Diversity of integron- and culture-associated antibiotic resistance genes in freshwater floc.

Clinically important antibiotic resistance genes were detected in culturable bacteria and class 1 integron gene cassettes recovered from suspended floc, a significant aquatic repository for microorganisms and trace elements, across freshwater systems variably impacted by anthropogenic activities. Antibiotic resistance gene cassettes in floc total community DNA differed appreciably in number and type from genes detected in bacteria cultured from floc. The number of floc antibiotic resistance gene cassette types detected across sites was positively correlated with total (the sum of Ag, As, Cu, and Pb) trace element concentrations in aqueous solution and in a component of floc readily accessible to bacteria. In particular, concentrations of Cu and Pb in the floc component were positively correlated with floc resistance gene cassette diversity. Collectively, these results identify suspended floc as an important reservoir, distinct from bulk water and bed sediment, for antibiotic resistance in aquatic environments ranging from heavily impacted urban sites to remote areas of nature reserves and indicate that trace elements, particularly Cu and Pb, are geochemical markers of resistance diversity in this environmental reservoir. The increase in contamination of global water supplies suggests that aquatic environments will become an even more important reservoir of clinically important antibiotic resistance in the future.

Inhibitors of bacterial cystathionine beta-lyase: leads for new antimicrobial agents and probes of enzyme structure and function.

The biosynthesis of methionine is an attractive antibiotic target given its importance in protein and DNA metabolism and its absence in mammals. We have performed a high-throughput screen of the methionine biosynthesis enzyme cystathionine beta-lyase (CBL) against a library of 50 000 small molecules and have identified several compounds that inhibit CBL enzyme activity in vitro. These hit molecules were of two classes: those that blocked CBL activity with mixed steady-state inhibition and those that covalently interacted with the enzyme at the active site pyridoxal phosphate cofactor with slow-binding inhibition kinetics. We determined the crystal structure of one of the slow-binding inhibitors in complex with CBL and used this structure as a guide in the synthesis of a small, focused library of analogues, some of which had improved enzyme inhibition properties. These studies provide the first lead molecules for antimicrobial agents that target cystathionine beta-lyase in methionine biosynthesis.

Cystathionine beta-lyase is important for virulence of Salmonella enterica serovar Typhimurium.

The biosynthesis of methionine in bacteria requires the mobilization of sulfur from Cys by the formation and degradation of cystathionine. Cystathionine beta-lyase, encoded by metC in bacteria and STR3 in Schizosaccharomyces pombe, catalyzes the breakdown of cystathionine to homocysteine, the penultimate step in methionine biosynthesis. This enzyme has been suggested to be the target for pyridinamine antimicrobial agents. We have demonstrated, by using purified enzymes from bacteria and yeast, that cystathionine beta-lyase is not the likely target of these agents. Nonetheless, an insertional inactivation of metC in Salmonella enterica serovar Typhimurium resulted in the attenuation of virulence in a mouse model of systemic infection. This result confirms a previous chemical validation of the Met biosynthetic pathway as a target for the development of antibacterial agents and demonstrates that cystathionine beta-lyase is important for bacterial virulence.