Activity of colistin combined with doripenem at clinically relevant concentrations against multidrug-resistant Pseudomonas aeruginosa in an in vitro dynamic biofilm model.

OBJECTIVES: Colistin combination therapy may be required to treat biofilm-associated infections. We evaluated bacterial killing and emergence of colistin resistance with colistin and doripenem combinations against biofilm-embedded and planktonic multidrug-resistant (MDR) Pseudomonas aeruginosa. METHODS: One colistin-susceptible reference strain (PAO1) and two colistin-susceptible MDR clinical isolates (HUB1 and HUB2; both carbapenem resistant) were investigated over 72 h in the CDC biofilm reactor, a dynamic biofilm model. Two colistin regimens (constant concentrations of 1.25 and 3.50 mg/L), one doripenem regimen (Cmax 25 mg/L 8 hourly) and their combination were employed. Microbiological response was examined as log changes and absolute bacterial counts. RESULTS: For biofilm-embedded bacteria, bactericidal activity was only observed with monotherapy with colistin at 3.50 mg/L. The emergence of colistin resistance occurred with colistin monotherapy against two strains (PAO1 and HUB1), but only with the colistin 3.50 mg/L regimen. Colistin 3.50 mg/L plus doripenem resulted in ∼2-3 log10 cfu/cm(2) initial killing against both clinical isolates and remained synergistic at 72 h. The emergence of colistin resistance was not observed in biofilm-embedded bacteria with either combination. For planktonic bacteria, bactericidal activity was not observed with any monotherapy regimen, although enhanced bacterial killing was observed with doripenem plus colistin 3.50 mg/L against all isolates. Colistin resistance was observed with colistin monotherapy against two isolates, but did not emerge with combination regimens. CONCLUSIONS: Doripenem enhanced killing by colistin of biofilm-embedded cells in both carbapenem-susceptible and -resistant strains, and the combination minimized the emergence of colistin resistance.

Polymyxin B Induces Apoptosis in Kidney Proximal Tubular Cells.

The nephrotoxicity of polymyxins is a major dose-limiting factor for treatment of infections caused by multidrug-resistant Gram-negative pathogens. The mechanism(s) of polymyxin-induced nephrotoxicity is not clear. This study aimed to investigate polymyxin B-induced apoptosis in kidney proximal tubular cells. Polymyxin B-induced apoptosis in NRK-52E cells was examined by caspase activation, DNA breakage, and translocation of membrane phosphatidylserine using Red-VAD-FMK [Val-Ala-Asp(O-Me) fluoromethyl ketone] staining, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and double staining with annexin V-propidium iodide (PI). The concentration dependence (50% effective concentration [EC50]) and time course for polymyxin B-induced apoptosis were measured in NRK-52E and HK-2 cells by fluorescence-activated cell sorting (FACS) with annexin V and PI. Polymyxin B-induced apoptosis in NRK-52E cells was confirmed by positive labeling from Red-VAD-FMK staining, TUNEL assay, and annexin V-PI double staining. The EC50 (95% confidence interval [CI]) of polymyxin B for the NRK-52E cells was 1.05 (0.91 to 1.22) mM and was 0.35 (0.29 to 0.42) mM for HK-2 cells. At lower concentrations of polymyxin B, minimal apoptosis was observed, followed by a sharp rise in the apoptotic index at higher concentrations in both cell lines. After treatment of NRK-52E cells with 2.0 mM polymyxin B, the percentage of apoptotic cells (mean ± standard deviation [SD]) was 10.9% ± 4.69% at 6 h and reached plateau (>80%) at 24 h, whereas treatment with 0.5 mM polymyxin B for 24 h led to 93.6% ± 5.57% of HK-2 cells in apoptosis. Understanding the mechanism of polymyxin B-induced apoptosis will provide important information for discovering less nephrotoxic polymyxin-like lipopeptides.

The combination of colistin and doripenem is synergistic against Klebsiella pneumoniae at multiple inocula and suppresses colistin resistance in an in vitro pharmacokinetic/pharmacodynamic model.

Multidrug-resistant (MDR) Klebsiella pneumoniae may require combination therapy. We systematically investigated bacterial killing with colistin and doripenem mono- and combination therapy against MDR K. pneumoniae and emergence of colistin resistance. A one-compartment in vitro pharmacokinetic/pharmacodynamic model was employed over a 72-h period with two inocula (∼10(6) and ∼10(8) CFU/ml); a colistin-heteroresistant reference strain (ATCC 13883) and three clinical isolates (colistin-susceptible FADDI-KP032 [doripenem resistant], colistin-heteroresistant FADDI-KP033, and colistin-resistant FADDI-KP035) were included. Four combinations utilizing clinically achievable concentrations were investigated. Microbiological responses were examined by determining log changes and population analysis profiles (for emergence of colistin resistance) over 72 h. Against colistin-susceptible and -heteroresistant isolates, combinations of colistin (constant concentration regimens of 0.5 or 2 mg/liter) plus doripenem (steady-state peak concentration [C(max)] of 2.5 or 25 mg/liter over 8 h; half-life, 1.5 h) generally resulted in substantial improvements in bacterial killing at both inocula. Combinations were additive or synergistic against ATCC 13883, FADDI-KP032, and FADDI-KP033 in 9, 9, and 14 of 16 cases (4 combinations at 6, 24, 48, and 72 h) at the 10(6)-CFU/ml inoculum and 14, 11, and 12 of 16 cases at the 10(8)-CFU/ml inoculum, respectively. Combinations at the highest dosage regimens resulted in undetectable bacterial counts at 72 h in 5 of 8 cases (4 isolates at 2 inocula). Emergence of colistin-resistant subpopulations in colistin-susceptible and -heteroresistant isolates was virtually eliminated with combination therapy. Against the colistin-resistant isolate, colistin at 2 mg/liter plus doripenem (C(max), 25 mg/liter) at the low inoculum improved bacterial killing. This investigation provides important information for optimization of colistin-doripenem combinations.

A novel polymyxin derivative that lacks the fatty acid tail and carries only three positive charges has strong synergism with agents excluded by the intact outer membrane.

Polymyxins are cationic lipopeptides (five cationic charges) and the last resort for the treatment of serious Gram-negative infections caused by multiresistant strains. NAB741 has a cyclic peptide portion identical to that of polymyxin B but carries in the linear peptide portion a threonyl-D-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). At the N terminus of the peptide, NAB741 carries an acetyl group instead of a mixture of methyl octanoyl and methyl heptanoyl residues. NAB741 sensitized Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Acinetobacter baumannii to antibiotics against which the intact outer membrane is an effective permeability barrier. When tested by using Etest strips on plates containing increasing concentrations of NAB741, the fractional inhibition concentration index (FICI) of the combination of NAB741 with rifampin ranged from

Influence of tissue fixation on depth-resolved birefringence of oral cavity tissue samples.

SIGNIFICANCE: To advance our understanding of the contrast observed when imaging with polarization-sensitive optical coherence tomography (PS-OCT) and its correlation with oral cancerous pathologies, a detailed comparison with histology provided via ex vivo fixed tissue is required. The effects of tissue fixation, however, on such polarization-based contrast have not yet been investigated. AIM: A study was performed to assess the impact of tissue fixation on depth-resolved (i.e., local) birefringence measured with PS-OCT. APPROACH: A PS-OCT system based on depth-encoded polarization multiplexing and polarization-diverse detection was used to measure the Jones matrix of a sample. A wide variety of ex vivo samples were measured freshly after excision and 24 h after fixation, consistent with standard pathology. Some samples were also measured 48 h after fixation. RESULTS: The tissue fixation does not diminish the birefringence contrast. Statistically significant changes were observed in 11 out of 12 samples; these changes represented an increase in contrast, overall, by 11% on average. CONCLUSIONS: We conclude that the fixed samples are suitable for studies seeking a deeper understanding of birefringence contrast in oral tissue pathology. The enhancement of contrast removes the need to image immediately postexcision and will facilitate future investigations with PS-OCT and other advanced polarization-sensitive microscopy methods, such as mapping of the local optic axis with PS-OCT and PS-optical coherence microscopy.

Pharmacokinetics of novel antimicrobial cationic peptides NAB 7061 and NAB 739 in rats following intravenous administration.

OBJECTIVES: To determine the disposition of novel antimicrobial cationic peptides NAB 7061 and NAB 739 following intravenous administration in rats. METHODS: Sprague-Dawley rats received a single intravenous bolus of 1.0 mg/kg NAB 7061 or NAB 739. Plasma concentrations of NAB 7061 or NAB 739 were determined by HPLC or liquid chromatography-mass spectrometry. The pharmacokinetic parameters of NAB 7061 and NAB 739 were calculated using non-compartmental analysis. RESULTS: Corresponding total body clearance, volume of distribution at steady state and terminal half-life of NAB 7061 and NAB 739 averaged 3.84 and 2.63 mL/min/kg, 339 and 222 mL/kg, and 66.2 and 69.0 min, respectively. Approximately 7.16% and 19.4% of the dose was eliminated in an unchanged form via the urine in 24 h for NAB 7061 and NAB 739, respectively. CONCLUSIONS: While both compounds had generally similar pharmacokinetics to colistin, even minor alterations in the chemical structures appear to have an impact on their pharmacokinetics, especially on their clearance by the kidney. There are also substantial differences in relation to the relative contributions of renal and non-renal clearance to overall elimination from the body.

In vitro pharmacodynamics of colistin against multidrug-resistant Klebsiella pneumoniae.

BACKGROUND: Resistance to colistin is emerging in multidrug-resistant Gram-negative bacteria and no solid pharmacodynamic data are available for colistin against Klebsiella pneumoniae. METHODS: Twenty-one multidrug-resistant clinical K. pneumoniae isolates from 16 different clinical sites worldwide were employed. The genetic relatedness of these isolates was examined with PFGE. In vitro pharmacodynamic properties of colistin (sulphate) were investigated by studying the MICs, mutation prevention concentrations, time-kill kinetics, population analysis profiles and the post-antibiotic effect (PAE). Time-kill was studied with three clinical isolates plus ATCC 13883 at concentrations ranging from 0.5 to 64x MIC. The PAE was examined after 20 min of exposure of these isolates. RESULTS: The 22 isolates belonged to 18 different PFGE groups. For susceptible isolates, colistin MICs ranged from 0.125 to 1 mg/L. Six isolates were colistin-resistant with MICs of >/=32 mg/L. Colistin heteroresistance was observed in 15 of 16 isolates considered colistin-susceptible based on MICs. For susceptible isolates, colistin showed extremely rapid killing; however, regrowth was observed as early as 2 h after treatment and substantial regrowth at 24 h even at concentrations up to 64x MIC for some isolates. Colistin exhibited no or very modest PAE against the isolates tested. CONCLUSIONS: The data suggest that monotherapy with colistin methanesulfonate, the parenteral form of colistin, and long dosage intervals may be problematic for the treatment of infections caused by multidrug-resistant K. pneumoniae, particularly for colistin-heteroresistant strains. Further investigation on combination therapy of colistin with other antibiotics is warranted.

Colistin hetero-resistance in multidrug-resistant Acinetobacter baumannii clinical isolates from the Western Pacific region in the SENTRY antimicrobial surveillance programme.

BACKGROUND: Multidrug-resistant Acinetobacter baumannii has presented a global medical problem. Emergence of colistin resistance, including hetero-resistance, has been increasingly reported. This study examined the susceptibility to colistin of multidrug-resistant A. baumannii from the Western Pacific region. METHODS: A total of 30 isolates were studied from 10 clinical centres in various countries. MICs were measured against 21 antibiotics by broth microdilution. Colistin population analysis profiles (PAPs) (0, 0.5, 1, 2, 3, 4, 5, 6, 8 and 10mg/L) were determined. Time-kill kinetics of colistin against 3 isolates (2 colistin-susceptible (one of which was colistin hetero-resistant) and 1 colistin-resistant) were studied over a wide range of concentrations and development of resistance was monitored by measurement of colistin PAPs after 24-h exposure. RESULTS: All the isolates were highly multiresistant. Colistin MICs were 0.5-2mg/L except one isolate which had an MIC of 128mg/L. Seven isolates were colistin hetero-resistant with subpopulations growing at >2mg/L. For the 2 colistin-susceptible isolates examined, >3log killing was observed within 3h even at 0.5x MIC. Interestingly, >3log killing was also observed with the colistin-resistant isolate within 1h even at 0.5mg/L. Regrowth occurred at 24h for both colistin-susceptible and -resistant isolates. Emergence of resistance to colistin after 24-h exposure was confirmed by PAP. CONCLUSION: Colistin was very active against A. baumannii based upon MICs and initial killing in time-kill studies. Hetero-resistance in this group of A. baumannii isolates was less common than in previous studies. Nevertheless, care is required with colistin monotherapy for A. baumannii infections.