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Infection and Immunity, July 2004, p. 3793-3802, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.3793-3802.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Use of a Continuous-Flow Anaerobic Culture To Characterize Enteric Virulence Gene Expression

Fernando Ruiz-Perez,1,2 Jalaluddin Sheikh,1,2 Suzanne Davis,1,2 Edgar C. Boedeker,1,2 and James P. Nataro1,2,3,4*

Center for Vaccine DevelopmentDepartments of,1 Medicine,2 Pediatrics,3 Microbiology & Immunology, University of Maryland School of Medicine, Baltimore, Maryland4

Received 16 February 2004/ Returned for modification 17 March 2004/ Accepted 24 March 2004

We developed an in vitro culture method to characterize the expression of bacterial genes under conditions mimicking the colonic environment. Our culture system (the intestinal simulator) comprised a continuous-flow anaerobic culture which was inoculated with fecal samples from healthy volunteers. As a test organism, we employed enteroaggregative Escherichia coli (EAEC), an emerging diarrheal pathogen that is thought to cause infection in both the small and large intestines. After the simulator culture achieved equilibrium conditions, we inoculated the system with prototype EAEC strain 042 and assessed the expression of three EAEC virulence-related genes. We focused particularly on expression of aggR, which encodes a global transcriptional regulator of EAEC virulence factors, and two AggR-regulated genes. By using real-time quantitative reverse transcription-PCR, we showed that aggR expression in the simulator is increased 3- to 10-fold when 042 is grown under low-pH (5.5 to 6.0) conditions, compared with results with neutral pH (7.0). Interestingly, however, this effect was seen only when the strain was grown in the presence of commensal bacteria. We also found that expression of aggR is 10- to 20-fold higher at low NaCl concentrations, and this effect was also observed only in the presence of commensal bacteria. Using coculture and conditioned-media experiments, we identified specific strains of Enterococcus and Clostridium that upregulated aggR expression; in contrast, strains of Lactobacillus and Veillonella downregulated aggR expression. Our data provide new insights into regulation of virulence genes in EAEC and suggest the utility of intestinal simulation cultures in characterizing enteric gene regulation.

* Corresponding author. Mailing address: Center for Vaccine Development, University of Maryland School of Medicine, 685 W. Baltimore St., Baltimore, MD 21201. Phone: (410) 706 5328. Fax: (410) 706 6205. E-mail: jnataro{at}medicine.umaryland.edu.

Editor: A. D. O'Brien

Infection and Immunity, July 2004, p. 3793-3802, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.3793-3802.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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