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1.  Pharmacodynamic Variability beyond That Explained by MICs 
Monte Carlo simulations (MCS) present a powerful tool to evaluate candidate regimens by determining the probability of target attainment. Although these assessments have traditionally incorporated variability in pharmacokinetic (PK) parameters and MICs, consideration of interstrain pharmacodynamic (PD) variability has been neglected. A population PK/PD model was developed for doripenem using murine thigh infection data based on 20 bacterial strains. PK data were fit to a linear two-compartment model with first-order input and elimination processes and an absorption lag time from a separate site (r2 > 0.96). PK parameters were utilized to simulate free-drug profiles for various regimens in PD studies, from which the percentage of the dosing interval for which free-drug concentrations exceed the MIC of the targeted strain (%fT>MIC) was calculated. Doripenem PD was excellently described with Hill-type models (r2 > 0.98); significant differences between mean PD estimates determined using a two-stage approach versus population analyses were not observed (P > 0.05); however, the variance in 50% effective concentration (EC50) and maximum effect (Emax) among strains was much greater using the two-stage approach. Even using the population approach, interstrain variability in EC50 (coefficient of variation expressed as a percentage [CV%] = 29.2%) and H (CV% = 46.1%) parameters was substantive, while the variability in Emax (CV% = 19.7%) was modest. This resulted in extensive variability in the range of %fT>MIC targets associated with stasis to those associated with a 2-log10 reduction in bacterial burden (CV% ∼ 50%). It appears that MCS, based on the assumption that PD variability is due to MIC alone, underestimates variability and may consequently underestimate treatment failures.
PMCID: PMC3623339  PMID: 23357773
2.  Molecular basis for the increased polymyxin susceptibility of Klebsiella pneumoniae strains with under-acylated lipid A 
Innate immunity  2012;19(3):265-277.
The impact of under-acylation of lipid A on the interaction between Klebsiella pneumoniae LPS and polymyxins B and E was examined with fluorometric and calorimetric methods, and by 1H NMR, using a paired wild type (WT) and the Δ6lpxM mutant strains B5055 and B5055 ΔlpxM, which predominantly express LPS with hexa- and penta-acylated lipid A structures respectively. LPS from B5055 ΔlpxM displayed a fourfold increased binding affinity for polymyxins B and E compared with the B5055 WT LPS. EC50 values were consistent with polymyxin minimum inhibitory concentration (MIC) values for each strain. Accordingly, polymyxin exposure considerably enhanced the permeability of the B5055 ΔlpxM OM. Analysis of the melting profiles of isolated LPS aggregates suggested that bactericidal polymyxin activity may relate to the acyl chains’ phase of the outer membrane (OM). The enhanced polymyxin susceptibility of B5055 Δ6lpxM may be attributable to the favorable insertion of polymyxins into the more fluid OM compared with B5055. Molecular models of the polymyxin B–lipid A complex illuminate the key role of the lipid A acyl chains for complexation of polymyxin. The data provide important insight into the molecular basis for the increased polymyxin susceptibility of K. pneumoniae strains with under-acylated lipid A. Under-acylation appears to facilitate the integration of the N-terminal fatty-acyl chain of polymyxin into the OM resulting in an increased susceptibility to its antimicrobial activity/activities.
PMCID: PMC4242410  PMID: 23008349
Polymyxin; Klebsiella pneumoniae; lipopolysaccharide
3.  Cell surface hydrophobicity of colistin-susceptible versus -resistant Acinetobacter baumannii determined by contact angles: methodological considerations and implications 
Journal of applied microbiology  2012;113(4):940-951.
Contact angle analysis of cell surface hydrophobicity (CSH) describes the tendency of a water droplet to spread across a lawn of filtered bacterial cells. Colistin-induced disruption of the Gram-negative outer membrane necessitates hydrophobic contacts with lipopolysaccharide (LPS). We aimed to characterize the CSH of Acinetobacter baumannii using contact angles, to provide insight into the mechanism of colistin resistance.
Contact angles were analysed for five paired colistin-susceptible and -resistant A. baumannii strains. Drainage of the water droplet through bacterial layers was demonstrated to influence results. Consequently, measurements were performed 0.66-sec after droplet deposition. Colistin-resistant cells exhibited lower contact angles (38.8±2.8° to 46.8±1.3°) compared to their paired-susceptible strains (40.7±3.0° to 48.0±1.4°; ANOVA; p<0.05). Contact angles increased at stationary phase (50.3±2.9° to 61.5±2.5° and 47.4±2.0° to 50.8±3.2°, susceptible and resistant, respectively, ANOVA; p<0.05), and in response to colistin 32-mgL−1 exposure (44.5±1.5° to 50.6±2.8° and 43.5±2.2° to 48.0±2.2°, susceptible and resistant, respectively; ANOVA; p<0.05). Analysis of complemented strains constructed with an intact lpxA gene, or empty vector, highlighted the contribution of LPS to CSH.
Compositional outer-membrane variations likely account for CSH differences between A. baumannii phenotypes, which influence the hydrophobic colistin-bacterium interaction.
Important insight into the mechanism of colistin resistance has been provided. Greater consideration of contact angle mehodology is nescessary to ensure accurate analyses are performed.
PMCID: PMC3434258  PMID: 22574702
Antimicrobials; Lipopolysaccharide; Mechanism of Action
4.  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 
Antimicrobial Agents and Chemotherapy  2012;56(10):5103-5112.
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 (∼106 and ∼108 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 [Cmax] 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 106-CFU/ml inoculum and 14, 11, and 12 of 16 cases at the 108-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 (Cmax, 25 mg/liter) at the low inoculum improved bacterial killing. This investigation provides important information for optimization of colistin-doripenem combinations.
PMCID: PMC3457376  PMID: 22802247
5.  Effect of colistin exposure and growth phase on the surface properties of live Acinetobacter baumannii cells examined by atomic force microscopy 
The diminishing antimicrobial development pipeline has forced the revival of colistin as a last line of defence against infections caused by multidrug-resistant Gram-negative ‘superbugs’ such as Acinetobacter baumannii. The complete loss of lipopolysaccharide (LPS) mediates colistin resistance in some A. baumannii strains. Atomic force microscopy was used to examine the surface properties of colistin-susceptible and -resistant A. baumannii strains at mid-logarithmic and stationary growth phases in liquid and in response to colistin treatment. The contribution of LPS to surface properties was investigated using A. baumannii strains constructed with and without the lpxA gene. Bacterial spring constant measurements revealed that colistin-susceptible cells were significantly stiffer than colistin-resistant cells at both growth phases (P < 0.01), whilst colistin treatment at high concentrations (32 mg/L) resulted in more rigid surfaces for both phenotypes. Multiple, large adhesive peaks frequently noted in force curves captured on colistin-susceptible cells were not evident for colistin-resistant cells. Adhesion events were markedly reduced following colistin exposure. The cell membranes of strains of both phenotypes remained intact following colistin treatment, although fine topographical details were illustrated. These studies, conducted for the first time on live A. baumannii cells in liquid, have contributed to our understanding of the action of colistin in this problematic pathogen.
PMCID: PMC3433558  PMID: 21925844
Atomic force microscopy; Colistin; Acinetobacter baumannii; Morphology; Surface properties
6.  Design, synthesis and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions 
Analytical biochemistry  2010;409(2):273-283.
Fluorescence assays employing semi-synthetic or commercial dansyl-polymyxin B, have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric-acid residues of polymyxin B are potentially derivatized with dansyl-cholride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra- dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]1polymyxinB3. The affinity of polymyxin for purified Gram-negative LPS, and whole bacterial cells was investigated. The affinity of dansyl[Lys]1polymyxinB3 for LPS was comparable to polymyxin B and colistin, and considerably greater (kd < 1 μM) than for whole cells (kd ~6 to 12 μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]1polymyxinB3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]1polymyxinB3-LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]1polymyxinB3-LPS complex due to steric hindrance from the dansyl[Lys]1 fluorophore; this corresponded with diminished antibacterial activity (MIC ≥ 16 μg/mL). Dansyl[Lys]1polymyxinB3 may prove useful as a screening tool for drug development.
PMCID: PMC3106281  PMID: 21050838
dansyl-polymyxin B; Gram-negative bacteria; binding affinity; polymyxin; colistin; lipopolysaccharide
7.  Different surface charge of colistin-susceptible and -resistant Acinetobacter baumannii cells measured with zeta potential as a function of growth phase and colistin treatment 
Electrostatic forces mediate the initial interaction between cationic colistin and Gram-negative bacterial cells. Lipopolysaccharide (LPS) loss mediates colistin resistance in some A. baumannii strains. Our aim was to determine the surface charge of colistin-susceptible and –resistant A. baumannii as a function of growth phase and in response to polymyxin treatment.
The zeta potential of A. baumannii ATCC 19606 and 10 clinical multidrug-resistant strains (MICs 0.5–2 mg/L) was assessed. Colistin-resistant derivatives (MIC >128 mg/L) of wild-type strains were selected in the presence of 10 mg/L colistin, including the LPS-deficient lpxA mutant, ATCC 19606R. To determine the contribution of LPS to surface charge, two complemented ATCC 19606R derivatives were examined, namely ATCC 19606R + lpxA (containing an intact lpxA gene) and ATCC 19606R + V (containing empty vector). Investigations were conducted as a function of growth phase and polymyxin treatment (1, 4 and 8 mg/L).
Wild-type cells exhibited a greater negative charge (−60.5 ± 2.36 to −26.2 ± 2.56 mV) thancolistin-resistant cells (−49.2 ± 3.09 to −19.1 ± 2.80 mV) at mid-log phase (ANOVA, P < 0.05). Opposing growth-phase trends were observed for both phenotypes: wild-type cells displayed reduced negative charge and colistin-resistant cells displayed increased negative charge at stationary compared with mid-logarithmic phase. Polymyxin exposure resulted in a concentration-dependent increase in zeta potential. Examination of ATCC 19606R and complemented strains supported the importance of LPS in determining surface charge, suggesting a potential mechanism of colistin resistance.
Zeta potential differences between A. baumannii phenotypes probably reflect compositional outer-membrane variations that impact the electrostatic component of colistin activity.
PMCID: PMC3001852  PMID: 21081544
physicochemical properties; Gram-negative; polymyxin
8.  Atomic Force Microscopy Investigation of the Morphology and Topography of Colistin-Heteroresistant Acinetobacter baumannii Strains as a Function of Growth Phase and in Response to Colistin Treatment▿  
Antimicrobial Agents and Chemotherapy  2009;53(12):4979-4986.
The prevalence of infections caused by multidrug-resistant gram-negative Acinetobacter baumannii strains and the lack of novel antibiotics under development are posing a global dilemma, forcing a resurgence of the last-line antibiotic colistin. Our aim was to use atomic force microscopy (AFM) to investigate the morphology and topography of paired colistin-susceptible and -resistant cells from colistin-heteroresistant A. baumannii strains as a function of bacterial growth phase and colistin exposure. An optimal AFM bacterial sample preparation protocol was established and applied to examine three paired strains. Images revealed rod-shaped colistin-susceptible cells (1.65 ± 0.27 μm by 0.98 ± 0.07 μm) at mid-logarithmic phase, in contrast to spherical colistin-resistant cells (1.03 ± 0.09 μm); the latter were also more diverse in appearance and exhibited a rougher surface topography (7.05 ± 1.3 nm versus 11.4 ± 2.5 nm for susceptible versus resistant, respectively). Cellular elongation up to ∼18 μm at stationary phase was more commonly observed in susceptible strains, although these “worm-like” cells were also observed occasionally in the resistant population. The effects of colistin exposure on the cell surface of colistin-susceptible and -resistant cells were found to be similar; topographical changes were minor in response to 0.5 μg/ml colistin; however, at 4 μg/ml colistin, a significant degree of surface disruption was detected. At 32 μg/ml colistin, cellular clumping and surface smoothening were evident. Our study has demonstrated for the first time substantial morphological and topographical differences between colistin-susceptible and -resistant cells from heteroresistant A. baumannii strains. These results contribute to an understanding of colistin action and resistance in regard to this problematic pathogen.
PMCID: PMC2786353  PMID: 19786595

Results 1-8 (8)