Fig 1.
MIC of E. coli and K. pneumoniae in CAMHB medium versus MIC in zinc-limited media.
MICs for 6 antibiotics (MEM, ETC, IPM, TZP, CRO, CAZ, A to F) were determined using either CAMHB or RPMI-1640 medium against 33 metallo-β-lactamase-producing clinical isolates: K. pneumoniae (n = 21), and E. coli (n = 12). (A) MIC distributions for MEM. (B) MIC distributions for ETC. (C) MIC distributions for IPM. (D) MIC distributions for TZP. (E) MIC distributions for CRO. (F) MIC distributions for CAZ. Breakpoints (μg/mL) are defined as follows: For MEM (S/I/R): E. coli/K. pneumoniae: ≤ 1, 2, ≥ 4; For ETC (S/I/R): E. coli/K. pneumoniae: ≤ 0.5, 1, ≥ 2; For IPM (S/I/R): E. coli/K. pneumoniae: ≤ 1, 2, ≥ 4; For TZP (S/I/R): E. coli/K. pneumoniae: ≤ 8, 16, ≥ 32; For CRO (S/I/R): E. coli/K. pneumoniae: ≤ 1, 2, ≥ 4; For CAZ (S/I/R): E. coli/K. pneumoniae: ≤ 4, 8, ≥ 16. The blue solid lines indicate the breakpoints. Everything to the right of the vertical line is predicted to be resistant in CAMHB and everything upon the horizontal line is predicted to be resistant in zinc-limited media. Right lower quadrant represents isolates resistant in CAMHB but susceptible in zinc-limited media. Abbreviations: imipenemase (IMP), New Delhi metallo-beta-lactamase (NDM), Verona integron-encoded metallo-beta-lactamase (VIM), metallo-β-lactamases (MBL).
Fig 2.
MIC of A. baumannii and P. aeruginosa in CAMHB medium versus MIC in zinc-limited media.
MICs for 6 antibiotics (MEM, ETC, IPM, TZP, CRO, CAZ, A to F) were determined using either CAMHB or CAMHB_EDTA medium against 23 metallo-β-lactamase-producing clinical isolates: A. baumannii (n = 4), and P. aeruginosa (n = 19). (A) MIC distributions for MEM. (B) MIC distributions for ETC. (C) MIC distributions for IPM. (D) MIC distributions for TZP. (E) MIC distributions for CRO. (F) MIC distributions for CAZ. Breakpoints (μg/mL) are defined as follows: For MEM (S/I/R): A. baumannii/P. aeruginosa: ≤ 2, 4, ≥ 8; For ETC (S/I/R): A. baumannii/P. aeruginosa: intrinsically resistant; for reference purposes, Enterobacterales breakpoints are shown; For IPM (S/I/R): A. baumannii/P. aeruginosa: ≤ 2, 4, ≥ 8; For TZP (S/I/R): A. baumannii: ≤ 16, 32-64, ≥ 128; P. aeruginosa: ≤ 16, 32, ≥ 64; For CRO (S/I/R): A. baumannii: ≤ 8, 16-32, ≥ 64; P. aeruginosa: intrinsically resistant; for reference purposes, A. baumannii breakpoints are shown; For CAZ (S/I/R): A. baumannii/P. aeruginosa: ≤ 8, 16, ≥ 32. The black dashed lines indicate the breakpoints for A. baumannii. The yellow solid lines indicate the breakpoints for P. aeruginosa. Everything to the right of the vertical line is predicted to be resistant in CAMHB and everything upon the horizontal line is predicted to be resistant in zinc-limited media. Right lower quadrant represents isolates resistant in CAMHB but susceptible in zinc-limited media. Abbreviations: imipenemase (IMP), New Delhi metallo-beta-lactamase (NDM), Verona integron-encoded metallo-beta-lactamase (VIM), metallo-β-lactamases (MBL).
Fig 3.
MICs for 6 antibiotics (MEM, ETC, IPM, TZP, CRO, CAZ) were determined using either CAMHB or zinc-limited media against 56 metallo-β-lactamase-producing clinical isolates: A. baumannii (n = 4), E. coli (n = 12), K. pneumoniae (n = 21), and P. aeruginosa (n = 19). Abbreviations: meropenem (MEM), ertapenem (ETC), imipenem and cilastatin (IPM), piperacillin and tazobactam (TZP), ceftriaxone (CRO), ceftazidime (CAZ), A. baumannii (AB), E. coli (EC), K. pneumoniae (KP), P. aeruginosa (PA).
Fig 4.
MICs were determined against 11 K. pneumoniae, and P. aeruginosa strains in CAMHB and CAMHB_EDTA media (A to B). (A). The therapeutic agent was IPM. (B). The therapeutic agent was a combination of IPM and AVI. The breakpoint was 8 mg/L. Statistical comparisons were made between the MICs values against two different drug groups when tested in zinc-limited media (Mann-Whitney; p < 0.001).
Table 1.
Summary of MICs. “S” = Susceptible, “I” = Intermediate, “R” = Resistant.
Table 2.
Carbapenem inactivation method (mCIM) and EDTA-modified carbapenem inactivation method (eCIM) results.
Table 3.
MIC values of selected strains for in vivo testing. KP49, KP158 and KP560 were IPM resistant in CAMHB while susceptible in RPMI-1640; KP49, KP68 and KP 153 were IPM resistant in both media.
Fig 5.
IPM efficacy in Galleria mellonella infection model.
Galleria mellonella larvae (n = 10 per group) were infected and treated with IPM and monitored for survival (A to F). KP49, KP158 and KP560 were resistant in CAMHB while susceptible in RPMI-1640 (A to C). KP46, KP68 and KP153 were resistant in both CAMHB and RPMI-1640 (D to F). The larvae were infected with lethal inoculum of KP49 (2.9E8 CFU/larva), KP158 (3.5E8 CFU/larva), KP560 (2E7 CFU/larva), KP46 (1.7E8 CFU/larva), KP68 (2.85E8 CFU/larva), KP153 (2.8E7 CFU/larva) before being treated with PBS or IPM. Statistical comparisons between PBS treatment and each IPM treatment group were made using the log-rank test to compare survival.
Fig 6.
IPM efficacy in neutropenic mouse thigh infection model.
Neutropenic mice were challenged with different K. pneumoniae clinical isolates. Each thigh of the mouse was inoculated with 0.1 mL 1E8 CFU/mL bacterial suspension in PBS; except for KP153, which received 1E3 CFU/mL inoculum. After infection, one mouse would be euthanized immediately, and another at 2 hours post-infection. The remaining mice were administered treatment subcutaneously with either 25 mg/kg imipenem or PBS at 2, 6, 10 and 22 hours post-infection. All the mice were euthanized at 26 hours for thigh collection. There were significant differences between PBS groups and treatment groups in susceptible strains (A-C; KP49: p = 0.017; KP158: p = 0.009; KP560: p = 0.004, Mann-Whitney), while not observed in strains resistant to imipenem (D-E).