The production of a novel carbapenem-hydrolysing lactamase in Aeromonas veronii biovar sobria , and its association with imipenem resistance

Sir, Species of the genus Aeromonas are widely dispersed in aquatic environments, but are now increasingly implicated in clinical infections that include gastroenteritis, wound infections and bacteraemia. Aeromonas veronii bv. sobria is a major pathogenic species of this genus and, as with most Aeromonas species, has the ability to produce a chromosomally encoded inducible carbapenemhydrolysing lactamase. This metallolactamase belongs to molecular class B; however, paradoxically, when standard in-vitro susceptibility testing is performed, these bacteria remain susceptible to the carbapenems. In this report we identify two such imipenem-resistant strains that produce a novel carbapenem-hydrolysing -lactamase, first described in 1997. A. veronii bv. sobria strains 13 and 99 were isolated from a reservoir in Vellore, July 1996, during a study on antibiotic resistance in environmental bacteria isolated from water sources in Southern India. Strains 13 and 99 were found to be resistant to imipenem (MIC 8 mg/L) by the agar dilution method, employing a conventional inoculum of 10 cfu/mL. To date, only a handful of naturally occurring carbapenem-resistant aeromonas isolates have been recorded when testing with a conventional sized inoculum. -Lactamase extracts from strains 13 and 99 demonstrated efficient hydrolysis of imipenem when assayed spectrophotometrically. -Lactamase could not be further induced and therefore, strains 13 and 99 constitutively produce a carbapenemase. Isoelectric focusing (IEF) demonstrated that both strain 13 and 99 possess two -lactamases, detectable with nitrocephin staining, with isoelectric point (pI) values of 5.84 and 8.3. An imipenem/agar overlay modification to IEF was applied and showed the presence of a single imipenem hydrolysing enzyme of pI 5.84 in both strains 13 and 99. The carbapenemase in these two strains is unlike all other previously described aeromonas carbapenemases. Furthermore, the pI value is considerably different from the aeromonas carbapenemases that have so far been reported (typically 8.0 or greater). IEF/inhibitor overlays facilitated further characterization of the 5.84 and 8.3 -lactamases. The gel was overlaid with 100 mM EDTA which is known to inhibit metallo-lactamases, and the serine -lactamase inhibitor BRL42715 (100 M), prior to staining with nitrocephin. A TEM-1 (serine-based) -lactamase and a cell extract from Stenotrophomonas maltophilia strain 511 that produces two -lactamases, (i) L1 metallo-lactamase (pI 6.4) and (ii) L2 serine-lactamase (pI 9.7), were included as controls. The pI 8.3 -lactamase of both A. veronii bv. sobria strains were found to be serine-based because they were inhibited by BRL42715 but not EDTA; however, the pI 5.84 carbapenemase is not inhibited by either EDTA or BRL42715, and therefore, cannot be classified as either a metalloor serine-lactamase (Figure). These results indicate the presence of a completely novel carbapenemase in A. veronii bv. sobria that may constitute a new -lactamase molecular class. The hyper-production of the carbapenemase can be clearly correlated with a decrease in sensitivity, which in a clinical setting could lead to therapeutic failure. The novel carbapenemhydrolysing -lactamase is to be designated AVS-1.