Impact of the immune response modification by lysophosphatidylcholine in the efficacy of antibiotic therapy of experimental models of peritoneal sepsis and pneumonia by Pseudomonas aeruginosa: LPC therapeutic effect in combined therapy.

INTRODUCTION: Immune response stimulation may be an adjuvant to antimicrobial treatment. Here, we evaluated the impact of immune response modification by lysophosphatidylcholine (LPC), combined with imipenem or ceftazidime, in murine models of peritoneal sepsis (PS) and pneumonia induced by Pseudomonas aeruginosa. METHODS: The imipenem and ceftazidime-susceptible strain (Pa39) and imipenem and ceftazidime-resistant strain (Pa238) were used. Ceftazidime pharmacokinetic and pharmacodynamic parameters were determined. The therapeutic efficacy and TNF-α and IL-10 levels were determined in murine models of PS and pneumonia induced by Pa39 and Pa238 and treated with LPC, imipenem or ceftazidime, alone or in combination. RESULTS: In the PS model, LPC+ceftazidime reduced spleen and lung Pa238 concentrations (-3.45 and -3.56log10CFU/g; P<0.05) to a greater extent than ceftazidime monotherapy, while LPC+imipenem maintained the imipenem efficacy (-1.66 and -1.45log10CFU/g; P>0.05). In the pneumonia model, LPC+ceftazidime or LPC+imipenem reduced the lung Pa238 concentrations (-2.37log10CFU/g, P=0.1, or -1.35log10CFU/g, P=0.75). For Pa39, no statistically significant difference was observed in the PS and pneumonia models between combined therapy and monotherapy. Moreover, LPC+imipenem and LPC+ceftazidime significantly decreased and increased the TNF-α and IL-10 levels, respectively, in comparison with the untreated controls and monotherapies. CONCLUSIONS: These results demonstrate the impact of immune response modification by LPC plus antibiotics on the prognosis of infections induced by ceftazidime-resistant P. aeruginosa.

Impact of the immune response modification by lysophosphatidylcholine in the efficacy of antibiotic therapy of experimental models of peritoneal sepsis and pneumonia by Pseudomonas aeruginosa: LPC therapeutic effect in combined therapy.

INTRODUCTION: Immune response stimulation may be an adjuvant to antimicrobial treatment. Here, we evaluated the impact of immune response modification by lysophosphatidylcholine (LPC), combined with imipenem or ceftazidime, in murine models of peritoneal sepsis (PS) and pneumonia induced by Pseudomonas aeruginosa. METHODS: The imipenem and ceftazidime-susceptible strain (Pa39) and imipenem and ceftazidime-resistant strain (Pa238) were used. Ceftazidime pharmacokinetic and pharmacodynamic parameters were determined. The therapeutic efficacy and TNF-α and IL-10 levels were determined in murine models of PS and pneumonia induced by Pa39 and Pa238 and treated with LPC, imipenem or ceftazidime, alone or in combination. RESULTS: In the PS model, LPC+ceftazidime reduced spleen and lung Pa238 concentrations (-3.45 and -3.56log10CFU/g; P<0.05) to a greater extent than ceftazidime monotherapy, while LPC+imipenem maintained the imipenem efficacy (-1.66 and -1.45log10CFU/g; P>0.05). In the pneumonia model, LPC+ceftazidime or LPC+imipenem reduced the lung Pa238 concentrations (-2.37log10CFU/g, P=0.1, or -1.35log10CFU/g, P=0.75). For Pa39, no statistically significant difference was observed in the PS and pneumonia models between combined therapy and monotherapy. Moreover, LPC+imipenem and LPC+ceftazidime significantly decreased and increased the TNF-α and IL-10 levels, respectively, in comparison with the untreated controls and monotherapies. CONCLUSIONS: These results demonstrate the impact of immune response modification by LPC plus antibiotics on the prognosis of infections induced by ceftazidime-resistant P. aeruginosa.

Efficacy of ceftaroline versus vancomycin in an experimental foreign-body and systemic infection model caused by biofilm-producing methicillin-resistant Staphylococcus epidermidis.

In this study, the efficacy of ceftaroline versus vancomycin against biofilm-producing methicillin-resistant Staphylococcus epidermidis (MRSE) in a murine model of foreign-body and systemic infection was compared. Two bacteraemic biofilm-producing MRSE strains were used (SE284 and SE385). The minimum inhibitory concentrations (MICs) for strains SE284 and SE385, were, respectively, 0.25 mg/L and 0.5 mg/L for ceftaroline and 4 mg/L and 2 mg/L for vancomycin. The in vitro bactericidal activities of ceftaroline and vancomycin were evaluated using time-kill curves. A foreign-body and systemic infection model in neutropenic female C57BL/6 mice was used to ascertain in vivo efficacy. Animals were randomly allocated into three groups (n = 15) without treatment (controls) or treated with ceftaroline 50 mg/kg every 8 h or vancomycin 110 mg/kg every 6 h. In vitro, ceftaroline showed concentration-dependent bactericidal activity, whilst vancomycin presented time-dependent activity. In the experimental in vivo model, ceftaroline and vancomycin decreased the liver and catheter bacterial concentrations (P <0.05) and increased survival (P <0.05) for both strains. In conclusion, ceftaroline is as effective as vancomycin in the treatment of experimental foreign-body and systemic infection caused by biofilm-producing MRSE.

Therapeutic efficacy of lysophosphatidylcholine in severe infections caused by Acinetobacter baumannii.

Due to the significant increase in antimicrobial resistance of Acinetobacter 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 by A. baumannii. A. baumannii ATCC 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-1ß, 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 of A. baumannii. The therapeutic efficacy of LPC against A. baumannii in 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 by A. 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 by A. baumannii.

Determining accurate vancomycin MIC values for methicillin-resistant Staphylococcus aureus by the microdilution method.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Association of the outer membrane protein Omp33 with fitness and virulence of Acinetobacter baumannii.

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.

Colistin resistance in a clinical Acinetobacter baumannii strain appearing after colistin treatment: effect on virulence and bacterial fitness.

The fitness and virulence costs associated with the clinical acquisition of colistin resistance by Acinetobacter baumannii were evaluated. The growth of strain CR17 (colistin resistant) was less than that of strain CS01 (colistin susceptible) when the strains were grown in competition (72-h competition index, 0.008). In a murine sepsis model, CS01 and CR17 reached spleen concentrations when coinfecting of 9.31 and 6.97 log10 CFU/g, respectively, with an in vivo competition index of 0.016. Moreover, CS01 was more virulent than CR17 with respect to mortality and time to death.

Rapid detection of antibiotic resistance in Acinetobacter baumannii using quantitative real-time PCR.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Platelet-activating factor receptor initiates contact of Acinetobacter baumannii expressing phosphorylcholine with host cells.

Adhesion is an initial and important step in Acinetobacter baumannii causing infections. However, the exact molecular mechanism of such a step between A. baumannii and the host cells remains unclear. Here, we demonstrated that the phosphorylcholine (ChoP)-containing outer membrane protein of A. baumannii binds to A549 cells through platelet-activating factor receptor (PAFR), resulting in activation of G protein and intracellular calcium. Upon A. baumannii expressing ChoP binding to PAFR, clathrin and ß-arrestins, proteins involved in the direction of the vacuolar movement, are activated during invasion of A. baumannii. PAFR antagonism restricts the dissemination of A. baumannii in the pneumonia model. These results define a role for PAFR in A. baumannii interaction with host cells and suggest a mechanism for the entry of A. baumannii into the cytoplasm of host cells.

Efficacy of linezolid versus a pharmacodynamically optimized vancomycin therapy in an experimental pneumonia model caused by methicillin-resistant Staphylococcus aureus.

OBJECTIVES: The British Thoracic Society, American Thoracic Society and Infectious Diseases Society of America guidelines recommend vancomycin for methicillin-resistant Staphylococcus aureus (MRSA) pneumonia, based on evidence suggesting that a vancomycin AUC0₋24/MIC ratio of 400 predicts clinical success against MRSA pneumonia. The aim of this study was the evaluation of an optimized dose of vancomycin in the treatment of MRSA experimental pneumonia versus linezolid. METHODS: In vitro activities of vancomycin and linezolid were tested using time-kill curves. Experimental pneumonia in neutropenic C57BL/6 mice was achieved using two clinical MRSA strains, MR30 and MR33 (vancomycin and linezolid MICs of 1 and 4 mg/L, respectively). In vivo dosages were 30 and 110 mg/kg vancomycin (obtaining an AUC0₋24/MIC ratio lower and higher than 400, respectively), and 30 mg/kg linezolid. RESULTS: Survival rates in controls, and in the groups treated with 120 mg/kg/day vancomycin, 440 mg/kg/day vancomycin and 120 mg/kg/day linezolid were 85.7%, 92.9%, 76.9% and 100%, and 66.7%, 100%, 75% and 100% for MR30 and MR33, respectively. Sterile blood cultures occurred at rates of 21.4%, 64.3%, 100% and 93.8%, and 40%, 66.7%, 100% and 93.3% for MR30 and MR33 strains, respectively. Finally, the respective bacterial lung concentrations (log10 cfu/g) were 8.93 ±â€Š0.78, 6.67 ±â€Š3.01, 3.25 ±â€Š1.59 and 2.87 ±â€Š1.86 for MR30, and 8.62 ±â€Š0.72, 5.76 ±â€Š2.43, 3.97 ±â€Š1.52 and 1.59 ±â€Š1.40 for MR33. CONCLUSIONS: These results support that a vancomycin AUC0₋24/MIC ratio >400 is necessary to obtain a high bacterial lung reduction in MRSA pneumonia, comparable to that achieved with linezolid and better than that with the low dose of vancomycin tested. Linezolid was more efficacious than the pharmacodynamically optimized vancomycin dose in the pneumonia caused by the most virulent strain (MR33).

Efficacies of colistin and tigecycline in mice with experimental pneumonia due to NDM-1-producing strains of Klebsiella pneumoniae and Escherichia coli.

New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Enterobacteriaceae have emerged as a global threat. The aim of this study was to assess the efficacies of colistin and tigecycline in an experimental model of pneumonia caused by NDM-1-producing Escherichia coli and Klebsiella pneumoniae. The susceptibilities of K. pneumoniae NDM, E. coli NDM and K. pneumoniae ATCC 29665 were determined using the broth microdilution technique. The pharmacokinetics of colistin and tigecycline in an experimental model of pneumonia were performed using immunocompetent C57BL/6 mice. Mice were treated with colistin (60 mg/kg/day) or tigecycline (10 mg/kg/day). Mortality, bacteraemia and lung bacterial concentrations were recorded. The strains were susceptible to colistin and tigecycline. The ratio of area under the concentration-time curve/minimum inhibitory concentration (AUC/MIC) for colistin was 158.5 (all three strains) and that for tigecycline was 18.5 (K. pneumoniae NDM) and 37 (K. pneumoniae ATCC 29665 and E. coli NDM). In vivo, colistin decreased bacterial lung concentrations of K. pneumoniae NDM and K. pneumoniae ATCC 29665 by 1.16 log colony-forming units (CFU)/g and 2.23 logCFU/g, respectively, compared with controls (not significant). Tigecycline reduced K. pneumoniae NDM and K. pneumoniae ATCC 29665 load by 2.67 logCFU/g and 4.62 logCFU/g (P<0.05). Colistin and tigecycline decreased lung concentrations of E. coli NDM by 2.27 logCFU/g and 4.15 logCFU/g (P<0.05), respectively, compared with controls, and was more active than colistin (P<0.05). In conclusion, these results suggest that colistin is inappropriate for treating pneumonia due to NDM-1-producing K. pneumoniae and its efficacy was suboptimal against NDM-1-producing E. coli. A high tigecycline dose was efficacious for treating experimental pneumonia due to NDM-1-producing E. coli and K. pneumoniae.

Rifampin protects human lung epithelial cells against cytotoxicity induced by clinical multi and pandrug-resistant Acinetobacter baumannii.

OBJECTIVE: Recently, it has become apparent that rifampin can act on eukaryotic cells modulating production of host mediators. We aimed to study the cytoprotective effect of rifampin against multidrug- and pandrug-resistant Acinetobacter baumannii-induced cell death using human lung epithelial cells. METHODS: We pretreated A549 cells with rifampin and infected them with 3 different A. baumannii strains (susceptible, multidrug-resistant, and pandrug-resistant) that induce cell death. Cellular viability, apoptosis and host mediators, free radicals, and proinflammatory cytokines associated with A. baumannii pathogenesis were studied. Moreover, bacterial concentrations in A549 cells culture were determined. RESULTS: Rifampin-pretreated A549 cells demonstrated decreases in apoptosis and cell death induced by A. baumannii. The oxidative stress and proinflammatory responses to A. baumannii were reduced in rifampin-pretreated A549 cells, as shown by decreased superoxide anion, tumor necrosis factor-α, and interleukin-6. Furthermore, bacterial count performed in A549 cell culture medium showed that rifampin did not reduce significantly the bacterial concentrations. CONCLUSION: These data demonstrate that rifampin is able to attenuate the cellular damage induced by multidrug- and pandrug-resistant A. baumannii clinical isolates without being relevantly bactericidal. Indeed, the cytoprotective effect of rifampin was observed on the decrease of dead cells induced by A. baumannii by reducing oxidative stress and proinflammatory cytokines release.

Impaired virulence and in vivo fitness of colistin-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.

Vaccination with outer membrane complexes elicits rapid protective immunity to multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii causes pneumonias, bacteremias, and skin and soft tissue infections, primarily in the hospitalized setting. The incidence of infections caused by A. baumannii has increased dramatically over the last 30 years, while at the same time the treatment of these infections has been complicated by the emergence of antibiotic-resistant strains. Despite these trends, no vaccines or antibody-based therapies have been developed for the prevention of A. baumannii infection. In this study, an outer membrane complex vaccine consisting of multiple surface antigens from the bacterial membrane of A. baumannii was developed and tested in a murine sepsis model. Immunization elicited humoral and cellular responses that were able to reduce postinfection bacterial loads, reduce postinfection proinflammatory cytokine levels in serum, and protect mice from infection with human clinical isolates of A. baumannii. A single administration of the vaccine was able to elicit protective immunity in as few as 6 days postimmunization. In addition, vaccine antiserum was used successfully to therapeutically rescue naïve mice with established infection. These results indicate that prophylactic vaccination and antibody-based therapies based on an outer membrane complex vaccine may be viable approaches to preventing the morbidity and mortality caused by this pathogen.

Efficacy of rifampin and its combinations with imipenem, sulbactam, and colistin in experimental models of infection caused by imipenem-resistant Acinetobacter baumannii.

There are currently no defined optimal therapies available for multidrug-resistant (MDR) Acinetobacter baumannii infections. We evaluated the efficacy of rifampin, imipenem, sulbactam, colistin, and their combinations against MDR A. baumannii in experimental pneumonia and meningitis models. The bactericidal in vitro activities of rifampin, imipenem, sulbactam, colistin, and their combinations were tested using time-kill curves. Murine pneumonia and rabbit meningitis models were evaluated using the A. baummnnii strain Ab1327 (with MICs for rifampin, imipenem, sulbactam, and colistin of 4, 32, 32, and 0.5 mg/liter, respectively). Mice were treated with the four antimicrobials and their combinations. For the meningitis model, the efficacies of colistin, rifampin and its combinations with imipenem, sulbactam, or colistin, and of imipenem plus sulbactam were assayed. In the pneumonia model, compared to the control group, (i) rifampin alone, (ii) rifampin along with imipenem, sulbactam, or colistin, (iii) colistin, or (iv) imipenem plus sulbactam significantly reduced lung bacterial concentrations (10.6 +/- 0.27 [controls] versus 3.05 +/- 1.91, 2.07 +/- 1.82, 2.41 +/- 1.37, 3.4 +/- 3.07, 6.82 +/- 3.4, and 4.22 +/- 2.72 log(10) CFU/g, respectively [means +/- standard deviations]), increased sterile blood cultures (0% versus 78.6%, 100%, 93.3%, 93.8%, 73.3%, and 50%), and improved survival (0% versus 71.4%, 60%, 46.7%, 43.8%, 40%, and 85.7%). In the meningitis model rifampin alone or rifampin plus colistin reduced cerebrospinal fluid bacterial counts (-2.6 and -4.4 log(10) CFU/ml). Rifampin in monotherapy or with imipenem, sulbactam, or colistin showed efficacy against MDR A. baumannii in experimental models of pneumonia and meningitis. Imipenem or sulbactam may be appropriate for combined treatment when using rifampin.

In vitro activity and in vivo efficacy of clavulanic acid against Acinetobacter baumannii.

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 log10 CFU/g (P < or = 0.001), respectively, in the experiments with Ab11 and 2.42 and 2.28 log10 CFU/g (P < or = 0.001), respectively, with Ab51. IPM significantly increased the sterility of blood cultures over that for the controls with both strains (P < or = 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.