1. D-739/181
50th ICAAC
Sept , 2010
Boston
Evaluation of the MBL Etest for detecting Metallo-ß-lactamase presence in Pseudomonas aeruginosa
L. DAVIES, M. WOOTTON, V. E. DANIEL, R. A. HOWE
Introduction
Pseudomonas aeruginosa (PAER) causes serious infection in immunosupressed patients and is a major cause of morbidity and mortality in cystic fibrosis patients. PAER are frequently resistant to many antimicrobials, and increasingly carbapenems (CBM). Recent reports of carbapenemases and in particular metallo--lactamases (MBL) in PAER have led to laboratories using the MBL Etest to detect MBL. However, it has been suggested that EDTA has antimicrobial properties or effects on other resistance mechanisms in PAER leading to false positive results. We examined detection of MBL in PAER using a variety of methods
Methods cont.
polymerase chain reaction using primers blaVIM, IMP. Isolates were considered to be ampC or OXA positive if a 3x Log2 drop of CBM MIC was seen when in the presence of 200mg/L cloxacillin and 0.2M salt respectively. A modified Hodge test was performed to detect carbapenemase activity. Resistance mechanism was assigned MBL if MBL PCR positive, porin loss/ampC/OXA if IMI R plus ampC/OXA present and porin loss/efflux if IMI/MER R plus no MBL/ampC or OXA
Table 2: Comparison of MBL-NEG, MBL-POS & MBL-FP
Results
Of the 90 PAER isolates submitted, MBL Etest results tested negative (MBL-NEG) for 60 (66.6%), positive for 30 (33.3%). Of the 30 PAER which tested positive with MBL Etest, 7 (23%) contained VIM genes (MBL-POS) and 23 (77%) were negative for MBL PCR. AmpC and OXA activity against CBM was confirmed and resistance mechanism assumed (Table 3). These isolates were classified as false positives (MBL-FP). Sensitivity, specificity, negative & positive predictive values for MBL Etest were 100%, 75%, 100% & 23.3%. The ranges of log drop in MIC for MBL Etest (IP/IPI) were >7 for MBL-POS, between 3 and 7 for MBL-FP and <3 for MBL-NEG (Table 1). MICs to EDTA were similar for both MBL-NEG and MBL-FP.
Results cont.
The prevalence of assumed efflux as a resistance mechanism contributing to CBM resistance is similar in MBL-NEG and MBL-FP groups.
As seen in Table 2, FP strains were negative by Hodge test (HT), frequently CAZ sensitive, and had lower MICs for MER and IMI and EDTA.
Fig 1: MBL Etest
a) MBL NEG b) MBL POS c) MBL FP 3 12
>256
1.5 24
Table 3: Prevalent resistance mechanisms
Methods
PAER sent to the SACU antimicrobial reference unit for carbapenem resistance confirmation over 18 months were tested. MBL Etests comparing imipenem (IMI) MIC +/- EDTA were performed using the manufacturer’s criteria. Imipenem (IMI), meropenem (MER) and ceftazidime (CAZ) MICs were determined by Etest and interpreted using BSAC breakpoints. The mechanism responsible for CBM resistance was investigated using a phenotypic assay for ampC & oxacillinase and
Conclusions
MBL Etest is a sensitive method for detecting MBL positive PAER. However, false positives can occur, particularly in isolates less resistant to CAZ or CBMs are tested. Hodge or molecular tests are necessary for confirmation.
Table 1: Number of isolates showing log drop in MBL Etest 12
Travel grant kindly funded by:
Specialist Antimicrobial Chemotherapy Unit, Public Health Wales, Cardiff, UK