C. William Keevil

Fluorescence in situ hybridization using peptide nucleic acid probes for rapid detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in potable-water biofilms

ABSTRACT Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.

A study of iron acquisition mechanisms of Legionella pneumophila grown in chemostat culture

Abstract. We recently demonstrated that the virulence of a clinical isolate of Legionella pneumophila is significantly attenuated when cultured in an iron-limited environment. In this study the influence of iron limitation on the expression of enzyme activities and iron-transport mechanisms was investigated. Expression of the important pathogenicity factor, the zinc metalloprotease, was reduced fivefold in response to iron limitation. Ferric citrate reductase activity was demonstrated in both iron-limited and replete cell fractions. Activity was located principally in the cytoplasm and periplasm, and was not enhanced by iron restriction. Optimum activity was observed with NADPH as reductant. Siderophores were not elaborated under these culture conditions. Iron-loaded transferrin enhanced the growth of steady-state, iron-limited cultures, demonstrating that transferrin represents a potentially important iron source for L. pneumophila in vivo. Although cell surface transferrin receptors were not detected, in vitro experiments demonstrated digestion of transferrin by the zinc metalloprotease activity of culture supernatants.