Biotransformation of chloramphenicol by white-rot-fungi Trametes versicolor under cadmium stress.

The recalcitrant chloramphenicol (CAP) combined with heavy metals cadmium (Cd) commonly co-existed in the environment, posing threat to environment health. The capacity of Trametes versicolor to remove/biodegrade CAP in air-pulse fluidized-bed reactor was evaluated, even under Cd stress. T. versicolor could remove 44 % CAP of 5 mg/L in 15 days, even 51 % CAP under 1 mg/L Cd stress. Sustained Cd stress inhibited CAP biodegradation and Cd removal in a 5-batches of a 5-days cycle sequential batch reactor. Nine transformation products and two novel pathways were proposed, with initial multi-step transformation reaction into CP2 and allylic alcohol, respectively. Furthermore, the main mechanism of Cd removal by T. versicolor was extracellular surface bioadsorption and intracellular accumulation. This study filled the gap of the mechanism of simultaneous CAP removal/biodegradation and Cd removal by white-rot fungi T. versicolor, which offer a theoretical basis for future application of biological removal of CAP containing wastewater.

Investigation on the uptake of ciprofloxacin, chloramphenicol and praziquantel by button mushrooms.

Button mushrooms are widely produced edible basidiomycetes. Commercially, they are cultivated on substrates containing fermented horse manure and chicken feces. Since pharmacologically active substances (PAS) might be introduced into the food chain via animal treatment, their residues may be present in manure used for mushroom growth. Previous studies in plants have demonstrated an uptake of PAS from the agricultural environment. The present study was performed to investigate the presence of PAS in button mushrooms. For analysis, a multi-analyte method for the detection of 21 selected PAS using liquid chromatography tandem-mass spectrometry was developed, successfully validated and applied to commercially available button mushrooms. Traces of chloramphenicol were detected in two of 20 samples. Additionally, in a mushroom cultivation experiment an uptake of ciprofloxacin, chloramphenicol and praziquantel was conducted. Throughout the whole experiment, praziquantel was present in quantifiable amounts in mushrooms and in high quantities in soil.

Dynamics of the context-specific translation arrest by chloramphenicol and linezolid.

Chloramphenicol (CHL) and linezolid (LZD) are antibiotics that inhibit translation. Both were thought to block peptide-bond formation between all combinations of amino acids. Yet recently, a strong nascent peptide context-dependency of CHL- and LZD-induced translation arrest was discovered. Here we probed the mechanism of action of CHL and LZD by using single-molecule Förster resonance energy transfer spectroscopy to monitor translation arrest induced by antibiotics. The presence of CHL or LZD does not substantially alter dynamics of protein synthesis until the arrest-motif of the nascent peptide is generated. Inhibition of peptide-bond formation compels the fully accommodated A-site transfer RNA to undergo repeated rounds of dissociation and nonproductive rebinding. The glycyl amino-acid moiety on the A-site Gly-tRNA manages to overcome the arrest by CHL. Our results illuminate the mechanism of CHL and LZD action through their interactions with the ribosome, the nascent peptide and the incoming amino acid, perturbing elongation dynamics.

The health risk for seafood consumers under future ocean acidification (OA) scenarios: OA alters bioaccumulation of three pollutants in an edible bivalve species through affecting the in vivo metabolism.

The current knowledge about the effect of pCO2-driven ocean acidification on the bioaccumulation of pollutants in marine species is still scarce, as only limited types of pollutants have been investigated. Therefore, to obtain a better understanding of the effect of ocean acidification on the process of bioaccumulation and subsequent food safety, the accumulation of benzo[a]pyrene (B[a]P), chloramphenicol (CAP), and nitrofurazone (NFZ) in an edible bivalve species, Tegillarca granosa, under present and near-future ocean acidification scenarios was investigated in the present study. The health risks associated with consuming contaminated blood clams were also assessed using target hazard quotient (THQ), lifetime cancer risk (CR), or margin of exposure (MoE). To explain the alterations in bioaccumulation of these pollutants, the expressions of genes encoding corresponding key metabolic proteins were analyzed as well. The results obtained showed that ocean acidification exerted a significant effect on the accumulation of B[a]P, NFZ, and CAP in the clams. After four-week exposure to B[a]P, NFZ, or CAP contaminated seawater acidified with CO2 at pH 7.8 and 7.4, significantly greater amounts of B[a]P and lower amounts of NFZ and CAP were accumulated in the clams compared to that in the control. Although no non-carcinogenic risk of consuming B[a]P-contaminated blood clams was detected using the THQ values obtained, the CR values obtained indicated a high life-time risk in all groups. In addition, according to the MoE values obtained, the health risks in terms of consuming NFZ- and CAP-contaminated clams were significantly reduced under ocean acidification scenarios but still cannot be ignored, especially for children. The gene expression results showed that the ability of clams to eliminate B[a]P may be significantly constrained, whereas the ability to eliminate NFZ and CAP may be enhanced under ocean acidification scenarios, indicating that the changes in the accumulation of these pollutants may be due to the altered in vivo metabolism.

A noncanonical binding site of chloramphenicol revealed via molecular dynamics simulations.

Chloramphenicol, an antibiotic belonging to the family of amphenicols, is an inhibitor of translation. On the basis of X-ray structural analysis of the binding of chloramphenicol to free bacterial ribosomes, the chloramphenicol action mechanism that consists in preventing the binding of aminoacyl-tRNA to the A-site of the large subunit of the ribosome was adopted. However, the known structures of chloramphenicol complexes with bacterial ribosomes poorly explain the results of the experiments on the chemical modification of 23S rRNA, the resistance to chloramphenicol caused by mutations in 23S rRNA and, which is particularly important, the selectivity of chloramphenicol in suppression of translation, depending on the amino acid sequence of the nascent peptide. In the present study the putative structure of the chloramphenicol complex with a bacterial ribosome in the A,A/P,P-state has been obtained by molecular dynamics simulations methods. The proposed structure of the complex allows us to explain the results of biochemical studies of the interaction of chloramphenicol with the bacterial ribosome.

Active Mediated Transport of Chloramphenicol and Thiamphenicol in a Calu-3 Lung Epithelial Cell Model.

Pulmonary administration enables high local concentrations along with limited systemic side effects but not all antibiotics could be good candidates. In this perspective, diffusion of the antibiotic chloramphenicol (CHL) and thiamphenicol (THA) through the lung has been evaluated to reassess their potential for pulmonary administration. The apparent permeability (Papp) was evaluated with the Calu-3 cell model. The influence of drug transporters was assessed with the PSC-833, MK-571, and KO-143 inhibitors. The influence of CHL and THA on the cell uptake of rhodamin 123 and fluorescein was also evaluated. Absorptive Papp of CHL and THA was concentration independent with CHL Papp 4 times higher than that of THA. Secretory Papp of CHL was concentration independent, whereas it was concentration dependent for THA with an efflux ratio of 3.6 for the lowest concentration. The use of inhibitors suggested that CHL and THA were substrates of efflux transporters but with a low affinity. In conclusion, the permeability results suggest that the pulmonary route may offer a biopharmaceutical advantage only for THA. Owing to the influence of drug transporters, a higher concentration in the lung than in the plasma is expected mostly for THA, whatever the route of administration.

Discovery of Potent ALK Inhibitors Using Pharmacophore-Informatics Strategy.

Anaplastic lymphoma kinase is a tyrosine kinase receptor protein belonging to insulin receptor superfamily. Gene fusions in anaplastic lymphoma kinase are associated with non-small cell lung cancer development. Hence, they are of immense importance in targeted therapies. Thus, for the treatment of non-small cell lung cancer, effective anaplastic lymphoma kinase inhibitors are of great significance. Therefore, our objective is to find hit compounds that could have better inhibitory activity than the existing anaplastic lymphoma kinase inhibitors. Keeping this in mind, in the present study pharmacophore based virtual screening was performed to identify possible anaplastic lymphoma kinase inhibitors. Initially, a five-point common pharmacophore hypothesis was generated based on twelve anaplastic lymphoma kinase inhibitors using PHASE module of Schrödinger. Subsequently, common pharmacophore hypothesis-based screening was conducted against in-trials subset of ZINC database and a total of 1000 hits were identified. The molecules obtained were further screened by three stages of docking using GLIDE software. The docking results reveal that six hit molecules showed higher glide score in comparison with the reference molecules. Finally, pharmacokinetic properties of the hit molecules were also analysed using QikProp programme. The results indicate that molecules namely videx, dexecadotril, chloramphenicol, naficillin were found to have good pharmacokinetic properties and human oral absorption. Moreover, videx, naficillin and chloramphenicol were found to have significant inhibitory activity for mutant (F1174L) anaplastic lymphoma kinase. It was also found that videx exhibited crucial interactions with the Met1199 residue of the native and mutant anaplastic lymphoma kinase protein. Furthermore, PASS algorithm predicted anti-neoplastic activity for all the four molecules. Thus these hits are found to be promising leads for anaplastic lymphoma kinase inhibitors. We believe that this study will be useful for the discovery and designing of more potent anaplastic lymphoma kinase inhibitors in the near future.

Preparation of Chloramphenicol/Amino Acid Combinations Exhibiting Enhanced Dissolution Rates and Reduced Drug-Induced Oxidative Stress.

Chloramphenicol is an old antibiotic agent that is re-emerging as a valuable alternative for the treatment of multidrug-resistant pathogens. However, it exhibits suboptimal biopharmaceutical properties and toxicity profiles. In this work, chloramphenicol was combined with essential amino acids (arginine, cysteine, glycine, and leucine) with the aim of improving its dissolution rate and reduce its toxicity towards leukocytes. The chloramphenicol/amino acid solid samples were prepared by freeze-drying method and characterized in the solid state by using Fourier transform infrared spectroscopy, powder X-ray diffraction, differential scanning calorimetry, scanning electron microscopy, and solid-state nuclear magnetic resonance. The dissolution properties, antimicrobial activity, reactive oxygen species production, and stability of the different samples were studied. The dissolution rate of all combinations was significantly increased in comparison to that of the pure active pharmaceutical ingredient. Additionally, oxidative stress production in human leukocytes caused by chloramphenicol was decreased in the chloramphenicol/amino acid combinations, while the antimicrobial activity of the antibiotic was maintained. The CAP:Leu binary combination resulted in the most outstanding solid system makes it suitable candidate for the development of pharmaceutical formulations of this antimicrobial agent with an improved safety profile.

A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel.

The ribosomal exit tunnel had recently become the centre of many functional and structural studies. Accumulated evidence indicates that the tunnel is not simply a passive conduit for the nascent chain, but a rather functionally important compartment where nascent peptide sequences can interact with the ribosome to signal translation to slow down or even stop. To explore further this interaction, we have synthesized short peptides attached to the amino group of a chloramphenicol (CAM) base, such that when bound to the ribosome these compounds mimic a nascent peptidyl-tRNA chain bound to the A-site of the peptidyltransferase center (PTC). Here we show that these CAM-peptides interact with the PTC of the ribosome while their effectiveness can be modulated by the sequence of the peptide, suggesting a direct interaction of the peptide with the ribosomal tunnel. Indeed, chemical footprinting in the presence of CAM-P2, one of the tested CAM-peptides, reveals protection of 23S rRNA nucleotides located deep within the tunnel, indicating a potential interaction with specific components of the ribosomal tunnel. Collectively, our findings suggest that the CAM-based peptide derivatives will be useful tools for targeting polypeptide chain mimics to the ribosomal tunnel, allowing their conformation and interaction with the ribosomal tunnel to be explored using further biochemical and structural methods.

Phosphorylation of chloramphenicol by a recombinant protein Yhr2 from Streptomyces avermitilis MA4680.

Although phosphorylation of chloramphenicol has been shown to occur in the chloramphenicol producer, Streptomyces venezuelae, there are no reports on the existence of chloramphenicol phosphorylase in other Streptomyces species. In the present study, we report the modification of chloramphenicol by a recombinant protein, designated as Yhr2 (encoded by SAV_877), from Streptomyces avermitilis MA4680. Recombinant Yhr2 was expressed in Escherichia coli BL21 (DE3) and the cells expressing this recombinant protein were shown to phosphorylate chloramphenicol to a 3'-O-phosphoryl ester derivative, resulting in an inactivated form of the antibiotic. Expression of yhr2 conferred chloramphenicol resistance to E. coli cells up to 25 µg/mL and in an in vitro reaction, adenosine triphosphate (ATP), guanosine triphosphate (GTP), adenosine diphosphate (ADP) and guanosine diphosphate (GDP) were shown to be the phosphate donors for phosphorylation of chloramphenicol. This study highlights that antibiotic resistance conferring genes could be easily expressed and functionalized in other organisms that do not produce the respective antibiotic.

Jadomycins derived from the assimilation and incorporation of norvaline and norleucine.

Streptomyces venezuelae ISP5230 is recognized for the production of chloramphenicol and the jadomycin family of natural products. The jadomycins are angucycline natural products containing a unique oxazolone ring incorporating an amino acid present in the minimal culture media. Substitution of different amino acids results in products of varying biological activity. Analysis of cultures of S. venezuelae ISP5230 incubated with l- and d-norvaline and l- and d-norleucine indicated that only the d-configured amino acids were incorporated into the natural products. Subsequently, jadomycin DNV and jadomycin DNL were isolated and characterized (titers 4 and 9 mg L(-1), respectively). The compounds were evaluated in the National Cancer Institute cell line cancer growth inhibition and cytotoxicity screens, for antimicrobial activity against selected Gram-positive and Gram-negative bacteria, and as DNA-cleavage agents in vitro.

Evidence of natural occurrence of the banned antibiotic chloramphenicol in herbs and grass.

Chloramphenicol (CAP), a broad-spectrum antibiotic, was detected in several herb and grass samples from different geographic origins. Due to its suspected carcinogenicity and linkages with the development of aplastic anemia in humans, CAP is banned for use in food-producing animals in the European Union (EU) and many other countries. However, products of animal origin originating from Asian countries entering the European market are still found noncompliant (containing CAP) on a regular basis, even when there is no history of chloramphenicol use in these countries. A possible explanation for the continued detection of these residues is the natural occurrence of CAP in plant material which is used as animal feed, with the consequent transfer of the substance to the animal tissues. Approximately 110 samples were analyzed using liquid chromatography coupled with mass spectrometric detection. In 26 samples, the presence of CAP was confirmed using the criteria for banned substances defined by the EU. Among other plant materials, samples of the Artemisia family retrieved from Mongolia and from Utah, USA, and a therapeutic herb mixture obtained from local stores in the Netherlands proved to contain CAP at levels ranging from 0.1 to 450 microg/kg. These findings may have a major impact in relation to international trade and safety to the consumer. The results of this study demonstrate that noncompliant findings in animal-derived food products may in part be due to the natural occurrence of chloramphenicol in plant material. This has implications for the application of current EU, USA, and other legislation and the interpretation of analytical results with respect to the consideration of CAP as a xenobiotic veterinary drug residue and the regulatory actions taken upon its detection in food.

[Diffusion and diffusion-osmosis models of the charged macromolecule transfer in barriers of biosystems].

Mathematical models of the transfer of charged macromolecules have been constructed on the basis of the classical equations of electromigration diffusion of Helmholtz-Smolukhovskii, Goldman, and Goldman-Hodgkin-Katz. It was shown that ion transfer in placental (mimicking lipid-protein barriers) and muscle barriers occurs by different mechanisms. In placental barriers, the electromigration diffusion occurs along lipid-protein channels formed due to the conformational deformation of phospholipid and protein molecules with the coefficients of diffusion D = (2.6-3.6) x 10(-8) cm2/s. The transfer in muscle barriers is due to the migration across charged interfibrillar channels with the negative diffusion activation energy, which is explained by changes in the structure of muscle fibers and expenditures of thermal energy for the extrusion of Cl- from channel walls with the diffusion coefficient D = (6.0-10.0) x 10(-6) cm2/s.

A structural study of the interaction between the Dr haemagglutinin DraE and derivatives of chloramphenicol.

Dr adhesins are expressed on the surface of uropathogenic and diffusely adherent strains of Escherichia coli. The major adhesin subunit (DraE/AfaE) of these organelles mediates attachment of the bacterium to the surface of the host cell and possibly intracellular invasion through its recognition of the complement regulator decay-accelerating factor (DAF) and/or members of the carcinoembryonic antigen (CEA) family. The adhesin subunit of the Dr haemagglutinin, a Dr-family member, additionally binds type IV collagen and is inhibited in all its receptor interactions by the antibiotic chloramphenicol (CLM). In this study, previous structural work is built upon by reporting the X-ray structures of DraE bound to two chloramphenicol derivatives: chloramphenicol succinate (CLS) and bromamphenicol (BRM). The CLS structure demonstrates that acylation of the 3-hydroxyl group of CLM with succinyl does not significantly perturb the mode of binding, while the BRM structure implies that the binding pocket is able to accommodate bulkier substituents on the N-acyl group. It is concluded that modifications of the 3-hydroxyl group would generate a potent Dr haemagglutinin inhibitor that would not cause the toxic side effects that are associated with the normal bacteriostatic activity of CLM.

Complete inhibition of the Pdr5p multidrug efflux pump ATPase activity by its transport substrate clotrimazole suggests that GTP as well as ATP may be used as an energy source.

The yeast Pdr5p transporter is a 160 kDa protein that effluxes a large variety of xenobiotic compounds. In this study, we characterize its ATPase activity and demonstrate that it has biochemical features reminiscent of those of other ATP-binding cassette multidrug transporters: a relatively high Km for ATP (1.9 mM), inhibition by orthovanadate, and the ability to specifically bind an azidoATP analogue at the nucleotide-binding domains. Pdr5p-specific ATPase activity shows complete, concentration-dependent inhibition by clotrimazole, which is also known to be a potent transport substrate. Our results indicate, however, that this inhibition is noncompetitive and caused by the interaction of clotrimazole with the transporter at a site that is distinct from the ATP-binding domains. Curiously, Pdr5p-mediated transport of clotrimazole continues at intracellular concentrations of substrate that should eliminate all ATPase activity. Significantly, however, we observed that the Pdr5p has GTPase and UTPase activities that are relatively resistant to clotrimazole. Furthermore, the Km(GTPase) roughly matches the intracellular concentrations of the nucleotide reported for yeast. Using purified plasma membrane vesicles, we demonstrate that Pdr5p can use GTP to fuel substrate transport. We propose that Pdr5p increases its multidrug transport substrate specificity by using more than one nucleotide as an energy source.

Surgical removal of intraocular antibiotic ointment after routine cataract phacoemulsification.

We report surgical removal of a droplet of intraocular chloramphenicol ointment in a 70-year-old man who had routine small-incision phacoemulsification. A spherical droplet of ointment adherent to the intraocular lens was noted 2 months postoperatively. The source was considered to be the immediate postoperative conjunctival fornix chloramphenicol ointment. Gonioscopy revealed tiny droplets adherent to the peripheral iris and angle. The droplet was surgically explanted using a minimally traumatic technique with a lens glide and ophthalmic viscosurgical device through an enlarged clear corneal incision and sent intact for laboratory biochemical analysis. The patient maintained good vision without evidence of uveitis or secondary glaucoma at 1 year of follow-up. This uncommon case also raises issues about the need for immediate postoperative antibiotic ointment for endophthalmitis prophylaxis and phacoemulsification thermal wound effects on the integrity of self-sealing clear corneal incisions.

Indole-3-carbinol inhibition of androgen receptor expression and downregulation of androgen responsiveness in human prostate cancer cells.

Indole-3-carbinol (I3C), a naturally occurring compound found in vegetables of the Brassica genus, such as broccoli and cabbage, is a promising anticancer agent previously shown to induce a G(1) cell-cycle arrest in the cells of human lymph node carcinoma of prostate (LNCaP) through regulation of specific G(1)-acting cell-cycle components. Since the androgen receptor (AR) mediates proliferation and differentiation in the prostate and is expressed in nearly all human prostate cancers, the effects of I3C on AR expression and function were examined in LNCaP cells. Immunoblot and quantitative RT-PCR assays revealed that I3C inhibited the expression of AR protein and mRNA levels within 12 h of indole treatment. I3C downregulated the reporter activity of LNCaP cells transiently transfected with an AR promoter-luciferase plasmid, demonstrating that a unique response to I3C is the inhibition of AR promoter activity. In contrast to I3C, the natural I3C dimerization product 3,3'-diindolylmethane, which acts as an androgen antagonist, had no effect on AR expression. To determine the functional significance of the I3C-inhibited expression of AR, the AR-regulated prostate specific antigen (PSA) was utilized as a downstream indicator. I3C downregulated the expression of PSA transcripts and protein levels and inhibited PSA promoter activity, as well as that of a minimal androgen responsive element containing reporter plasmid. Expression of exogenous AR prevented the I3C disruption of androgen-induced PSA expression. Taken together, our results demonstrate that I3C represses AR expression and responsiveness in LNCaP cells as a part of its antiproliferative mechanism.

JAK2 is necessary and sufficient for interferon-gamma-induced transcription of the gene encoding gp91PHOX.

During the inflammatory response, interferon-gamma (IFN-gamma) increases transcription of the gene encoding gp91PHOX, a respiratory burst oxidase component. This gene (referred to as the CYBB gene) is transcribed in phagocytic cells differentiated beyond the promyelocyte stage, and transcription continues until cell death. Previous investigations identified a positive regulatory element in the proximal CYBB promoter referred to as the hematopoiesis-associated factor 1 (HAF1)-cis element. This element is activated by a multiprotein complex, which includes the IFN consensus sequence-binding protein (ICSBP). Interaction of this complex with the HAF1-cis element requires ICSBP tyrosine phosphorylation, which is induced by IFN-gamma stimulation of phagocytic cells. Previous studies also identified a negative cis element in the CYBB promoter. This element is repressed by the homeodomain protein HoxA10. HoxA10 tyrosine phosphorylation, which occurs in response to IFN-gamma, decreases HoxA10 DNA binding and therefore repression of CYBB transcription. In these studies, we determine Janus tyrosine kinase 2 (JAK2) activation is necessary and sufficient for IFN-gamma-induced CYBB transcription in phagocytic cells and also for ICSBP and HoxA10 tyrosine phosphorylation. Consistent with these results, we find JAK2 activation is sufficient to induce ICSBP interaction with the HAF1 element and abolish HoxA10 binding to the CYBBrepressor element. Therefore, these findings provide direct demonstration of JAK2 dependence of IFN-gamma-induced CYBB transcription. In addition, these results identify a mechanism mediating this effect.

Salicylate induces an antibiotic efflux pump in Burkholderia cepacia complex genomovar III (B. cenocepacia).

An antibiotic efflux gene cluster that confers resistance to chloramphenicol, trimethoprim, and ciprofloxacin has been identified in Burkholderia cenocepacia (genomovar III), an important cystic fibrosis pathogen. Five open reading frames have been identified in the cluster. There is apparently a single transcriptional unit, with llpE encoding a lipase-like protein, ceoA encoding a putative periplasmic linker protein, ceoB encoding a putative cytoplasmic membrane protein, and opcM encoding a previously described outer membrane protein. A putative LysR-type transcriptional regulatory gene, ceoR, is divergently transcribed upstream of the structural gene cluster. Experiments using radiolabeled chloramphenicol and salicylate demonstrated active efflux of both compounds in the presence of the gene cluster. Salicylate is an important siderophore produced by B. cepacia complex isolates, and both extrinsic salicylate and iron starvation appear to upregulate ceoR promoter activity, as does chloramphenicol. These results suggest that salicylate is a natural substrate for the efflux pump in B. cenocepacia and imply that the environment of low iron concentration in the cystic fibrosis lung can induce efflux-mediated resistance, even in the absence of antibiotic selective pressure.