Juan Domínguez-Herrera

Due to the significant increase in antimicrobial resistance ofAcinetobacter baumannii, immune system stimulation to block infection progression may be a therapeutic adjuvant to antimicrobial treatment. Lysophosphatidylcholine (LPC), a major component of phospholipids in eukaryotic cells, is involved in immune cell recruitment and modulation. The aim of this study was to show if LPC could be useful for treating infections caused byA. baumannii. A. baumanniiATCC 17978 was used in this study. Levels of serum LPC and levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-1β, and IL-10 were determined by spectrophotometric assay and enzyme-linked immunosorbent assay (ELISA), respectively, using a murine peritoneal sepsis model in which mice were inoculated with 5.3 log CFU/ml ofA. baumannii. The therapeutic efficacy of LPC againstA. baumanniiin murine peritoneal sepsis and pneumonia models was assessed for 48 h after bacterial infection. At early time points in the murine model of peritoneal sepsis caused byA. baumannii, LPC was depleted and was associated with an increase of inflammatory cytokine release. Preemptive therapy with LPC in murine peritoneal sepsis and pneumonia models markedly enhanced spleen and lung bacterial clearance and reduced the numbers of positive blood cultures and the mouse mortality rates. Moreover, treatment with LPC reduced proinflammatory cytokine production. These data demonstrate that LPC is efficacious as a preemptive treatment in experimental models of peritoneal sepsis and pneumonia caused byA. baumannii.

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.

To model the standard broth microdilution method, based on a modified Gompertz function, to obtain accurate vancomycin MIC values for methicillin-resistant Staphylococcus aureus (MRSA). The effect of these MIC values on the vancomycin therapeutic target of AUC_0–24/MIC ≥400 was evaluated. Three clinical isolates of MRSA with different vancomycin MIC values were used in this model. The optical densities (OD) of each MIC determination were modelled by a non-linear regression method using an F-test. The OD data were adjusted to the Gompertz equation to obtain the MIC values. The mean vancomycin AUC_0–24 obtained with a 30 mg/kg/day dosing schedule was calculated using a Monte Carlo simulation over 5000 subjects, using the pharmacokinetic data obtained in vancomycin-treated patients in our hospital. Although the MIC values obtained with this model were lower than those of the diffusion method (Etest) in all three cases, this did not affect the AUC_0–24/MIC ratio for the strains with MICs of 1 mg/L by Etest. However, in those strains with MIC values >1 mg/L, the confidence intervals obtained for this ratio included values <400. The inherent variability of the broth microdilution method could explain the differences in the clinical outcome in MRSA-infected patients treated with vancomycin, mainly in those due to strains with MIC values of 1.5–2 mg/L by Etest, because the corresponding MIC values would range from 0.84 to 1.52 mg/L by the microdilution method, which could affect the therapeutic target.

Outer membrane protein 33 (Omp33) is an outer membrane porin of Acinetobacter baumannii associated with carbapenem resistance. However, the role of Omp33 in the fitness and virulence of A. baumannii remains unknown. In the present study, we investigated the role of Omp33 in fitness and virulence of A. baumannii by using an isogenic knockout strain deficient in the omp33 gene (JPAB02), derived from the ATCC 17978 wild-type (wt). Both in vitro and in vivo defect in the growth rate was found in the JPAB02 strain in competition with the ATCC 17978 wt, highlighting the effect of Omp33 on the metabolic fitness. A significant reduction was observed both in adherence and invasion of human lung epithelial cells and in cytotoxicity of these cells and macrophages with JPAB02. In a murine peritoneal sepsis model, the JPAB02 strain exhibited lower lethal dose 0 (LD₀), LD₅₀, and LD₁₀₀, and dissemination in mice, with reduced bacterial concentration in spleen and lungs. From these data, we concluded that Omp33 plays an important role for fitness and virulence of A. baumannii.

Escherichia coli is commonly involved in infections with a heavy bacterial burden. Piperacillin-tazobactam and carbapenems are among the recommended empirical treatments for health care-associated complicated intra-abdominal infections. In contrast to amoxicillin-clavulanate, both have reduced in vitro activity in the presence of high concentrations of extended-spectrum β-lactamase (ESBL)-producing and non-ESBL-producing E. coli bacteria. Our goal was to compare the efficacy of these antimicrobials against different concentrations of two clinical E. coli strains, one an ESBL-producer and the other a non-ESBL-producer, in a murine sepsis model. An experimental sepsis model {∼5.5 log ₁₀ CFU/g [low inoculum concentration (LI)] or ∼7.5 log ₁₀ CFU/g [high inoculum concentration (HI)]} using E. coli strains ATCC 25922 (non-ESBL producer) and Ec1062 (CTX-M-14 producer), which are susceptible to the three antimicrobials, was used. Amoxicillin-clavulanate (50/12.5 mg/kg given intramuscularly [i.m.]), piperacillin-tazobactam (25/3.125 mg/kg given intraperitoneally [i.p.]), and imipenem (30 mg/kg i.m.) were used. Piperacillin-tazobactam and imipenem reduced spleen ATCC 25922 strain concentrations (−2.53 and −2.14 log ₁₀ CFU/g [ P < 0.05, respectively]) in the HI versus LI groups, while amoxicillin-clavulanate maintained its efficacy (−1.01 log ₁₀ CFU/g [no statistically significant difference]). Regarding the Ec1062 strain, the antimicrobials showed lower efficacy in the HI than in the LI groups: −0.73, −1.89, and −1.62 log ₁₀ CFU/g ( P < 0.05, for piperacillin-tazobactam, imipenem, and amoxicillin-clavulanate, respectively, although imipenem and amoxicillin-clavulanate were more efficacious than piperacillin-tazobactam). An adapted imipenem treatment (based on the time for which the serum drug concentration remained above the MIC obtained with a HI of the ATCC 25922 strain) improved its efficacy to −1.67 log ₁₀ CFU/g ( P < 0.05). These results suggest that amoxicillin-clavulanate could be an alternative to imipenem treatment of infections caused by ESBL- and non-ESBL-producing E. coli strains in patients with therapeutic failure with piperacillin-tazobactam.

The aim of this study was to evaluate the impact of qnrA1, qnrB1 and qnrS1 on the in vivo efficacies of ciprofloxacin and levofloxacin in an experimental model of pneumonia caused by Escherichia coli. Two isogenic groups of E. coli transformants, based on two ATCC 25922 strains, with or without the GyrA mutation Ser83Leu, and carrying qnrA1, qnrB1 or qnrS1, were used in an experimental pneumonia model. The efficacies of ciprofloxacin (40 mg/kg/day) and levofloxacin (50 and 150 mg/kg/day) were evaluated. For the pneumonia caused by the parental strains lacking qnr genes, both fluoroquinolones significantly (P < 0.05) reduced the bacterial lung concentration by >7 log₁₀ cfu/g against E. coli ATCC/pBK and between 5.09 and 6.34 log₁₀ cfu/g against E. coli ATCC-S83L/pBK. The presence of any qnr genes in the strains of both isogenic groups diminished the reduction of bacterial lung concentration with any therapy (P < 0.05). Furthermore, all therapeutic schemes reduced the percentage of positive blood cultures in both isogenic groups (P < 0.05). Finally, the survival results suggest a higher mortality with the strains expressing qnr genes. The presence of qnrA1, qnrB1 and qnrS1 in E. coli reduced the efficacy of ciprofloxacin and levofloxacin in a murine pneumonia model.

The rapid detection of antibiotic resistance in clinical isolates of Acinetobacter baumannii would shorten the period during which patients receive empirical therapy and facilitate the early initiation of directed antibiotic therapy. The objective of this study was to evaluate the ability of a real-time PCR assay to detect antibiotic resistance to four clinically relevant antibiotics from different antibiotic classes in clinical isolates of A. baumannii. The growth of 48 clinical isolates of A. baumannii with a broad range of MICs of imipenem, ciprofloxacin, colistin and amikacin was evaluated using a real-time PCR assay targeting a highly conserved region of the ompA gene. Fold changes in the number of copies of genomic DNA after 6 h of growth were used to determine resistance and the results were compared with those obtained using broth microdilution. The results obtained using the real-time PCR assay were concordant with broth microdilution for 184 of 192 determinations (95.8%). The global values for specificity (97.5%), sensitivity (92.9%), positive predictive value (95.6%) and negative predictive value (96.0%) indicated that the real-time PCR assay was able to reliably differentiate between resistant and non-resistant strains. The use of real-time PCR to monitor bacterial growth in the presence of antibiotics is effective for rapidly identifying antibiotic resistance in A. baumannii.