The development of anaerobic methods for bacterial mutation assays: aerobic and anaerobic fluctuation tests of human faecal extracts and reference mutagens

Abstract

Methods for performing microtiter fluctuation tests in an anaerobic enclosure were devised in order to study the mutagenic activity of human fecal extracts under conditions of strict anaerobiosis. Under these conditions (monitored by growth of obligate anaerobes), 2 mutagens (4-chloromethylbiphenyl (4CMB) and potassium dichromate) were reproducibly mutagenic to Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA(pKM101), respectively. The mutagenicity was investigated of aqueous extracts of feces, prepared aerobically and anaerobically, collected from a single donor. Bioassay of 5 fecal extracts showed that 4 contained substances which promoted auxotrophic growth of his- and tryp- bacteria, and were therefore unsuitable for mutagenicity assay. The remaining extract (half of which was prepared aerobically, the other anaerobically) was assayed for mutagenicity in S. typhimurium TA 100. Reproducible dose-response curves were obtained with the aerobic extract and with the anaerobic extract when both were assayed under aerobic conditions. Negative results were obtained when the anaerobic or aerobic extracts were assayed anaerobically, under conditions where concurrent assays of 4CMB were positive. Apparently bacterial fluctuation tests using reversion from auxotrophy to prototrophy as the genetic marker can be seriously compromised by the presence of substances (including amino acids) in the test material which promote auxotrophic growth. The results and interpretation of mutagenicity tests of fecal extracts are very dependent on whether air is present or absent during both extraction and assay. [The idea that colorectal cancer might be initiated by carcinogens produced endogenously by gut bacteria is currently under test in several laboratories.].