Metallo-β-lactamase mediated carbapenem resistance in gram negative bacilli

Abstract

Carbapenem antibiotics were thought to be the ultimate ß- lactams, combining the action of a ß- lactam with that of a ß- lactamase inhibitor in a single molecule. These compounds are very effective antimicrobials and are currently licensed for use as last line treatments in Intensive Therapy Units (ITUs) and remain active against organisms producing extended spectrum TEM- and SHV- derived enzymes which are able to hydrolyse third generation cephalosporins.

In this thesis a study of imipenem resistant Bacteroides fragilis clinical isolates from several sources including the Public Health Laboratory Service in Cardiff and the Scottish Anaerobe Reference Laboratory was undertaken. Three of these isolates were found to have Minimum Inhibitory Concentrations (MIC's) of imipenem greater than the breakpoint set by the NCCLS of 16mg/l. In all cases, the isolates were found to produce an imipenem hydrolysing ß- lactamase. Biochemical analysis has shown that these strains each produce a single ß- lactamase with a substrate and inhibitor profile similar to that of the CfiA metallo- ß- lactamase previously described from a Bacteroides fragilis isolate

Whilst attempting to optimise the assay conditions for these enzymes, it was discovered that the concentration of zinc sulphate present during the preparation procedure and during the assay significantly affected the specific activity and substrate profile of these enzymes. By splitting a single culture of a metallo -ß- lactamase producing strain, it was possible to demonstrate that this effect was a direct result of the zinc sulphate concentration present during the preparation and assay of these enzymes. It was noted that these enzymes were inhibited by the presence of excess zinc sulphate, and that this inhibition was reversible and substrate specific, affecting carbapenem hydrolysis more than any other substrate. This observation was found to extend to several other metallo- ß- lactamases.

A survey of Bacteroides clinical isolates from the Edinburgh Royal Infirmary was also undertaken to determine the frequency of resistance to several antimicrobials. The rate of imipenem resistance was found to be 0.6% (n = 166).

Imipenem resistant isolates from several species including Burkholderria, Enterobacter and Stenotrophomonas were examined for carbapenemase production. Only the Stenotrophomonas isolates were found to produce a ß- lactamase capable of hydrolysing imipenem.

Ten clinical isolates from the genus Flavobacterium were examined for carbapenemase production. All isolates were found to hydrolyse imipenem, although in three isolates, the rate was very low. The addition of the metal ion chelator EDTA was found to inhibit both the nitrocephin and imipenem hydrolysis of all but two of these isolates. Two strains were also identified each of which produced a novel metallo -ß- lactamase, and the ß- lactamases of one of these ( Flavobacterium spiritivorum NCTC 11388) were characterised in greater detail.