Anhydrous edta

Pea aphids (Acrythosiphon pisum strain LSR1) were grown as an all-female clone on Fava bean (Vicia faba) seedlings on 16h/8h light/dark cycles at 20°C. Once reaching adulthood, apterous adults were raised on Fava bean plants on 16h/8h light cycles and allowed to reproduce overnight. After seven days, all aphids, in the 4^th larval instar and typically amounting to 5g, were removed from the Fava bean plants. Aphids were weighed and surface-sterilized in 0.5% NaClO solution, then rinsed twice in Ultrapure water (MilliporeSigma), each 30 s. Aphids were gently ground in a mortar and pestle in 40mL sterile Buffer A (25mM KCl (Sigma-Aldrich), 35mM Tris base (Sigma-Aldrich), 10mM MgCl₂ (Sigma-Aldrich), 250mM anhydrous EDTA (Sigma-Aldrich), and 500mM Sucrose (Sigma-Aldrich) at pH 7.5). Aphid homogenate was vacuum filtered to 100μm, then centrifuged at 1500g for 10 min at 4°C. Supernatant was discarded, and the resulting pellet was resuspended in 20mL Buffer A and vacuum-filtered three times from 20μm, to 10μm, and finally to 5μm. The resulting filtrate was spun at 1500g for 30 min at 4°C and supernatant discarded. The resulting pellet was resuspended in 10mL Sucrose solution (300mM Sucrose (Sigma-Aldrich) and 100mM Tris base (Sigma-Aldrich)) then checked on a brightfield microscope for intact Buchnera cells. Buchnera cells remain intact while at 4°C for a maximum of 24h.

MBL production was detected by performing combined disc test described by Franklin et al. [11 (link)] in all carbapenemase screening positive isolates. In this test, two imipenem discs (10 μg), one containing 10 μl of 0.1 M (292 μg) anhydrous EDTA (Sigma Chemicals, St. Louis, MO) were used. They were placed on a MHA plate inoculated with 0.5 McFarland suspension of the test isolate. Plates were incubated for 16–18 hours at 35°C. After incubation, the diameter of inhibition zones was measured. An increase in zone diameter of >4 mm around the imipenem-EDTA disc compared to that of the imipenem disc alone was considered positive for MBL production (Figure 2).Figure 2

Positive combined disc test for detection of MBL producer by use of EDTA. The test isolate shows a zone diameter of >4 mm around the imipenem-EDTA disc compared to that of the imipenem disc alone.

E. coli strain NDM-1 EC27 (positive for NDM-1; kindly provided by Prof. Giasuddin Ahmed) was used as positive control in MHT and combined disc test. E. coli ATCC 25922 was used as negative control for both the tests.

The phenotypic detection of the carbapenemase production was performed by the modified Hodge test by using an imipenem disc (10 μg) as was described by CLSI. The detection of metallo-β- lactamase production was also performed by the combined-disc test by using two imipenem discs (10 μg), one containing 10 μL of 0.1 M (292 μg) anhydrous EDTA (Sigma Chemicals, St. Louis, MO), which were placed 25 mm apart on a MH agar plate. An increase in the zone diameter of >4 mm around the imipenem-EDTA disc as compared to that of the imipenem disc alone was considered as positive for metallo-β-lactamase production.