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Five aerobic intestinal bacterial strains that utilized chloramphenicol (CAP) as sole carbon and energy source were isolated from fecal samples collected from healthy volunteers. Based on 16S rRNA gene sequence analysis, four of the five strains were identified as Klebsiella pneumoniae and one as Escherichia fergusonii. The degradation rate of strain I-10-CHL (E. fergusonii) varied with the initial concentration of CAP. The pH value also had an effect on the degradation rate of CAP and bacterial growth. A pH of 6.5 was optimal for CAP degradation and growth of strain I-10-CHL (E. fergusonii). In mixed substrate batch cultivations, where CAP was one of the components, glucose, acetamide and CAP were utilized simultaneously. The presence of glucose and acetamide increased the growth and substrate degradation rates of CAP. During incubation with E. fergusonii cells, reduction intermediates (1-p-nitrophenyl-2-amino-1,3-propanediol) were observed. The strains capable of utilizing CAP were CAP-susceptible, which indicates that the mechanism of CAP assimilation by the strains is independent on that of CAP resistance. The role of this CAP reduction pathway in the physiology of K. pneumoniae and E. fergusonii is unknown., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)