Zinc Chelator N,N,N′,N′-Tetrakis(2-Pyridylmethyl)Ethylenediamine Reduces the Resistance of Mycobacterium abscessus to Imipenem

Figures & data

Figure 1 MIC of imipenem for 194 clinical M. abscessus isolates.

Table 1 Imipenem and TPEN Act Synergistically to Inhibit the Growth of Imipenem-Resistant M. abscessus*

Isolates	Imipenem	Imipenem + TPEN	TPEN	TPEN + Imipenem	FICI	Percent Synergy
Resistant (n=30)	32–128	4–16	32–256	8–32	0.1875–0.5	100% (30/30)
Intermediate (n=24)	8–16	1–4	16–128	4–32	0.25–0.75	79.2% (19/24)
	8	1–4	16–128	4–32	0.375–0.75	50% (5/10)
	16	2–4	32–128	4–16	0.25–0.5	100% (14/14)
Sensitive (n=8)	4	1–4	16–64	4–32	0.75–1.25	0% (0/8)

Notes: *Reference strain ATCC19977 and 61 clinical M. abscessus isolates were divided into imipenem-resistant, -resistance intermediate and -sensitive groups, and the ability of TPEN to synergize with imipenem to inhibit growth was determined. Minimum inhibitory concentrations (or range of concentrations) shown in columns are: imipenem tested alone; imipenem tested in combination with TPEN; TPEN tested alone; TPEN tested in combination with imipenem. FICI ≤0.5 = synergy; FICI >0.5–4.0 = no drug interaction.

Figure 2 Susceptibility of M. abscessus isolates (n=62) to imipenem with or without TPEN. The data above each bar are the percentage of isolates.

Figure 3 Time–kill kinetic assays. In vitro TK assays were conducted with reference strain ATCC19977 (A) and an imipenem-resistant clinical isolate, A189 (B). The bacteria were incubated in the presence of either imipenem or TPEN alone, or in combination at the various concentrations shown. The MIC of imipenem for ATCC19977 and A189 was 16 and 32 mg/L, respectively; the MIC of TPEN was 64 mg/L for both. A bactericidal effect was defined as ≥2 log reduction in CFUs compared to 2×MIC imipenem alone in TK assays. *≥2 log reduction in CFUs.

Table 2 Zone of Inhibition Surrounding Imipenem Disk with or without EDTA*

Strains	Imipenem (mm)		Imipenem + EDTA (mm)		Mean Increase
	Range	Mean	Range	Mean	(mm)
Resistant	6–10	8	24–28	26	18
Intermediate (16mg/L)	17–19	18	28–30	29	11
Intermediate (8 mg/L)	24–27	26	28–31	30	4
Sensitive	26–28	27	29–32	30	3

Notes: *ATCC19977 and 61 clinical isolates classified as imipenem-resistant, -resistance intermediate or -sensitive were spread onto agar plates. Ten microgram imipenem disks impregnated with or without 0.03125M EDTA were placed on the surface and the plates were incubated for 5 days at 37°C. A zone ≥5 mm diameter surrounding the EDTA-impregnated disk compared to the non-impregnated disk was considered MBLs positive.

Figure 4 Detection of metallo-β-lactamases by imipenem-EDTA combined disk method. (A) Zones of growth inhibition are shown surrounding imipenem disks impregnated or not impregnated with EDTA on plates inoculated with imipenem-resistant, -resistance intermediate, or -sensitive M. abscessus strains. EDTA, ethylenediaminetetraacetic acid; IMI, imipenem. (B) The abilities of EDTA, sulbactam, avibactam and relebactam to synergize with imipenem and inhibit the growth of imipenem-resistant (A247) M. abscessus were determined and compared. Imipenem disks were impregnated with or without the compound listed; the surrounding zones of growth inhibition are shown. Bacterial growth surrounding disks impregnated with EDTA, sulbactam, avibactam or relebactam alone (plate on the right) serves as the negative control. ^⋆Indicates a zone of growth inhibition ≥5 mm larger than the zone surrounding a disk impregnated with imipenem alone.