Rafael López-Rojas

Two novel IMP-type metallo-β-lactamase variants, IMP-43 and IMP-44, were identified in multidrug-resistant Pseudomonas aeruginosa isolates obtained in medical settings in Japan. Analysis of their predicted amino acid sequences revealed that IMP-43 had an amino acid substitution (Val67Phe) compared with IMP-7 and that IMP-44 had two substitutions (Val67Phe and Phe87Ser) compared with IMP-11. The amino acid residue at position 67 is located at the end of a loop close to the active site, consisting of residues 60 to 66 in IMP-1, and the amino acid residue at position 87 forms a hydrophobic patch close to the active site with other amino acids. An Escherichia coli strain expressing bla_IMP-43 was more resistant to doripenem and meropenem but not to imipenem than one expressing bla_IMP-7. An E. coli strain expressing bla_IMP-44 was more resistant to doripenem, imipenem and meropenem than one expressing bla_IMP-11. IMP-43 had more efficient catalytic activities against all three carbapenems than IMP-7, indicating that the Val67Phe substitution contributed to increased catalytic activities against carbapenems. IMP-44 had more efficient catalytic activities against all carbapenems tested than IMP-11, as well as increased activities compared with IMP-43, indicating that both the Val67Phe and Phe87Ser substitutions contributed to increased catalytic activities against carbapenems.

Pan-resistant Acinetobacter baumannii have prompted the search for therapeutic alternatives. We evaluate the efficacy of four cecropin A-melittin hybrid peptides (CA-M) in vivo. Toxicity was determined in mouse erythrocytes and in mice (lethal dose parameters were LD₀, LD₅₀, LD₁₀₀). Protective dose 50 (PD₅₀) was determined by inoculating groups of ten mice with the minimal lethal dose of A. baumannii (BMLD) and treating with doses of each CA-M from 0.5 mg/kg to LD₀. The activity of CA-Ms against A. baumannii was assessed in a peritoneal sepsis model. Mice were sacrificed at 0 and 1, 3, 5, and 7-h post-treatment. Spleen and peritoneal fluid bacterial concentrations were measured. CA(1–8)M(1–18) was the less haemolytic on mouse erythrocytes. LD₀ (mg/kg) was 32 for CA(1–8)M(1–18), CA(1–7)M(2–9), and Oct-CA(1–7)M(2–9), and 16 for CA(1–7)M(5–9). PD₅₀ was not achieved with non-toxic doses (≤LD₀). In the sepsis model, all CA-Ms were bacteriostatic in spleen, and decreased bacterial concentration (p < 0.05) in peritoneal fluid, at 1-h post-treatment; at later times, bacterial regrowth was observed in peritoneal fluid. CA-Ms showed local short-term efficacy in the peritoneal sepsis model caused by pan-resistant Acinetobacter baumannii.

Acinetobacter baumannii (American Type Culture Collection strain 19606) acquires mutations in the pmrB gene during the in vitro development of resistance to colistin. The colistin-resistant strain has lower affinity for colistin, reduced in vivo fitness (competition index, .016), and decreased virulence, both in terms of mortality (0% lethal dose_, 6.9 vs 4.9 log colony-forming units) and survival in a mouse model of peritoneal sepsis. These results may explain the low incidence and dissemination of colistin resistance in A. baumannii in clinical settings.

Clavulanic acid (CLA) exhibits low MICs against some Acinetobacter baumannii strains. The present study evaluates the efficacy of CLA in a murine model of A. baumannii pneumonia. For this purpose, two clinical strains, Ab11 and Ab51, were used; CLA MICs for these strains were 2 and 4 mg/liter, respectively, and the imipenem (IPM) MIC was 0.5 mg/liter for both. A pneumonia model in C57BL/6 mice was used. The CLA dosage (13 mg/kg of body weight given intraperitoneally) was chosen to reach a maximum concentration of the drug in serum similar to that in humans and a time during which the serum CLA concentration remained above the MIC equivalent to 40% of the interval between doses. Six groups ( n = 15) were inoculated with Ab11 or Ab51 and were allocated to IPM or CLA therapy or to the untreated control group. In time-kill experiments, CLA was bactericidal only against Ab11 whereas IPM was bactericidal against both strains. CLA and IPM both decreased bacterial concentrations in lungs, 1.78 and 2.47 log ₁₀ CFU/g ( P ≤ 0.001), respectively, in the experiments with Ab11 and 2.42 and 2.28 log ₁₀ CFU/g ( P ≤ 0.001), respectively, with Ab51. IPM significantly increased the sterility of blood cultures over that for the controls with both strains ( P ≤ 0.005); CLA had the same effect with Ab51 ( P < 0.005) but not with Ab11 ( P = 0.07). For the first time, we suggest that CLA may be used for the treatment of experimental severe A. baumannii infections.