Unravelling the Hepatic Elimination Mechanisms of Colistin.

PURPOSE: Colistin is an antibiotic which is increasingly used as a last-resort therapy in critically-ill patients with multidrug resistant Gram-negative infections. The purpose of this study was to evaluate the mechanisms underlying colistin's pharmacokinetic (PK) behavior and to characterize its hepatic metabolism. METHODS: In vitro incubations were performed using colistin sulfate with rat liver microsomes (RLM) and with rat and human hepatocytes (RH and HH) in suspension. The uptake of colistin in RH/HH and thefraction of unbound colistin in HH (fu,hep) was determined. In vitro to in vivo extrapolation (IVIVE) was employed to predict the hepatic clearance (CLh) of colistin. RESULTS: Slow metabolism was detected in RH/HH, with intrinsic clearance (CLint) values of 9.34± 0.50 and 3.25 ± 0.27 mL/min/kg, respectively. Assuming the well-stirred model for hepatic drug elimination, the predicted rat CLh was 3.64± 0.22 mL/min/kg which could explain almost 70% of the reported non-renal in vivo clearance. The predicted human CLh was 91.5 ± 8.83 mL/min, which was within two-fold of the reported plasma clearance in healthy volunteers. When colistin was incubated together with the multidrug resistance-associated protein (MRP/Mrp) inhibitor benzbromarone, the intracellular accumulation of colistin in RH/HH increased significantly. CONCLUSION: These findings indicate the major role of hepatic metabolism in the non-renal clearance of colistin, while MRP/Mrp-mediated efflux is involved in the hepatic disposition of colistin. Our data provide detailed quantitative insights into the hereto unknown mechanisms responsible for non-renal elimination of colistin.

Exploring Cluster-Dependent Antibacterial Activities and Resistance Pathways of NOSO-502 and Colistin against Enterobacter cloacae Complex Species.

The Enterobacter cloacae complex (ECC) is a group of diverse environmental and clinically relevant bacterial species associated with a variety of infections in humans. ECC have emerged as one of the leading causes of nosocomial infections worldwide. The purpose of this paper is to evaluate the activity of NOSO-502 and colistin (CST) against a panel of ECC clinical isolates, including different Hoffmann's clusters strains, and to investigate the associated resistance mechanisms. NOSO-502 is the first preclinical candidate of a novel antibiotic class, the odilorhabdins (ODLs). MIC50 and MIC90 of NOSO-502 against ECC are 1 µg/mL and 2 µg/mL, respectively, with a MIC range from 0.5 µg/mL to 32 µg/mL. Only strains belonging to clusters XI and XII showed decreased susceptibility to both NOSO-502 and CST while isolates from clusters I, II, IV, and IX were only resistant to CST. To understand this phenomenon, E. cloacae ATCC 13047 from cluster XI was chosen for further study. Results revealed that the two-component system ECL_01761-ECL_01762 (ortholog of CrrAB from Klebsiella pneumoniae) induces NOSO-502 hetero-resistance by expression regulation of the ECL_01758 efflux pump component (ortholog of KexD from K. pneumoniae) which could compete with AcrB to work with the multidrug efflux pump proteins AcrA and TolC. In E. cloacae ATCC 13047, CST-hetero-resistance is conferred via modification of the lipid A by addition of 4-amino-4-deoxy-l-arabinose controlled by PhoPQ. We identified that the response regulator ECL_01761 is also involved in this resistance pathway by regulating the expression of the ECL_01760 membrane transporter.

Delineation of the molecular mechanisms underlying Colistin-mediated toxicity using metabolomic and transcriptomic analyses.

The mechanisms underlying colistin-induced toxicity are not fully understood. This study used untargeted metabolomics and transcriptomics to elucidate the molecular processes occurring in the liver and kidney of rats after treatment with colistin methanesulfonate (CMS). Rats were treated with 50 mg/kg CMS (high-dose), 25 mg/kg CMS (low-dose), or vehicle control, either as a single dose or once daily for 1 or 4 weeks. We found that metabolic alterations were dose- and treatment duration-dependent in the kidney, whereas mild changes were noted in the liver. Metabolic profiles in the high-dose, low-dose, and control groups of both tissues could be classified using partial least-squares discriminant analysis. Metabolic alterations were associated with the citric acid cycle and related processes, disrupted balance between pro-oxidants and antioxidants, inflammatory responses, and amino acid and nucleic acid metabolism. Gene expression profiles further showed that high-dose treatment was associated with disrupted metabolism, oxidative stress, and proinflammatory signals in the kidney. The expression levels of genes related to the cell cycle, DNA replication, and programmed cell death were also predominantly upregulated. These findings suggested that high-dose treatment was associated with a dramatic increase in cellular kidney injury, while only minor effects were observed in the low-dose group. Almost no significant gene expression was changed in the liver, even with high-dose CMS. In conclusion, untargeted metabolomics and transcriptomics provided better insights into the biological mechanisms underlying colistin-induced nephrotoxicity.

Role of silymarin (Silybum marianum) in the prevention of colistin-induced acute nephrotoxicity in rats.

Silymarin (Silybum marianum) has some protective effects against drug toxicity (cisplatin, acetaminophen, adriamycin, gentamicin etc.). Colistin is a strong antimicrobial, which is frequently used in the treatment of resistant gram-negative bacterial infections in recent years although it has nephrotoxic potential. This study was aimed to determine the role of silymarin against colistin-induced acute nephrotoxicity (CIN). Rats were randomly divided into four groups. The control group was treated with tap water whereas groups 2 and 3 received silymarin (orally, 100 mg/kg/day) and colistin (intraperitoneally, 750.000 IU/kg/day) for seven days, respectively. Group 4 received both 750,000 IU/kg/day colistin and 100 mg/kg/day silymarin for seven days. After euthanasia, histopathological and biochemical examinations were completed for the kidney tissue specimens and blood samples. All parameters of the control and silymarin groups were similar. Severe weight loss was seen in the groups receiving colistin (groups 3 and 4). Silymarin significantly increased glutathione peroxidase and superoxide dismutase levels when administered with colistin in group 4 only. Acute tubular injury, tubular necrosis, meduller congestion, interstitial inflammation and apoptotic indices of colistin group were significantly higher than the control group. The administration of colistin with silymarin (group 4) was able to make some improvements in tubular necrosis and significant increase in antioxidant capacity. Silymarin increased antioxidant enzyme activity only when used in combination with colistin. The effects of silymarin may become more pronounced when used at higher doses or with a longer duration of treatment and may prevent nephrotoxicity.

Chlorhexidine Promotes Psl Expression in Pseudomonas aeruginosa That Enhances Cell Aggregation with Preserved Pathogenicity Demonstrates an Adaptation against Antiseptic.

Because Pseudomonas aeruginosa is frequently in contact with Chlorhexidine (a regular antiseptic), bacterial adaptations are possible. In comparison with the parent strain, the Chlorhexidine-adapted strain formed smaller colonies with metabolic downregulation (proteomic analysis) with the cross-resistance against colistin (an antibiotic for several antibiotic-resistant bacteria), partly through the modification of L-Ara4N in the lipopolysaccharide at the outer membrane. Chlorhexidine-adapted strain formed dense liquid-solid interface biofilms with enhanced cell aggregation partly due to the Chlorhexidine-induced overexpression of psl (exopolysaccharide-encoded gene) through the LadS/GacSA pathway (c-di-GMP-independence) in 12 h biofilms and maintained the aggregation with SiaD-mediated c-di-GMP dependence in 24 h biofilms as evaluated by polymerase chain reaction (PCR). The addition of Ca2+ in the Chlorhexidine-adapted strain facilitated several Psl-associated genes, indicating an impact of Ca2+ in Psl production. The activation by Chlorhexidine-treated sessile bacteria demonstrated a lower expression of IL-6 and IL-8 on fibroblasts and macrophages than the activation by the parent strain, indicating the less inflammatory reactions from Chlorhexidine-exposed bacteria. However, the 14-day severity of the wounds in mouse caused by Chlorhexidine-treated bacteria versus the parent strain was similar, as indicated by wound diameters and bacterial burdens. In conclusion, Chlorhexidine induced psl over-expression and colistin cross-resistance that might be clinically important.

The effects of hesperidin on colistin-induced reproductive damage, autophagy, and apoptosis by reducing oxidative stress.

This study has been conducted to investigate the effect of hesperidin on colistin-induced reproductive damage in male rats. Twenty-four adult male Sprague Dawley rats were used as animal material. They were divided into four groups: control group, received physiological saline for 7 days by oral gavage; hesperidin group, received 300 mg/kg day hesperidin for 7 days; colistin group, received 73 mg/kg (total dose) colistin during 7 days; and colistin + hesperidin group, received 300 mg/kg day hesperidin following the colistin treatment. At the end of the study, routine spermatological parameters and biochemical evaluations were assayed. Also, apoptosis and autophagy biomarkers in testes were evaluated. Colistin increased oxidative stress, apoptosis and autophagy expression levels in testis. Hesperidin supplementation significantly decreased the oxidative stress levels in the testes of the colistin + hesperidin group when compared to the colistin group. The highest apoptosis and autophagy expression levels were detected in the colistin group. These values were statistically lower in the colistin + hesperidin group when compared to the colistin group. Colistin treatment decreased the percentage of sperm motility and increased sperm abnormality. Hesperidin supplementation mitigated significantly mentioned side effects compared to the colistin group. In conclusion, hesperidin supplementation can be a good strategy to mitigate colistin-induced testicular toxicity.

Subinhibitory concentration stress of colistin enhanced PhoPQ expression in Escherichia coli harboring mcr-1.

The increased and inappropriate use of colistin led to the emergence of its resistance among Gram-negative bacterial isolates and the most common mechanism of colistin resistance in Gram-negative bacteria is the modification of the lipopolysaccharide mediated by two-component regulatory systems, PhoPQ and PmrAB. The aim of the present study was to investigate the transcriptional expression of the PhoPQ system against colistin stress in clinical isolates of Escherichia coli with colistin-resistant phenotype. Six colistin-resistant E. coli isolates were obtained from Silchar Medical College and Hospital, Silchar that were of clinical origin and received for routine culture and sensitivity testing. Screening for colistin resistance was done by broth microdilution method and further screened for the presence of the different types of plasmid-mediated colistin resistance mcr genes namely, mcr-1 to mcr-10 by polymerase chain reaction (PCR). The screened positive isolates were subjected to PCR assay targeting phoP and phoQ genes and their expression was measured by quantitative real-time PCR. The results of this study revealed that two E. coli isolates (TS2 and TS4) were found to carry the mcr-1 gene. PhoP and PhoQ gene amplification was observed in all the isolates. Transcriptional analysis showed that the isolates harboring the mcr-1 gene showed an enhanced level of expression in the PhoP, PhoQ genes in the presence of a subinhibitory concentration of colistin whereas no significant expression was observed for the isolates which were devoid of the mcr gene. This study demonstrates the involvement of mcr-1 in the PhoPQ system in clinical isolates of colistin-resistant E. coli which will help in designing a molecular marker for detecting colistin-resistant E. coli and contribute to the assessment of resistance burden and infection control strategy.

Evaluation of Chemical Changes in Laboratory-Induced Colistin-Resistant Klebsiella pneumoniae.

This study evaluates the electrical potential and chemical alterations in laboratory-induced colistin-resistant Klebsiella pneumoniae, as compared to the susceptible strain using spectroscopic analyses. The minimal inhibitory concentration (MIC) of colistin, ζ-potential and chemical composition analysis of K. pneumoniae strains are determined. The results obtained for the K. pneumoniaeCol-R with induced high-level colistin resistance (MIC = 16.0 ± 0.0 mg/L) are compared with the K. pneumoniaeCol-S strain susceptible to colistin (MIC = 0.25 ± 0.0 mg/L). Fourier transform infrared (FTIR) and Raman spectroscopic studies revealed differences in bacterial cell wall structures and lipopolysaccharide (LPS) of K. pneumoniaeCol-R and K. pneumoniaeCol-S strains. In the beginning, we assumed that the obtained results could relate to a negative charge of the bacterial surface and different electrostatic interactions with cationic antibiotic molecules, reducing the affinity of colistin and leading to its lower penetration into K. pneumoniaeCol-R cell. However, no significant differences in the ζ-potential between the K. pneumoniaeCol-R and K. pneumoniaeCol-S strains are noticed. In conclusion, this mechanism is most probably associated with recognisable changes in the chemical composition of the K. pneumoniaeCol-R cell wall (especially in LPS) when compared to the susceptible strain.

Metabolic mechanism of colistin resistance and its reverting in Vibrio alginolyticus.

Colistin is a last-line antibiotic against Gram-negative multidrug-resistant bacteria, but the increased resistance poses a huge challenge to this drug. However, the mechanisms underlying such resistance are largely unexplored. The present study first identified the mutations of two genes encoding AceF subunit of pyruvate dehydrogenase (PDH) and TetR family transcriptional regulator in colistin-resistant Vibrio alginolyticus (VA-RCT ) through genome sequencing. Then, gas chromatography-mass spectroscopy-based metabolomics was adopted to investigate metabolic responses since PDH plays a role in central carbon metabolism. Colistin resistance was associated with the reduction of the central carbon metabolism and energy metabolism, featuring the alteration of the pyruvate cycle, a recently characterized energy-producing cycle. Metabolites in the pyruvate cycle reprogramed colistin-resistant metabolome to colistin-sensitive metabolome, resulting in increased gene expression, enzyme activity or protein abundance of the cycle and sodium-translocating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase. This reprogramming promoted the production of the proton motive force that enhances the binding between colistin and lipid A in lipopolysaccharide. Moreover, this metabolic approach was effective against VA-RCT in vitro and in vivo as well as other clinical isolates. These findings reveal a previously unknown mechanism of colistin resistance and develop a metabolome-reprogramming approach to promote colistin efficiency to combat with colistin-resistant bacteria.

Inactivation of Polymyxin by Hydrolytic Mechanism.

Polymyxins are nonribosomal peptide antibiotics used as the last-resort drug for treatment of multidrug-resistant Gram-negative bacteria. However, strains that are resistant to polymyxins have emerged in many countries. Although several mechanisms for polymyxin resistance have been well described, there is little knowledge on the hydrolytic mechanism of polymyxin. Here, we identified a polymyxin-inactivating enzyme from Bacillus licheniformis strain DC-1 which was produced and secreted into the medium during entry into stationary phase. After purification, sequencing, and heterologous expression, we found that the alkaline protease Apr is responsible for inactivation of polymyxins. Analysis of inactivation products demonstrated that Apr cleaves polymyxin E at two peptide bonds: one is between the tripeptide side chain and the cyclic heptapeptide ring, the other between l-Thr and l-α-γ-diaminobutyric acid (l-Dab) within the cyclic heptapeptide ring. Apr is highly conserved among several genera of Gram-positive bacteria, including Bacillus and Paenibacillus It is noteworthy that two peptidases S8 from Gram-negative bacteria shared high levels of sequence identity with Apr. Our results indicate that polymyxin resistance may result from inactivation of antibiotics by hydrolysis.

Plasmid-Mediated mcr-1 Colistin Resistance in Escherichia coli from a Black Kite in Russia.

The gene mcr-1 conferring resistance to last-line antibiotic colistin has been reported globally. Here, we describe the first detection of plasmid-mediated colistin resistance in Russian wildlife, an isolate of Escherichia coli sequence type 2280 from a black kite (Milvus migrans) scavenging raptor. Whole-genome sequencing and plasmid transferability experiments revealed that mcr-1.1 was located on conjugative IncI2 plasmid pDR164 (59891 bp). Migratory black kites may contribute to the global spread of mobile colistin resistance.

Polymer Masked-Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin-Colistin Conjugates.

Polymer masked-unmasked protein therapy (PUMPT) uses conjugation of a biodegradable polymer, such as dextrin, hyaluronic acid, or poly(l-glutamic acid), to mask a protein or peptide's activity; subsequent locally triggered degradation of the polymer at the target site regenerates bioactivity in a controllable fashion. Although the concept of PUMPT is well established, the relationship between protein unmasking and reinstatement of bioactivity is unclear. Here, we used dextrin-colistin conjugates to study the relationship between the molecular structure (degree of unmasking) and biological activity. Size exclusion chromatography was employed to collect fractions of differentially degraded conjugates and ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) employed to characterize the corresponding structures. Antimicrobial activity was studied using a minimum inhibitory concentration (MIC) assay and confocal laser scanning microscopy of LIVE/DEAD-stained biofilms with COMSTAT analysis. In vitro toxicity of the degraded conjugate was assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. UPLC-MS revealed that the fully "unmasked" dextrin-colistin conjugate composed of colistin bound to at least one linker, whereas larger species were composed of colistin with varying lengths of glucose units attached. Increasing the degree of dextrin modification by succinoylation typically led to a greater number of linkers bound to colistin. Greater antimicrobial and antibiofilm activity were observed for the fully "unmasked" conjugate compared to the partially degraded species (MIC = 0.25 and 2-8 µg/mL, respectively), whereas dextrin conjugation reduced colistin's in vitro toxicity toward kidney cells, even after complete unmasking. This study highlights the importance of defining the structure-antimicrobial activity relationship for novel antibiotic derivatives and demonstrates the suitability of LC-MS to aid the design of biodegradable polymer-antibiotic conjugates.

Colistin induced peripheral neurotoxicity involves mitochondrial dysfunction and oxidative stress in mice.

Polymyxin is a critical antibiotic against the infection caused by multidrug-resistant gram-negative bacteria. Neurotoxicity is one of main dose-limiting factors. The present study aimed to investigate the underlying molecular mechanism on colistin induced peripheral neurotoxicity using a mouse model. Forty mice were divided into control, colistin 1-, 3- and 7-day groups, the mice were intravenously injected with saline or colistin (sulfate) at the dose of 15 mg/kg/day for 1, 3 and 7 days, respectively. The results showed that, colistin treatment for 7 days markedly resulted in the demyelination, axonal degeneration and mitochondria swelling in the mice's sciatic tissues. Colistin treatment induces oxidative stress as well as the increases of mitochondrial permeability transition, decreases of membrane potential (ΔΨm) and activities of mitochondrial respiratory chain in the mice's sciatic nerve tissues. Furthermore, in the colistin-7 day group, adenosine-triphosphate (ATP) level Na+/K+-ATPase activity decreased to 75.2% (p < 0.01) and 80.1% (p < 0.01), respectively. Meanwhile, colistin treatment down-regulates the expression of protein kinase B (Akt) and mammalian target of rapamycin (mTOR) mRNAs and up-regulates the expression of Bax and caspase-3 mRNAs. Our results reveal that colistin induced sciatic nerves damage involves oxidative stress, mitochondrial dysfunction and the inhibition of Akt/mTOR pathway.

Selective Interaction of Colistin with Lipid Model Membranes.

Although colistin's clinical use is limited due to its nephrotoxicity, colistin is considered to be an antibiotic of last resort because it is used to treat patients infected with multidrug-resistant bacteria. In an effort to provide molecular details about colistin's ability to kill Gram-negative (G(-)) but not Gram-positive (G(+)) bacteria, we investigated the biophysics of the interaction between colistin and lipid mixtures mimicking the cytoplasmic membrane of G(+), G(-) bacteria as well as eukaryotic cells. Two different models of the G(-) outer membrane (OM) were assayed: lipid A with two deoxy-manno-octulosonyl sugar residues, and Escherichia coli lipopolysaccharide mixed with dilaurylphosphatidylglycerol. We used circular dichroism and x-ray diffuse scattering at low and wide angle in stacked multilayered samples, and neutron reflectivity of single, tethered bilayers mixed with colistin. We found no differences in secondary structure when colistin was bound to G(-) versus G(+) membrane mimics, ruling out a protein conformational change as the cause of this difference. However, bending modulus KC perturbation was quite irregular for the G(-) inner membrane, where colistin produced a softening of the membranes at an intermediate lipid/peptide molar ratio but stiffening at lower and higher peptide concentrations, whereas in G(+) and eukaryotic mimics there was only a slight softening. Acyl chain order in G(-) was perturbed similarly to KC. In G(+), there was only a slight softening and disordering effect, whereas in OM mimics, there was a slight stiffening and ordering of both membranes with increasing colistin. X-ray and neutron reflectivity structural results reveal colistin partitions deepest to reach the hydrocarbon interior in G(-) membranes, but remains in the headgroup region in G(+), OM, and eukaryotic mimics. It is possible that domain formation is responsible for the erratic response of G(-) inner membranes to colistin and for its deeper penetration, which could increase membrane permeability.

A Gcn5-Related N-Acetyltransferase (GNAT) Capable of Acetylating Polymyxin B and Colistin Antibiotics in Vitro.

Deeper exploration of uncharacterized Gcn5-related N-acetyltransferases has the potential to expand our knowledge of the types of molecules that can be acylated by this important superfamily of enzymes and may offer new opportunities for biotechnological applications. While determining native or biologically relevant in vivo functions of uncharacterized proteins is ideal, their alternative or promiscuous in vitro capabilities provide insight into key active site interactions. Additionally, this knowledge can be exploited to selectively modify complex molecules and reduce byproducts when synthetic routes become challenging. During our exploration of uncharacterized Gcn5-related N-acetyltransferases from Pseudomonas aeruginosa, we identified such an example. We found that the PA3944 enzyme acetylates both polymyxin B and colistin on a single diaminobutyric acid residue closest to the macrocyclic ring of the antimicrobial peptide and determined the PA3944 crystal structure. This finding is important for several reasons. (1) To the best of our knowledge, this is the first report of enzymatic acylation of polymyxins and thus reveals a new type of substrate that this enzyme family can use. (2) The enzymatic acetylation offers a controlled method for antibiotic modification compared to classical promiscuous chemical methods. (3) The site of acetylation would reduce the overall positive charge of the molecule, which is important for reducing nephrotoxic effects and may be a salvage strategy for this important class of antibiotics. While the physiological substrate for this enzyme remains unknown, our structural and functional characterization of PA3944 offers insight into its unique noncanonical substrate specificity.

A Population WB-PBPK Model of Colistin and its Prodrug CMS in Pigs: Focus on the Renal Distribution and Excretion.

PURPOSE: The objective was the development of a whole-body physiologically-based pharmacokinetic (WB-PBPK) model for colistin, and its prodrug colistimethate sodium (CMS), in pigs to explore their tissue distribution, especially in kidneys. METHODS: Plasma and tissue concentrations of CMS and colistin were measured after systemic administrations of different dosing regimens of CMS in pigs. The WB-PBPK model was developed based on these data according to a non-linear mixed effect approach and using NONMEM software. A detailed sub-model was implemented for kidneys to handle the complex disposition of CMS and colistin within this organ. RESULTS: The WB-PBPK model well captured the kinetic profiles of CMS and colistin in plasma. In kidneys, an accumulation and slow elimination of colistin were observed and well described by the model. Kidneys seemed to have a major role in the elimination processes, through tubular secretion of CMS and intracellular degradation of colistin. Lastly, to illustrate the usefulness of the PBPK model, an estimation of the withdrawal periods after veterinary use of CMS in pigs was made. CONCLUSIONS: The WB-PBPK model gives an insight into the renal distribution and elimination of CMS and colistin in pigs; it may be further developed to explore the colistin induced-nephrotoxicity in humans.

Evaluation of colistin stability in agar and comparison of four methods for MIC testing of colistin.

Susceptibility testing for colistin remains challenging primarily due to its inherent properties. We evaluated colistin stability in agar and reproducibility of colistin MICs obtained by agar dilution, broth macro- and micro-dilution and MIC gradient strips on 3-7 iterations of each method using clinical Klebsiella pneumoniae (susceptible-CS, and resistant-CR, n = 2 each), mcr-harboring Escherichia coli (n = 2), and reference strains E. coli ATCC25922 and Pseudomonas aeruginosa ATCC27853. MICs for reference strains were not in the given range using Etest and broth microdilution (ATCC25922, 0.125 and 4 µg/ml, respectively). MICs of CR-1 and CR-2, and of the mcr-harboring E. coli showed high concordance between agar and broth dilution varying up to one 2-fold dilution. However, remarkable variations were observed on broth dilution with CS-1 and CS-2 (MIC range 0.25-32 and 0.5-64 µg/ml, respectively); whereas for agar dilution the MIC for both CS strains was 0.5 µg/ml in all the runs. MICs obtained by MIC gradient strips were lower than those obtained by dilution methods (1-2 dilutions for CS and mcr strains, and up to five dilutions for CR strains). To confirm uniform distribution of colistin in agar, a single strain was spotted in five different regions of the same plate. All spots showed concordant growth with maximum one dilution difference. No effect on MIC was found due to storage of colistin-containing agar plates for 7 days at 4 °C. In our hands, agar dilution was superior in terms of reproducibility and robustness, compared to broth dilution methods, for colistin MIC determination.

Colistin is substrate of the carnitine/organic cation transporter 2 (OCTN2, SLC22A5).

Colistin is a polycation antibiotic used for the treatment of multidrug-resistance (MDR) gram-negative infections; nevertheless, its use is often limited by the high incidence of renal damage. The mechanism underlying colistin-induced nephrotoxicity is not known, but perhaps related to its accumulation in the renal cortex upon extensive reabsorption from the nascent urine. Because little is known about the membrane transport of colistin, the purpose of the present study was to characterize better the transport system involved in colistin renal handling by using HEK293 cells stably transfected with the main organic cation transporters expressed at the apical membrane of the proximal tubule. [14C]Colistin was transported by the carnitine/organic cation transporter 2 (OCTN2, SLC22A5) but not by the organic cation transporter 1 (OCT1) and N1 (OCTN1). Non-labeled colistin inhibited the OCTN2-mediated transport of [3H]L-carnitine in a non-competitive manner and that of [14C]tetraethylammonium bromide ([14C]TEA) in a competitive manner. Unlike that of [3H]L-carnitine, the [14C]colistin OCTN2-mediated uptake was Na+-independent. When endogenous OCTN2-mediated colistin transport was inhibited by co-incubation with L-carnitine, primary mouse proximal tubular cells were fully protected from colistin toxicity, suggesting that colistin toxicity occurred upon intracellular accumulation.

Altered susceptibility to the bactericidal effect of photocatalytic oxidation by TiO2 is related to colistin resistance development in Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii is a well-documented pathogen associated with hospital-acquired infections. In addition to multidrug resistance, A. baumannii can also become resistant to colistin, the antibiotic treatment of last resort, by the loss of the lipopolysaccharide from its outer membrane. Here, we demonstrate that the development of colistin resistance also increases the resistance of A. baumannii to titanium dioxide (TiO2) photocatalysis. Both colistin-sensitive A. baumannii (CSAB) and colistin-resistant A. baumannii (CRAB) were inactivated by TiO2 when irradiated by ultraviolet A (UV-A). The resistance of CRAB to TiO2 photocatalysis was 1.5 times higher than that of CSAB, as determined by either culture assay or quantification of leaked proteins after photocatalysis (p < 0.05). The results of two-dimensional gel electrophoresis led to the speculation that the high resistance of CRAB may be associated with a lack of sensitive targets and oxidative enzymes. This hypothesis was confirmed by antimicrobial assays with 25 mM hydrogen peroxide (H2O2) and 1.07 mM sodium hypochlorite (NaClO). CRAB was significantly more resistant to H2O2 and NaClO treatment than CSAB (p < 0.01), consistent with the results of the TiO2 inactivation experiment. Therefore, the antibiotic resistance profiles of bacterial strains should be considered before the use of strains as indicators to represent sanitary quality after TiO2 photocatalysis.

Mucin Binding Reduces Colistin Antimicrobial Activity.

Colistin has found increasing use in treating drug-resistant bacterial lung infections, but potential interactions with pulmonary biomolecules have not been investigated. We postulated that colistin, like aminoglycoside antibiotics, may bind to secretory mucin in sputum or epithelial mucin that lines airways, reducing free drug levels. To test this hypothesis, we measured binding of colistin and other antibiotics to porcine mucin, a family of densely glycosylated proteins used as a surrogate for human sputum and airway mucin. Antibiotics were incubated in dialysis tubing with or without mucin, and concentrations of unbound antibiotics able to penetrate the dialysis tubing were measured over time using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The percentage of antibiotic measured in the dialysate after 4 h in the presence of mucin, relative to the amount without mucin, was 15% for colistin, 16% for polymyxin B, 19% for tobramycin, 52% for ciprofloxacin, and 78% for daptomycin. Antibiotics with the strongest mucin binding had an overall polybasic positive charge, whereas those with comparatively little binding were less basic. When comparing MICs measured with or without added mucin, colistin and polymyxin B showed >100-fold increases in MICs for multiple Gram-negative bacteria. Preclinical evaluation of mucin binding should become a standard procedure when considering the potential pulmonary use of new or existing antibiotics, particularly those with a polybasic overall charge. In the airways, mucin binding may reduce the antibacterial efficacy of inhaled or intravenously administered colistin, and the presence of sub-MIC effective antibiotic concentrations could result in the development of antibiotic resistance.