Inhibition of Vancomycin-Resistant Enterococcus by Continuous-Flow Cultures of Human Stool Microflora With and Without Anaerobic Gas Supplementation Michael E. Hume, 1 Toni L. Poole, 1 Nicole J. Pultz, 2 Jennifer A. Hanrahan, 3 Curtis J. Donskey 2 1 United States Department of Agriculture, Agricultural Research Service, Southern Plains Agricultural Research Center, Food and Feed Safety Research Unit, College Station, TX 77845, USA 2 Infectious Diseases Section, Louis Stokes Cleveland Veterans Affairs Medical Center, 10701 East Blvd., Cleveland, OH 44106, USA 3 Division of Infectious Diseases, MetroHealth Medical Center, Cleveland, OH 44106, USA Received: 18 March 2003 / Accepted: 3 September 2003 Abstract. A continuous-flow competitive exclusion (CFCE) culture model of human stool microflora was used to examine whether supplemental anaerobic gas is necessary for maintenance of anaerobes and inhibition of vancomycin-resistant Enterococcus (VRE). CFCE cultures of human stool microflora were maintained with supplemental nitrogen, without supplemental nitrogen, or with percolated room air. Cultures with or without supplemental nitrogen maintained 9 log 10 CFU mL 1 of obligate anaerobes and eliminated 10 6 CFU mL 1 of VRE. When room air was percolated into the culture, anaerobes were detected at 2 log 10 CFU mL 1 , and the same VRE inoculum was not eliminated (P  0.001). These data demonstrate that human stool CFCE cultures maintain high levels of obligate anaerobes and inhibit VRE without the addition of supplemental anaerobic gas. The anaerobic, continuous-flow culture model developed by Freter [7] closely reproduced a variety of bacterial interactions that occur in the intestinal tracts of mice. This culture technique has been utilized to develop con- tinuous-flow competitive exclusion (CFCE) cultures de- rived from cecal contents of adult chickens, which inhibit colonization by enteric pathogens in newly hatched chicks [8, 9, 11, 12]. Anaerobic bacteria are essential components of an effective CFCE culture, so conditions supportive of anaerobic growth are maintained in CFCE culture systems by providing a constant flow of oxygen- free nitrogen or carbon dioxide in the culture vessel and medium reservoir [8]. We observed serendipitously that a CFCE culture containing human stool microflora elim- inated vancomycin-resistant Enterococcus (VRE) de- spite the loss of external anaerobic culture conditions when the nitrogen supply was depleted. In nature, obligate anaerobes may be present in high concentrations in areas exposed to air (e.g., the orophar- ynx) [6]. The consumption of oxygen by co-existing aerobic and facultative organisms provides a microenvi- ronment at these sites supportive of obligate anaerobes [6]. In addition, elimination of all oxygen is not required for growth of many obligate anaerobes; obligate anaer- obes include strict anaerobes incapable of growth in oxygen levels above 0.5% and moderate obligate anaer- obes capable of growth in oxygen levels from 2% to 8%) [6, 10, 14]. Based on the hypothesis that facultative organisms would deplete sufficient oxygen from the cul- ture to allow growth of moderate anaerobes, the objec- tive of the current study was to determine whether obli- gate anaerobes would persist in the CFCE culture system with no added anaerobic gas and whether the culture would continue to inhibit VRE. Materials and Methods VRE strains. Clinical isolates of Enterococcus faecium VRE (C68, a VanB-type isolate [4]; C24, a VanB-type isolate; and C67, a VanA- type isolate) were used in the study. Vancomycin resistance was stable in each of the strains through multiple culture passages. CFCE cultures. Three human stool CFCE cultures were derived from a parent culture [4] by transferring effluent collected anaerobically at 4°C to additional culture vessels (New Brunswick Scientific Company, Edison, NJ) and were maintained at culture volumes of 385 mL. The Correspondence to: C.J. Donskey; email: curtisd123@yahoo.com CURRENT MICROBIOLOGY Vol. 48 (2004), pp. 364 –367 DOI: 10.1007/s00284-003-4112-7 Current Microbiology An International Journal © Springer-Verlag New York LLC 2004