Novel nalidixic acid derivatives targeting topoisomerase II enzyme; Design, synthesis, anticancer activity and effect on cell cycle profile.

AIM: Design and synthesis of novel nalidixic acid derivatives of potent anticancer and topoisomerase II inhibitory activities were our major aim. MATERIALS & METHODS: All the newly synthesized nalidixic acid derivatives were submitted to the National Cancer Institute (NCI), Bethesda, USA and were accepted for single dose screening. Further investigation via IC50 determination of the most potent compound 6a against K-562 and SR leukemia cell lines. Finally, the topoisomerase II inhibitory activity, the cell cycle analysis and molecular docking of 6a were performed in order to identify the possible mechanism of the anticancer activity. RESULTS: Compound 6a showed interesting selectivity against leukemia especially K-562 and SR subpanels with IC50 35.29 µM and 13.85 µM respectively. Moreover, compound 6a revealed potent topoisomerase IIα and topoisomerase IIß inhibitory activity compared with known topoisomerase inhibitors such as doxorubicin and topotecan with IC50 1.30 µM and 0.017 µM respectively. Cell cycle analysis indicated that compound 6a induced cell cycle arrest at G2-M phase leading to inhibition of cell proliferation and apoptosis. Molecular modeling demonstrated that the potent topoisomerase inhibitory activity of 6a was due to the interaction with the topoisomerase II enzyme through coordinate bonding with the magnesium ion Mg2+, hydrogen bonding with Asp 545 and arene cation interaction with His 759.

Optimization of Light Intensity and NaNO3 Concentration in Amazon Cyanobacteria Cultivation to Produce Biodiesel.

The objective of this study, for the first time, was to optimize Amazonian cyanobacterial culture conditions for improving cell productivity and lipid content, by analyzing the effect of light intensity and nitrogen concentration, for empirically evaluating biodiesel quality parameters. The strains Synechocystis sp. CACIAM05, Microcystis aeruginosa CACIAM08, Pantanalinema rosaneae CACIAM18, and Limnothrix sp. CACIAM25, were previously identified by morphological and molecular analysis (16S rRNA) and were selected based on their production of chlorophyll a and dry cell weight. Then, factorial planning (22) with central points was applied, with light intensity and NaNO3 concentration as independent variables. As response variables, cell productivity and lipid content were determined. Statistical analysis indicated that for all strains, the independent variables were statistically significant for cell productivity. Analysis of the fatty acid composition demonstrated diversity in the composition of the fatty acid profile from the experimental planning assays of each strain. The Biodiesel Analyzer software predicted the biodiesel quality parameters. CACIAM05 and CACIAM25 obtained better parameters with low levels of light intensity and NaNO3 concentration, whereas CACIAM08 and CACIAM18 obtained better parameters with low NaNO3 concentrations and high luminous intensity.

A strain of Streptomyces sp. isolated from rhizospheric soil of Crataegus oxycantha producing nalidixic acid, a synthetic antibiotic.

AIM: Exploration of microbes isolated from rhizospheric soil of Crataegus oxycantha for bioactive natural products. METHODS AND RESULTS: A strain of Streptomyces sp. (C-7) was isolated from rhizospheric soil of C. oxycantha. The 16S rRNA gene sequence of strain C-7 displayed 99% sequence similarity with different Streptomyces species. The highest score was displayed for Streptomyces sp. strain Chy2-8 followed by Streptomyces violarus strain NBRC13104 and Streptomyces arenae strain ISP5293. The position of C-7 in the phylogenetic tree suggested uniqueness of the strain. Nalidixic acid (1), a quinolone antibiotic, was isolated from Streptomyces sp. strain (C-7) for the first time and characterized by NMR and chemically analysed. Compound 1 exhibited antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. The production of compound 1 was also validated by repeating fermentation of strain C-7 and compound isolation in a separate natural product laboratory with no prior information. Furthermore, Compound 1 showed a cytotoxic effect against human prostate cancer cell line PC3 with an IC50 11 µg ml-1 . CONCLUSION: To the best of our knowledge, this is the first report showing production of nalidixic acid naturally by a strain of Streptomyces sp. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we isolated a strain of Streptomyces sp. producing nalidixic acid, which was otherwise only obtained through chemical synthesis.

Differential Interaction of Dantrolene, Glafenine, Nalidixic Acid, and Prazosin with Human Organic Anion Transporters 1 and 3.

In renal proximal tubule cells, the organic anion transporters 1 and 3 (OAT1 and OAT3) in the basolateral membrane and the multidrug resistance-associated protein 4 (MRP4) in the apical membrane share substrates and co-operate in renal drug secretion. We hypothesized that recently identified MRP4 inhibitors dantrolene, glafenine, nalidixic acid, and prazosin also interact with human OAT1 and/or OAT3 stably transfected in human embryonic kidney 293 cells. These four drugs were tested as possible inhibitors of p-[3H]aminohippurate (PAH) and [14C]glutarate uptake by OAT1, and of [3H]estrone-3-sulfate (ES) uptake by OAT3. In addition, we explored whether these drugs decrease the equilibrium distribution of radiolabeled PAH, glutarate, or ES, an approach intended to indirectly suggest drug/substrate exchange through OAT1 and OAT3. With OAT3, a dose-dependent inhibition of [3H]ES uptake and a downward shift in [3H]ES equilibrium were observed, indicating that all four drugs bind to OAT3 and may possibly be translocated. In contrast, the interaction with OAT1 was more complex. With [14C]glutarate as substrate, all four drugs inhibited uptake but only glafenine and nalidixic acid shifted glutarate equilibrium. Using [3H]PAH as a substrate of OAT1, nalidixic acid inhibited but dantrolene, glafenine, and prazosin stimulated uptake. Nalidixic acid decreased equilibrium content of [3H]PAH, suggesting that it may possibly be exchanged by OAT1. Taken together, OAT1 and OAT3 interact with the MRP4 inhibitors dantrolene, glafenine, nalidixic acid, and prazosin, indicating overlapping specificities. At OAT1, more than one binding site must be assumed to explain substrate and drug-dependent stimulation and inhibition of transport activity.

Listeria phage and phage tail induction triggered by components of bacterial growth media (phosphate, LiCl, nalidixic acid, and acriflavine).

The detection of Listeria monocytogenes from food is currently carried out using a double enrichment. For the ISO methodology, this double enrichment is performed using half-Fraser and Fraser broths, in which the overgrowth of L. innocua can occur in samples where both species are present. In this study, we analyzed the induction of phages and phage tails of Listeria spp. in these media and in two brain heart infusion (BHI) broths (BHIM [bioMérieux] and BHIK [Biokar]) to identify putative effectors. It appears that Na2HPO4 at concentrations ranging from 1 to 40 g/liter with an initial pH of 7.5 can induce phage or phage tail production of Listeria spp., especially with 10 g/liter of Na2HPO4 and a pH of 7.5, conditions present in half-Fraser and Fraser broths. Exposure to LiCl in BHIM (18 to 21 g/liter) can also induce phage and phage tail release, but in half-Fraser and Fraser broths, the concentration of LiCl is much lower (3 g/liter). Low phage titers were induced by acriflavine and/or nalidixic acid. We also show that the production of phages and phage tails can occur in half-Fraser and Fraser broths. This study points out that induction of phages and phage tails could be triggered by compounds present in enrichment media. This could lead to a false-negative result for the detection of L. monocytogenes in food products.

A diaminopimelic acid auxotrophic Escherichia coli donor provides improved counterselection following intergeneric conjugation with actinomycetes.

Considering the medical, biotechnological, and economical importance of actinobacteria, there is a continuous need to improve the tools for genetic engineering of a broad range of these microorganisms. Intergeneric conjugation has proven to be a valuable yet imperfect tool for this purpose. The natural resistance of many actinomycetes to nalidixic acid (Nal) is generally exploited to eliminate the sensitive Escherichia coli donor strain following conjugation. Nevertheless, Nal can delay growth and have other unexpected effects on the recipient strain. To provide an improved alternative to antibiotics, we propose a postconjugational counterselection using a diaminopimelic acid (DAP) auxotrophic donor strain. The DAP-negative phenotype was obtained by introducing a dapA deletion into the popular methylase-negative donor strain E. coli ET12567/pUZ8002. The viability of ET12567 and its ΔdapA mutant exposed to DAP deprivation or Nal selection were compared in liquid pure culture and after mating with Streptomyces coelicolor. Results showed that death of the E. coli ΔdapA Nal-sensitive donor strain occurred more efficiently when subjected to DAP deprivation than when exposed to Nal. Our study shows that postconjugational counterselection based on DAP deprivation circumvents the use of antibiotics and will facilitate the transfer of plasmids into actinomycetes with high biotechnological potential, yet currently not accessible to conjugative techniques.

Synthesis and evaluation of uniformly sized nalidixic acid-imprinted nanospheres based on precipitation polymerization method for analytical and biomedical applications.

For the first time in this work, uniform molecularly imprinted polymer (MIP) nanoparticles were prepared using nalidixic acid as a template. The MIP nanoparticles were successfully synthesized by precipitation polymerization applying methacrylic acid (MAA) as a functional monomer and trimethylolpropane trimethacrylate (TRIM) as a cross-linking monomer at different mole ratios. The morphology, binding, recognition, selectivity, and in vitro release behaviors of obtained particles were studied. The produced polymers were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetric. Furthermore, their morphology was analyzed accurately by scanning electron microscopy, photon correlation spectroscopy, and Brunauer-Emmett-Teller analysis. The nanospheres and microspheres with mean diameter values of 94 nm, 256 nm, and 1.2 µm were obtained using nalidixic acid-MAA-TRIM various mole ratios. Among the MIPs, the product with nalidixic acid-MAA-TRIM mole ratio of 1:12:12 established nanospheres with the lowest polydispersity index (0.003), an average pore diameter (12 nm), and the highest specific surface area (280 m(2) g(-1)) and selectivity factor (10.4). Results from binding experiments demonstrated that the imprinted nanospheres with a 94-nm mean diameter and a binding capacity of 28 mg of nalidixic acid per gram of polymer had higher specific affinity to nalidixic acid in contrast with the other imprinted nanospheres, microspheres, and nonimprinted particles. However, the binding performance of imprinted nanospheres in human serum was estimated using high-performance liquid chromatography analysis (binding approximately 98% of nalidixic acid). In addition, release experiments proved to be successful in the controlled release of nalidixic acid during a long period. The 20% of loaded nalidixic acid was released from the imprinted nanospheres within the first 20 h, whereas the remaining 80% was released in the after 120 h. The nalidixic acid release kinetics from the MIPs was highly affected by properties of the particles.

Genetic Determinants of Hydrogen Sulfide Biosynthesis in Fusobacterium nucleatum Are Required for Bacterial Fitness, Antibiotic Sensitivity, and Virulence.

The Gram-negative anaerobe Fusobacterium nucleatum is a major producer of hydrogen sulfide (H2S), a volatile sulfur compound that causes halitosis. Here, we dissected the genetic determinants of H2S production and its role in bacterial fitness and virulence in this important member of the oral microbiome. F. nucleatum possesses four enzymes, CysK1, CysK2, Hly, and MegL, that presumably metabolize l-cysteine to H2S, and CysK1 was previously shown to account for most H2S production in vitro, based on correlations of enzymatic activities with gene expression at mid-log phase. Our molecular studies showed that cysK1 and megL were highly expressed at the late exponential growth phase, concomitant with high-level H2S production, while the expression levels of the other genes remained substantially lower during all growth phases. Although the genetic deletion of cysK1 without supplementation with a CysK1-catalyzed product, lanthionine, caused cell death, the conditional ΔcysK1 mutant and a mutant lacking hly were highly proficient in H2S production. In contrast, a mutant devoid of megL showed drastically reduced H2S production, and a cysK2 mutant showed only minor deficiencies. Intriguingly, the exposure of these mutants to various antibiotics revealed that only the megL mutant displayed altered susceptibility compared to the parental strain: partial sensitivity to nalidixic acid and resistance to kanamycin. Most significantly, the megL mutant was attenuated in virulence in a mouse model of preterm birth, with considerable defects in the spread to amniotic fluid and the colonization of the placenta and fetus. Evidently, the l-methionine γ-lyase MegL is a major H2S-producing enzyme in fusobacterial cells that significantly contributes to fusobacterial virulence and antibiotic susceptibility. IMPORTANCE Fusobacterium nucleatum is a key commensal anaerobe of the human oral cavity that plays a significant role in oral biofilm development and contributes to additional pathologies at extraoral sites, such as promoting preterm birth and colorectal cancer. Although F. nucleatum is known as a major producer of hydrogen sulfide (H2S), its genetic determinants and physiological functions are not well understood. By a combination of bacterial genetics, biochemical methods, and in vivo models of infection, here, we demonstrate that the l-methionine γ-lyase MegL not only is a major H2S-producing enzyme of F. nucleatum but also significantly contributes to the antibiotic susceptibility and virulence of this organism.

Quinolone-resistant Salmonella Typhi in South Africa, 2003-2007.

In South Africa, for the years 2003-2007, the Enteric Diseases Reference Unit received 510 human isolates of Salmonella Typhi, of which 27 were nalidixic acid-resistant [minimum inhibitory concentrations (MICs) 128-512 microg/ml] with reduced susceptibility to ciprofloxacin (MICs 0.125-0.5 microg/ml). Pulsed-field gel electrophoresis analysis of 19 available isolates differentiated them into five DNA pattern types; multiple-locus variable-number tandem repeat analysis differentiated the isolates into 10 types. This level of genetic diversity suggested that resistant strains usually emerged independently of one another. A 16- to 32-fold decrease in nalidixic acid MIC and a 2- to 8-fold decrease in ciprofloxacin MIC, was observed in the presence of an efflux pump inhibitor. All isolates were negative by PCR screening for qnr genes. Seven resistant isolates were further analysed for mutations in the quinolone resistance-determining region of gyrA, gyrB, parC and parE. No amino-acid mutations were identified in GyrB and ParE; all isolates showed amino-acid mutations in both GyrA and ParC. We conclude that amino-acid mutations in GyrA and ParC in combination with active efflux of antibiotic out of the bacterial cell are the probable mechanisms conferring quinolone resistance.

Interaction of nalidixic acid and ciprofloxacin with wild type and mutated quinolone-resistance-determining region of DNA gyrase A.

The quinolones exert their anti-bacterial activity by binding to DNA gyrase A (GyrA), an essential enzyme in maintenance of DNA topology within bacterial cell. The mutations conferring resistance to quinolones arise within the quinolone-resistance-determining region (QRDR) of GyrA. Therefore, quinolones interaction with wild and mutated GyrA can provide the molecular explanation for resistance. Resistant strains of Salmonella enterica of our hospital have shown mutations in the QRDR of GyrA of serine 83 (to phenylalanine or tyrosine) or aspartic acid 87 (to glycine or tyrosine). In order to understand the association between observed resistance and structural alterations of GyrA with respect to quinolone binding, we have studied the interaction of mutated QRDR of GyrA with nalidixic acid and ciprofloxacin by molecular modeling using GLIDE v4. Analysis of interaction parameters like G-score has revealed reduced interaction between nalidixic acid/ciprofloxacin with QRDR of GyrA in all four mutated cases of resistant strains. The mutation of Ser83 to Phe or Tyr shows least binding for nalidixic acid, while Asp87 to Gly or Tyr exhibits minimal binding for ciprofloxacin. The study also highlights the important role of arginines at 21, 91 and His at 45, which form strong hydrogen bonds (at < 3 A) with quinolones. The hydrophilic OH group of Serine 83, which is in close proximity to the quinolone binding site is replaced by aromatic moieties of Tyr or Phe in mutated GyrA. This replacement leads to steric hindrance for quinolone binding. Therefore, quinolone resistance developed by Salmonella appears to be due to the decreased selectivity and affinity of nalidixic acid/ciprofloxacin to QRDR of GyrA.

Ceftibuten-containing agar plate for detecting group B streptococci with reduced penicillin susceptibility (PRGBS).

Penicillins remain first-line agents for treatment of group B Streptococcus (Streptococcus agalactiae; GBS) infections; however, several reports have confirmed the existence of GBS with reduced penicillin susceptibility (PRGBS). Because no selective agar plates for detection of PRGBS are available to date, in this investigation, we developed the selective agar plate for detection of PRGBS. We used 19 genetically well-confirmed PRGBS isolates and 38 penicillin-susceptible GBS isolates identified in Japan. For preparation of trial PRGBS-selective agar plates, we added 1 of antimicrobial agents (among oxacillin, ceftizoxime, and ceftibuten) to a well-established GBS-selective agar plate. Among 12 trial PRGBS-selective agar plates, Muller-Hinton agar containing 128 µg/mL ceftibuten with 5% sheep blood, 8 µg/mL gentamicin, and 12 µg/mL nalidixic acid was the most appropriate selective agar for PRGBS, showing 100% sensitivity and 81.6% specificity. In cases of potential nosocomial spread of PRGBS, the selective agar plate could be useful and reliable.

Mutations that Separate the Functions of the Proofreading Subunit of the Escherichia coli Replicase.

The dnaQ gene of Escherichia coli encodes the ε subunit of DNA polymerase III, which provides the 3' → 5' exonuclease proofreading activity of the replicative polymerase. Prior studies have shown that loss of ε leads to high mutation frequency, partially constitutive SOS, and poor growth. In addition, a previous study from our laboratory identified dnaQ knockout mutants in a screen for mutants specifically defective in the SOS response after quinolone (nalidixic acid) treatment. To explain these results, we propose a model whereby, in addition to proofreading, ε plays a distinct role in replisome disassembly and/or processing of stalled replication forks. To explore this model, we generated a pentapeptide insertion mutant library of the dnaQ gene, along with site-directed mutants, and screened for separation of function mutants. We report the identification of separation of function mutants from this screen, showing that proofreading function can be uncoupled from SOS phenotypes (partially constitutive SOS and the nalidixic acid SOS defect). Surprisingly, the two SOS phenotypes also appear to be separable from each other. These findings support the hypothesis that ε has additional roles aside from proofreading. Identification of these mutants, especially those with normal proofreading but SOS phenotype(s), also facilitates the study of the role of ε in SOS processes without the confounding results of high mutator activity associated with dnaQ knockout mutants.

Environmental phosphate differentially affects virulence phenotypes of uropathogenic Escherichia coli isolates causative of prostatitis.

K-12 Escherichia coli cells grown in static media containing a critical phosphate (Pi) concentration ≥25 mM maintained a high polyphosphate (polyP) level in stationary phase, impairing biofilm formation, a phenomenon that is triggered by polyP degradation. Pi concentration in human urine fluctuates according to health state. Here, the influence of environmental Pi concentration on the occurrence of virulence traits in uropathogenic E. coli (UPEC) isolated from acute prostatitis patients was evaluated. After a first screening, 3 isolates were selected according to differential biofilm formation profiles depending on media Pi concentration. For each isolate, biofilm positive and negative conditions were established. Regardless of the isolate, biofilm formation capacity was accompanied with curli and cellulose production and expression of some key virulence factors associated with adhesion. When the selected isolates were grown in their non-biofilm-forming condition, low concentrations of nalidixic acid and ciprofloxacin induced biofilm formation. Interestingly, similar to laboratory strains, polyP degradation induced biofilm formation in the selected isolates. Data demonstrated the complexity of UPEC responses to environmental Pi and the importance of polyP metabolism in the virulence of clinical isolates.

Nalidixic acid kinetics after single and repeated oral doses.

The kinetics of nalidixic acid (NA) and its two metabolites, 7-hydroxynalidixic acid (HNA) and 7-carboxynalidixic acid (CNA) were studied after a single oral dose and after two doses a day for 7 days. Total unconjugated NA, HNA, and CNA concentrations in plasma and urine samples were assayed by a new high-pressure liquid chromatographic (HPLC) technique. NA was rapidly absorbed and hydroxylated to HNA. Both were rapidly excreted in urine with an apparent elimination half-life (t1/2) of 6 to 7 hr. CNA was never detected in the plasma, although it accounted for about 25% of total urinary NA and metabolites. No major sex-related differences in the kinetics and the metabolic pattern were observed. During the 7-day treatment there was no significant cumulation of NA and HNA. The study demonstrated that the concentration of the active compounds, unconjugated NA and HNA, was more than five times the minimum bacteria inhibiting concentration for 8 hr after drug and slightly above this concentration during the next 4 hr.

Pharmacokinetics of nalidixinic acid and oxolinic acid in healthy women.

The pharmacokinetics of oral nalidixic acid (NA, 1 gm 4 times a day) and oxolinic acid (OA, 750 mg 2 times a day) administered for 7 days were studied in the same 10 healthy women on the first, third, and seventh days of the treatment. The peak concentrations of NA + OH-NA (hydroxynalidixinic acid) in serum at 2 to 3 hr were 34 mug/ml (total) and 23 mug/ml (unconjugated) on the first day and nearly two times higher on the third and seventh days; 82% to 85% of these amounts were NA. The protein-free fraction of NA + OH-NA was 8.8% to 18.3%. The total concentration of NA + OH-NA in urine was 1,220 to 2,700 mug/ml, the unconjugated concentration, 250 to 350 mug/ml, and the chemotherapeutically active concentration, 55 to 75 mug/ml. In steady state the 24-hr recovery of the total drug was 79% of the daily dose. The excretion rate in urine was 591 to 853 mg/6 hr. The OA concentration in serum was very low on the first day of the treatment, but increased to 4- to 5-fold on the third and seventh days: 6.2 to 6.4 mug/ml (total) and 3.3 to 3.6 mug/ml (unconjugated). The protein-free OA represented 19% to 23% of the total amount. The modest initial serum concentrations of OA were confirmed by the low urine concentrations on the first day. In steady state the OA concentration in urine was 570 mug/ml (total) and 35 mug/ml (unconjugated), and the 24-hr recovery, 49% and 3%, respectively. The microbiologic assay gave somewhat higher concentrations of the active drug than did the chemical assay. When taken with food, the excretion of OA in urine was retarded by 6 hr but the 48-hr recovery was not decreased.

A prodrug approach toward the development of water soluble fluoroquinolones and structure--activity relationships of quinoline-3-carboxylic acids.

A fluoroquinolone prodrug, PA2808, was prepared and shown to convert to the highly active parent drug PA2789. In vitro and in vivo activation of PA2808 by alkaline phosphatase was demonstrated using disk diffusion and rat lung infection models. The water solubility of PA2808 showed a marked increase compared to PA2789 over a pH range suitable for aerosol drug delivery. A total of 48 analogues based on PA2789 were prepared and screened against a panel of Gram-positive and Gram-negative pathogens. Incorporating a cyclopropane-fused pyrrolidine (amine) at C-7 resulted in some of the most active analogues.

Pharmacokinetics of nalidixic acid in old and young volunteers.

Plasma levels of active naphthyridine were measured in healthy old (mean age 77.3 years) and young (mean age 25.4 years) subjects following oral administration of 1 Gm nalidixic acid. The plasma levels of active naphthyridine, i.e., nalidixic acid and its metabolite 7-hydroxynalidixic acid, were generally higher in the old group, particularly in the elimination phase. Significant variations in the pharmacokinetic parameters were obtained between the two groups of volunteers. The plasma half-life of active naphthyridine averaged 11.5 hours in old subjects and 2.7 hours in the younger group. Diminished renal function in the geriatric group may explain the slower elimination of the drug in the old subjects. The mean values of the other parameters calculated were: apparent volume of distribution, 0.55 (old) and 0.47 (young) liter/kg body weight; area under the curve, 327.7 (old) and 156.3 (young) micrograms/ml x hr; total body clearance 2.9 (old) and 8.3 (young) liters/hr; absorption rate constant, 0.29 (old) and 0.52 (young) hr. -1

OmpF channel permeability of quinolones and their comparison with beta-lactams.

The diffusion rates of nalidixic acid, ofloxacin and ofloxacin's two optically active isomers through OmpF channels were measured in proteoliposomes and compared with the rates of beta-lactams. The four quinolones showed high diffusion rates, exceeding that of cephaloridine and being comparable to imipenem. There was no significant difference in diffusion rate between nalidixic acid and ofloxacin, or between the two optically active isomers. The diffusion rates of enoxacin and norfloxacin were also estimated to be higher than many beta-lactams.

TbtABM, a multidrug efflux pump associated with tributyltin resistance in Pseudomonas stutzeri.

Tributyltin (TBT) is a toxic agent used in marine antifouling paints. Among the bacterial flora of a polluted harbor, TBT-resistant strains of Pseudomonas stutzeri have been isolated. In the strain 5MP1 (TBT minimum inhibitory concentration (MIC) > or =1000 mg l(-1)), TBT resistance was found to be associated with the presence of the operon tbtABM, homologous to the resistance-nodulation-cell division (RND) efflux pump family, as demonstrated by cloning in Escherichia coli. TbtABM exhibited the greatest homology (60.9-84.9%) with the TtgDEF and SrpABC systems, both involved in aromatic compound tolerance in P. putida. TbtABM conferred multidrug resistance (MDR) including to n-hexane, nalidixic acid, chloramphenicol, and sulfamethoxazole (antibiotic MICsx4 for the E. coli host strain carrying the operon). By polymerase chain reaction amplification and hybridization experiments, the presence of tbtABM was detected in the TBT-sensitive P. stutzeri 3MP1 (TBT MIC 25 mg l(-1)). However, the latter strain did not seem to express TbtABM. This is the first description of a MDR efflux pump in P. stutzeri, and of a new kind of substrate, TBT, for the RND family of transporters.

DNA breakdown by the 4-quinolones and its significance.

DNA breakdown occurred in Escherichia coli KL16 exposed to nalidixic acid, ciprofloxacin or norfloxacin. However DNA breakdown does not seem to be the cause of the lethality of the 4-quinolones because it still occurred under conditions which abolished the lethality of nalidixic acid. Furthermore, no correlation was found between the amount of DNA breakdown and the rate of death of bacteria caused by the three 4-quinolones. Similarly, DNA breakdown did not occur when recB or recC mutants were treated with nalidixic acid despite both mutants being killed by the drug, again suggesting that DNA breakdown is not the cause of bacterial death. Since recB and recC mutants lack exonuclease V, this enzyme may be responsible for the DNA breakdown observed in bacteria treated with 4-quinolones.