, and named according to the nomenclature suggested by the Oligonucleotide Probe Database (OPD) (1). The probe sequences have also been deposited in the ProbeBase data bank (15). The specificity of the newly designed probes was tested by whole-cell in situ hybridization against a panel of 120 reference strains derived from the human and animal gastrointestinal tract as described by Schwiertz et al. (22) and also against the new target butyrate-producing strains. Hybridization temperatures (TH) are given in Table 1. All newly designed probes hybridized only to the respective target organisms but not to any of the other organisms tested. The exception was the L1-83 probe, which showed weak cross-reactivity with Eubacterium sp. strain A2-194.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Probes used during the study

Fresh fecal samples from 10 healthy volunteers of both sexes, aged 28 to 56, who had consumed a Western diet and had not received any antibiotic treatment at least 3 months prior to the study, were collected and fixed as described elsewhere (24). Hybridization and enumeration were performed as described previously (22), with the lower limit of detection of 107 cells g-1 of dry feces. In addition to analysis with the newly designed probes described above, the fecal samples were analyzed with 11 Eubacterium species-specific probes described previously (22-24). Broad-specificity probes or probe mixes that targeted all eubacteria were applied (14), along with the Ruminococcus-Eubacterium-Clostridium cluster probe (Erec482) (10), the Clostridium lituseburense group probe (Clit135) (13), the Clostridium histolyticum group probe (Chis150) (10), and the Eubacterium cylindroides group probe (Ecyl387) (11).

Cell counts for the target organisms in the 10 subjects are summarized in Table 2. Each subject harbored at least three groups of butyrate producers, with a mean of 7.7% of the total fecal microbiota identified as butyrate producers in this study. The Fpra655 (F. prausnitzii) probe detected between 1.4 and 5.9% (mean, 3.8%) of the total fecal microbiota in all 10 subjects, which is in agreement with previous evidence, by using a different probe, indicating that this is one of the most abundant species in human feces (26). Also found in all 10 subjects was the R. intestinalis cluster (by using the Rint603 probe), which accounted for 0.9 to 5.0% (mean, 2.3%) of the total microbiota. Thus, the R. intestinalis and F. prausnitzii groups, which are likely to consist largely if not wholly of butyrate-producing strains, together accounted for not less than 3% and up to 10.9% (mean, 6.1%) of total eubacterial cells in the subjects studied. Organisms detected by the Ehal578 (E. hallii) probe were also widespread, being found in nine subjects and accounting for up to 2.4% (average of 0.6%) of the total microbiota. Recent work by Harmsen et al. (11) with a different probe showed that E. hallii and its close relatives can account for up to 3.6% of the total human fecal microbiota.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Quantification of the various bacterial components known to produce butyric acid within the human fecal microbiotaa

The species-specific probes for R. intestinalis and Eubacterium hadrum were positive for eight subjects. Bacteria related to Eubacterium sp. strains L1-83 and A2-194 and E. ramulus were each found in six subjects, while relatives of E. rectale sp. strain A1-86 were detected in four subjects. Eubacterium biforme and Eubacterium ventriosum were detected in only two subjects, and Coprococcus sp. strain L2-50 was detected in only one subject. In a PCR-based analysis the Coprococcus cluster was shown to account for up to 8% of total bacterial diversity in one human individual (25). Species probes for Anaerostipes caccae, Eubacterium barkeri, E. cylindroides, Eubacterium dolichum, Eubacterium saburreum, Eubacterium limosum, Eubacterium moniliforme, and Eubacterium multiforme failed to detect cells above the limit of detection in samples from any of the 10 subjects. Negative results were also obtained with the cluster probes Chis150 and Clit135, but the Ecyl387 group probe detected significant numbers in two individuals.

The Erec482 probe used for the detection of the whole Ruminococcus-Eubacterium-Clostridium cluster (cluster XIVa) detected between 5.2 and 26.4% of total fecal bacteria in the 10 volunteers tested here. These numbers are essentially in agreement with previously published data (10, 22, 23). Nonoverlapping probes designed to recognize butyrate-producing species within the Erec482 cluster, namely Rint603, Ehal578, Ehad579, and Event66, together accounted for between 10.2 and 85% (mean, 43%) of the Erec482 signal. Thus, in some subjects (subjects 2, 4, and 7) almost all of the Erec482 representatives were closely related to known butyrate producers, while in others the remaining Erec482 diversity may correspond to species that do not produce butyrate (e.g., Ruminococcus sp.) but might also include groups of butyrate producers that have yet to be targeted.

We have now designed and validated probes that target most of the presently known butyrate-producing species or groups from the human gut within the Clostridium clusters IV and XIVa. The main conclusion is that butyrate producers accounted for, on average, 7.7% of the bacteria in the 10 subjects studied, with the most abundant groups by far being R. intestinalis and F. prausnitzii. Interestingly, the proportion of bacterial cells belonging to Clostridium cluster XIVa was lower in this set of volunteers than for those of previously published data sets (10, 18). This highlights interindividual differences possibly due to diet or geographic location. Also, the fact that the narrower strain or species-specific probes tested here did not detect bacteria in all fecal samples further emphasizes the diversity of the colonic microbiota at the strain level.


arrow
ACKNOWLEDGMENTS
 
This work was supported in part by SEERAD (Scottish Executive Environment and Rural Affairs Department).

We are grateful to Jennifer Martin and Sylvia Duncan for supplying many of the bacterial isolates used in this work. This work was also carried out with financial support from the Commission of the European Communities, specific RTD program "Quality of Life and Management of Living Resources," QLK1-2000-108, "Microbe Diagnostics." It does not necessarily reflect its views and in no way anticipates the commission’s future policy in this area.


arrow
FOOTNOTES
 
* Corresponding author. Present address: Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom. Phone: 44 (0) 1224 555980. Fax: 44 (0) 1224 554761. E-mail: g.l.hold{at}abdn.ac.uk. Back

{dagger} Present address: SymbioHerborn Group, D-35745 Herborn, Germany. Back


arrow
REFERENCES
 
    1
  1. Alm, E. W., D. B. Oerther, N. Larsen, D. A. Stahl, and L. Raskin. 1996. The oligonucleotide probe database. Appl. Environ. Microbiol. 62:3557-3559.[Medline]
    2. 2
  2. Barcenilla, A., S. E. Pryde, J. C. Martin, S. H. Duncan, C. S. Stewart, C. Henderson, and H. J. Flint. 2000. Phylogenetic relationships of butyrate-producing bacteria from the human gut. Appl. Environ. Microbiol. 66:1654-1661.[Abstract/Free Full Text]
    3. 3
  3. Bradburn, D. M., J. C. Mathers, A. Gunn, J. Burn, P. D. Chapman, and I. D. Johnston. 1993. Colonic fermentation of complex carbohydrates in patients with familial adenomatous polyposis. Gut 34:630-636.[Abstract/Free Full Text]
    4. 4
  4. Chapman, M. A., M. F. Grahn, M. A. Boyle, M. Hutton, J. Rogers, and N. S. Williams. 1994. Butyrate oxidation is impaired in the colonic mucosa of sufferers of quiescent ulcerative colitis. Gut 35:73-76.[Abstract/Free Full Text]
    5. 5
  5. Chapman, M. A., M. F. Grahn, M. Hutton, and N. S. Williams. 1995. Butyrate metabolism in the terminal ileal mucosa of patients with ulcerative colitis. Br. J. Surg. 82:36-38.[Medline]
    6. 6
  6. Collins, M. D., P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. Farrow. 1994. The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int. J. Syst. Bacteriol. 44:812-826.[Abstract/Free Full Text]
    7. 7
  7. Cummings, J. H., and G. T. Macfarlane. 1997. Colonic microflora: nutrition and health. Nutrition 13:476-478.[CrossRef][Medline]
    8. 8
  8. Duncan, S. H., G. L. Hold, A. Barcenilla, C. Stewart, and H. Flint. 2002. Roseburia intestinalis sp. nov., a novel saccharolytic butyrate-producing bacteria from human faeces. Int. J. Syst. Evol. Microbiol. 52:1615-1620.[Abstract]
    9. 9
  9. Duncan, S. H., G. L. Hold, H. J. Harmsen, C. Stewart, and H. Flint. 2002. Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify the species into the new genus Faecalibacterium gen. nov. Int. J. Syst. Evol. Microbiol. 52:2141-2146.[Abstract]
    10. 10
  10. Franks, A. H., H. J. Harmsen, G. C. Raangs, G. J. Jansen, F. Schut, and G. W. Welling. 1998. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol. 64:3336-3345.[Abstract/Free Full Text]
    11. 11
  11. Harmsen, H. J., G. C. Raangs, T. He, J. E. Degener, and G. W. Welling. 2002. Extensive set of 16S rRNA-based probes for detection of bacteria in human feces. Appl. Environ. Microbiol. 68:2982-2990.[Abstract/Free Full Text]
    12. 12
  12. Hold, G. L., S. E. Pryde, V. J. Russell, E. Furrie, and H. J. Flint. 2002. Assessment of microbial diversity in human colonic samples by 16S rDNA sequence analysis. FEMS Microbiol. Ecol. 39:33-39.[CrossRef]
    13. 13
  13. Jansen, G. J., A. C. Wildeboer-Veloo, R. H. Tonk, A. H. Franks, and G. W. Welling. 1999. Development and validation of an automated, microscopy-based method for enumeration of groups of intestinal bacteria. J. Microbiol. Methods 37:215-221.[CrossRef][Medline]
    14. 14
  14. Kruse, H. P., B. Kleessen, and M. Blaut. 1999. Effects of inulin on faecal bifidobacteria in human subjects. Br. J. Nutr. 82:375-382.[Medline]
    15. 15
  15. Loy, A., M. Horn, and M. Wagner. 2003. probeBase: an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res. 31:514-516.[Abstract/Free Full Text]
    16. 16
  16. Ludwig, W., O. Strunk, S. Klugbauer, N. Klugbauer, M. Weizenegger, J. Neumaier, M. Bachleitner, and K. H. Schleifer. 1998. Bacterial phylogeny based on comparative sequence analysis. Electrophoresis 19:554-568.[CrossRef][Medline]
    17. 17
  17. Pryde, S. E., S. H. Duncan, G. L. Hold, C. Stewart, and H. Flint. 2002. The microbiology of butyrate formation in the human colon. FEMS Microbiol. Lett. 17:133-139.
    18. 18
  18. Rigottier-Gois, L., A. G. Le Bourhis, G. Gramet, V. Rochet, and J. Dore. 2003. Fluorescent hybridisation combined with flow cytometry and hybridisation of total RNA to analyse the composition of microbial communities in human faeces using 16S rRNA probes. FEMS Microbiol. Ecol. 43:237-245.[CrossRef]
    19. 19
  19. Scheppach, W. 1994. Effects of short chain fatty acids on gut morphology and function. Gut 35:S35-S38.
    20. 20
  20. Scheppach, W., H. P. Bartram, and F. Richter. 1995. Role of short-chain fatty acids in the prevention of colorectal cancer. Eur. J. Cancer 31A:1077-1080.
    21. 21
  21. Schwiertz, A., G. L. Hold, S. H. Duncan, B. Gruhl, M. D. Collins, P. A. Lawson, H. J. Flint, and M. Blaut. 2002. Anaerostipes caccae gen. nov., sp. nov., a new saccharolytic, acetate-utilising, butyrate-producing bacterium from human faeces. Syst. Appl. Microbiol. 25:46-51.[CrossRef][Medline]
    22. 22
  22. Schwiertz, A., G. Le Blay, and M. Blaut. 2000. Quantification of different Eubacterium spp. in human fecal samples with species-specific 16S rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol. 66:375-382.[Abstract/Free Full Text]
    23. 23
  23. Schwiertz, A., U. Lehmann, G. Jacobasch, and M. Blaut. 2002. Influence of resistant starch on the SCFA production and cell counts of butyrate-producing Eubacterium spp. in the human intestine. J. Appl. Microbiol. 93:157-162.[CrossRef][Medline]
    24. 24
  24. Simmering, R., B. Kleessen, and M. Blaut. 1999. Quantification of the flavonoid-degrading bacterium Eubacterium ramulus in human fecal samples with a species-specific oligonucleotide hybridization probe. Appl. Environ. Microbiol. 65:3705-3709.[Abstract/Free Full Text]
    25. 25
  25. Suau, A., R. Bonnet, M. Sutren, J. J. Godon, G. R. Gibson, M. D. Collins, and J. Dore. 1999. Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl. Environ. Microbiol. 65:4799-4807.[Abstract/Free Full Text]
    26. 26
  26. Suau, A., V. Rochet, A. Sghir, G. Gramet, S. Brewaeys, M. Sutren, L. Rigottier-Gois, and J. Dore. 2001. Fusobacterium prausnitzii and related species represent a dominant group within the human fecal flora. Syst. Appl. Microbiol. 24:139-145.[CrossRef][Medline]
    27. 27
  27. Topping, D. L., and P. M. Clifton. 2001. Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiol. Rev. 81:1031-1064.[Abstract/Free Full Text]

Applied and Environmental Microbiology, July 2003, p. 4320-4324, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.4320-4324.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Falony, G., Verschaeren, A., De Bruycker, F., De Preter, V., Verbeke, K., Leroy, F., De Vuyst, L. (2009). In Vitro Kinetics of Prebiotic Inulin-Type Fructan Fermentation by Butyrate-Producing Colon Bacteria: Implementation of Online Gas Chromatography for Quantitative Analysis of Carbon Dioxide and Hydrogen Gas Production. Appl. Environ. Microbiol. 75: 5884-5892 [Abstract] [Full Text]  
  • Falony, G., Lazidou, K., Verschaeren, A., Weckx, S., Maes, D., De Vuyst, L. (2009). In Vitro Kinetic Analysis of Fermentation of Prebiotic Inulin-Type Fructans by Bifidobacterium Species Reveals Four Different Phenotypes. Appl. Environ. Microbiol. 75: 454-461 [Abstract] [Full Text]  
  • Nadal, I., Donant, E., Ribes-Koninckx, C., Calabuig, M., Sanz, Y. (2007). Imbalance in the composition of the duodenal microbiota of children with coeliac disease. J Med Microbiol 56: 1669-1674 [Abstract] [Full Text]  
  • Belenguer, A., Duncan, S. H., Holtrop, G., Anderson, S. E., Lobley, G. E., Flint, H. J. (2007). Impact of pH on Lactate Formation and Utilization by Human Fecal Microbial Communities. Appl. Environ. Microbiol. 73: 6526-6533 [Abstract] [Full Text]  
  • Duncan, S. H., Belenguer, A., Holtrop, G., Johnstone, A. M., Flint, H. J., Lobley, G. E. (2007). Reduced Dietary Intake of Carbohydrates by Obese Subjects Results in Decreased Concentrations of Butyrate and Butyrate-Producing Bacteria in Feces. Appl. Environ. Microbiol. 73: 1073-1078 [Abstract] [Full Text]  
  • Falony, G., Vlachou, A., Verbrugghe, K., Vuyst, L. D. (2006). Cross-Feeding between Bifidobacterium longum BB536 and Acetate-Converting, Butyrate-Producing Colon Bacteria during Growth on Oligofructose. Appl. Environ. Microbiol. 72: 7835-7841 [Abstract] [Full Text]  
  • Duncan, S. H., Aminov, R. I., Scott, K. P., Louis, P., Stanton, T. B., Flint, H. J. (2006). Proposal of Roseburia faecis sp. nov., Roseburia hominis sp. nov. and Roseburia inulinivorans sp. nov., based on isolates from human faeces.. Int. J. Syst. Evol. Microbiol. 56: 2437-2441 [Abstract] [Full Text]  
  • Aminov, R. I., Walker, A. W., Duncan, S. H., Harmsen, H. J. M., Welling, G. W., Flint, H. J. (2006). Molecular Diversity, Cultivation, and Improved Detection by Fluorescent In Situ Hybridization of a Dominant Group of Human Gut Bacteria Related to Roseburia spp. or Eubacterium rectale. Appl. Environ. Microbiol. 72: 6371-6376 [Abstract] [Full Text]  
  • Scott, K. P., Martin, J. C., Campbell, G., Mayer, C.-D., Flint, H. J. (2006). Whole-Genome Transcription Profiling Reveals Genes Up-Regulated by Growth on Fucose in the Human Gut Bacterium "Roseburia inulinivorans".. J. Bacteriol. 188: 4340-4349 [Abstract] [Full Text]  
  • Maukonen, J., Satokari, R., Matto, J., Soderlund, H., Mattila-Sandholm, T., Saarela, M. (2006). Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome in relation to predominant faecal bacteria.. J Med Microbiol 55: 625-633 [Abstract] [Full Text]  
  • Mueller, S., Saunier, K., Hanisch, C., Norin, E., Alm, L., Midtvedt, T., Cresci, A., Silvi, S., Orpianesi, C., Verdenelli, M. C., Clavel, T., Koebnick, C., Zunft, H.-J. F., Dore, J., Blaut, M. (2006). Differences in Fecal Microbiota in Different European Study Populations in Relation to Age, Gender, and Country: a Cross-Sectional Study. Appl. Environ. Microbiol. 72: 1027-1033 [Abstract] [Full Text]  
  • Walker, A. W., Duncan, S. H., McWilliam Leitch, E. C., Child, M. W., Flint, H. J. (2005). pH and Peptide Supply Can Radically Alter Bacterial Populations and Short-Chain Fatty Acid Ratios within Microbial Communities from the Human Colon. Appl. Environ. Microbiol. 71: 3692-3700 [Abstract] [Full Text]  
  • Duncan, S. H., Louis, P., Flint, H. J. (2004). Lactate-Utilizing Bacteria, Isolated from Human Feces, That Produce Butyrate as a Major Fermentation Product. Appl. Environ. Microbiol. 70: 5810-5817 [Abstract] [Full Text]  
  • Schoefer, L., Mohan, R., Schwiertz, A., Braune, A., Blaut, M. (2003). Anaerobic Degradation of Flavonoids by Clostridium orbiscindens. Appl. Environ. Microbiol. 69: 5849-5854 [Abstract] [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 Hold, G. L.
Right arrow Articles by Flint, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hold, G. L.
Right arrow Articles by Flint, H. J.
Agricola
Right arrow Articles by Hold, G. L.
Right arrow Articles by Flint, H. J.

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