In-situ enumeration and probing of pyrene-degrading soil bacteria

Inferences about which microorganisms degrade polycyclic aromatic hydrocarbons in contaminated soils have largely been obtained using culture-based techniques, despite the low percentage of microorganisms in soil that are believed to be culturable. We used a substrate-responsive direct viable count method to identify and quantify potential polycyclic aromatic hydrocarbon-degrading bacteria in a soil containing petroleum wastes. Bacteria were extracted and their response to substrates determined in the presence of DNA gyrase inhibitors, which cause viable and active cells to elongate. When yeast extract, a widely used carbon source, was added as a growth substrate, together with nalidixic acid, piromidic acid and ciprofloxacin, a significant increase in elongated cells to 47%, 37% and 22%, respectively, was observed within 24 h. With pyrene as the main substrate, 10 mg L(-1) of nalidixic acid or piromidic acid caused 18-22% and 8-12%, respectively, of the cells to elongate within 24 h; whereas the effect of 0.5 mg L(-1) ciprofloxacin was not significant until 53 h later. Enlarged cells were identified and enumerated by fluorescent in situ hybridization, using Alpha-, Beta- and Gammaproteobacteria, and domain Bacteria-specific probes. The Bacteria-specific probe detected 35-71% of the total microorganisms detected by the DNA-binding dye 4,6-diamidino-2-phenylindole. Initially, 44%, 13% and 5% of the total bacteria in the soil extract were Alpha-, Beta- and Gammaproteobacteria, respectively. Without pyrene or a gyrase inhibitor, these subgroups decreased to 30% of the total population but were predominant with piromidic acid or unchanged with ciprofloxacin when pyrene was the main substrate. The proportion of elongated Alpha- and Betaproteobacteria (potential pyrene degraders) increased significantly (P<0.05). This approach links phylogenetic information with physiological function in situ without the conventional cultivation of bacteria and can be used to probe and enumerate degradative groups at even a finer level of discrimination.     

Real-time PCR detection of Pseudomonas aeruginosa in clinical and municipal wastewater and genotyping of the ciprofloxacin-resistant isolates

Real-time quantification of Pseudomonas aeruginosa was performed in various wastewater systems including clinical, municipal wastewaters and inflow from a wastewater treatment plant. The highest concentrations of P. aeruginosa-specific targets were detected in clinical wastewaters. Limitations of the detection system resulting from inhibition or cross-reaction were identified. Ciprofloxacin-resistant P. aeruginosa strains were isolated after specific enrichment from clinical and municipal wastewaters. In some cases they were also cultivated from effluent of a wastewater treatment plant, and from its downstream river water. A total of 119 isolates were phenotypically characterized as ciprofloxacin-resistant via antibiogram testing. Subsequently, the fluoroquinolone-resistance-mediating mutations in the genes gyrA codon positions 83 and 87, gyrB codon position 466 and parC codon positions 87 and 91 were determined by mini-sequencing. Ciprofloxacin resistance was mainly associated with mutations in gyrA codon position 83 and parC mutation in codon positions 87 or 91 of the bacterial gyrase and topoisomerase II genes. All ciprofloxacin-resistant P. aeruginosa strains were compared with genotypes from clinical data of fluoroquinolone-resistant P. aeruginosa infections. The results were in agreement with data from clinical analyses, with the exception that no gyrA 87 and no gyrB mutations were found in ciprofloxacin-resistant P. aeruginosa wastewater isolates.     

4-Quinolones cause a selective loss of mitochondrial DNA from mouse L1210 leukemia cells

The 4-quinolone antibiotics nalidixic acid and ciprofloxacin and potent inhibitors of the bacterial type II topoisomerase DNA gyrase. Treatment of mouse L1210 leukemia cells with these drugs resulted in a delayed inhibition of cell proliferation. Prior to inhibition of cell proliferation, there was a time-dependent decrease in the cellular content of mitochondrial DNA (mtDNA). The decrease in mtDNA was associated with a decrease in the rate of mitochondrial respiration and an increase in the concentration of lactate in the growth medium. Inhibition of cell proliferation by 4-quinolones was reversible upon drug washout. However, there was a 2- to 4-day lag before the growth rate returned to normal levels. This was preceeded by an increase in mtDNA content and mitochondrial respiration. These studies suggest that inhibition of mammalian cell proliferation by 4-quinolone drugs is related to the selective depletion of mtDNA. © 1993 Wiley-Liss, Inc.     

Antimicrobial and antifouling efficacy of urinary catheters impregnated with a combination of macrolide and fluoroquinolone antibiotics against Pseudomonas aeruginosa

The incidence of catheter associated urinary tract infections (CAUTIs) is increasing worldwide. This study was designed to modify a biomaterial by impregnating a silicone urinary catheter with combination of a macrolide, azithromycin (AZM) and a fluoroquinolone, ciprofloxacin (CIP). Drug release profiles showed slow yet continuous release of antibiotics from catheters for one month. In vitro efficacy testing showed that group B catheters [3% (w v^?1) CIP + 6% (w v^?1) AZM] outperformed group A catheters [2% (w v^?1) CIP + 5% (w v^?1) AZM] by (1) showing larger zones of inhibition (>31 mm) compared to group A (<28 mm) for up to 30 days against Pseudomonas aeruginosa PAO1; (2) killing adhered bacteria in 24 h compared to 24–48 h in group A; (3) showing longer antimicrobial durability for four weeks; and (4) exhibiting a stable real-time shelf life of one year, suggesting that these catheters can be explored in clinical settings, especially in long-term CAUTI.     

Synthesis of amides from (E)-3-(1-chloro-3,4-dihydronaphthalen-2-yl)acrylic acid and substituted amino acid esters as NorA efflux pump inhibitors of Staphylococcus aureus

Inhibitors for NorA efflux pump of Staphylococcus aureus have attracted the attention of many researchers towards the discovery and development of novel efflux pump inhibitors (EPIs). In an attempt to find specific potent inhibitors of NorA efflux pump of S. aureus, a total of 15 amino acid conjugates of 3-(1-chloro-3,4-dihydronaphthalen-2-yl)acrylic acid (4–18) were synthesized using a simple convenient synthetic approach and bioevaluated against NorA efflux pump. Two compounds 7 and 8 (each having MEC of 1.56?µg/mL) were found to restore the activity of ciprofloxacin through reduction of the MIC elucidated by comparing the ethidium bromide efflux in dose dependent manner in addition to ethidium bromide efflux inhibition and accumulation study using NorA overexpressing strain SA-1199B. Most potent compounds among these were able to restore the antibacterial activity of ciprofloxacin completely against SA-1199B. Structure activity relationship (SAR) studies and docking study of potent compounds 7 and 8 could elucidate the structural requirements necessary for interaction with the NorA efflux pumps. On the whole, compounds 7 and 8 have ability to reverse the NorA efflux mediated resistance and could be further optimized for development of potent efflux pump inhibitors.     

Pseudomonas aeruginosa deficient in 8-oxodeoxyguanine repair system shows a high frequency of resistance to ciprofloxacin

The 8-oxodeoxyguanine (8-oxodG) repair system participates in the prevention and correction of mutations generated by oxidative DNA damage in prokaryotes and eukaryotes. In this study, we report that Pseudomonas aeruginosa strains deficient in this repair mechanism by inactivation of the mutT, mutM and mutY genes generate a high frequency of cells resistant to the antibiotic ciprofloxacin. In the mutT strain, the increase in ciprofloxacin resistance achieved at threefold minimal inhibitory concentration was about 1600-fold over the wild-type (WT) level, similar to the frequency achieved by the mismatch repair-deficient mutS strain. Molecular analysis of WT, mutT and mutY clones resistant to ciprofloxacin indicated that the nfxB gene was mutated in the majority of the cases, while mutS- derived resistant clones were mainly mutated in gyrA and parC genes. Cell viability analysis after treatment with paraquat or hydrogen peroxide indicated that 8-oxodG repair-deficient strains were considerably more susceptible to oxidative stress than the parental strain. Finally, it is shown that the ciprofloxacin resistance frequency of WT and repair-deficient strains increased significantly after cell exposure to paraquat. Thus, oxidative stress is strongly implicated in the emergence of ciprofloxacin-resistant mutants in P. aeruginosa , and the 8-oxodG repair pathway plays an important role in the prevention of these mutations.     

Enzyme structural plasticity and the emergence of broad-spectrum antibiotic resistance

The emergence of multi-resistant pathogenic bacteria is a worldwide health issue. Recently, clinical variants of a single antibiotic-modifying acetyltransferase, AAC(6')-Ib-a variant of aminoglycoside 6'-N-acetyltransferase-have been identified that confer extended resistance to most aminoglycosides and, more surprisingly, to structurally unrelated fluoroquinolones. The corresponding gene is carried by mobile genetic elements and is present in most multi-resistant pathogenic strains, hence making it a serious threat to current therapies. Here, we report the crystal structures of both narrow- and broad-spectrum resistance variants of this enzyme, which reveal the structural basis for the emergence of extended resistance. The active site shows an important plasticity and has adapted to new substrates by a large-scale gaping process. We have also obtained co-crystals with both substrates, and with a simple transition state analogue, which provides new clues for the design of inhibitors of this resistance mechanism.     

Molecular properties of ciprofloxacin-indion 234 complexes

The purpose of this research was to formulate tasteless complexes of ciprofloxacin with Indion 234 and to evaluate molecular properties of drug complexes. The effect of batch and column process, complexation time, temperature, and pH on ciprofloxacin loading on Indion 234 is reported. Drug resin complexes (DRC) were characterized by infrared spectroscopy, thermal analysis, and x-ray diffraction pattern. Ciprofloxacin release from DRC is obtained at salivary and gastric pH and in the presence of electrolytes. The efficient drug loading was evident in batch process using activated Indion 234 with a drug-resin ratio of 1∶1.3. Drug complexation enhanced with pH from 1.2 to 6, while temperature did not affect the complexation process. Infrared spectroscopy revealed complexation of—NH (drug) with Indion 234. DRC are amorphous in nature. Drug release from DRC in salivary pH was insufficient to impart bitter taste. Volunteers rated the complex as tasteless and agreeable. Complete drug release was observed at gastric pH in 2 hours. The drug release was accelerated in the presence of electrolytes. Indion 234 is inexpensive, and the simple technique is effective for bitterness masking of ciprofloxacin.     

Bcl-2 down modulation in WEHI-3B/CTRES cells resistant to Cholera Toxin （CT）-induced apoptosis

The very different effects of Cholera Toxin （CT） on cell growth and proliferation may depend on the type of ganglioside receptors in cell membranes and different signal transduction mechanisms triggered, but other functions related to the drug resistance mechanisms can not be excluded. The effect of CT treatment on the ＂in vitro＂ clonogenicity, the Population Doubling Time （PDT）, apoptosis, PKA activation and Bax and Bcl-2 expression was evaluated in WEHI-3B cell line and its CT-resistant subclone （WEHI-3B/CTRES）. In WEHI-3B parental cells the dramatic accumulation of cAMP induced by CT correlated well with PKA activation, increased PDT value, inhibition of clonogenicity and apoptosis. H-89 treatment inhibited PKA activation by CT but did not protect the cells from apoptosis and growth inhibition. In WEHI-3B/CTRES no significant CT-dependent accumulation of cAMP occurred with any increase of PKA activity and PDT. In CT resistant cells （WEHI-3B/CTRES）, Bcl-2 expression was down regulated by both CT or drug treatment （eg, ciprofloxacin, CPX） although these cells were protected from CT-dependent apoptosis but not from drug-induced apoptosis. Differently from other cell models described, down regulation of Bcl-2 is proved to be independent on cAMP accumulation and PKA activation. Our observations support the implication of cAMP dependent kinase （PKA） in the inhibition of WEHI-3B cells growth and suggest that, in WEHI-3B/CTRES, Bcl-2 expression could be modulated by CT in the absence of cAMP accumulation. Also in consideration of many contradictory data reported in literature, our cell models （of one sensitive parental cell strain and two clones with different uncrossed specific resistance to CT and CPX） provides a new and interesting tool for better investigating the relationship between the CT signal transduction mechanisms and Bcl-2 expression and function.     

Quercetin protection against ciprofloxacin induced liver damage in rats

Ciprofloxacin is a common, broad spectrum antibacterial agent; however, evidence is accumulating that ciprofloxacin may cause liver damage. Quercetin is a free radical scavenger and antioxidant. We investigated histological changes in hepatic tissue of rats caused by ciprofloxacin and the effects of quercetin on these changes using histochemical and biochemical methods. We divided 28 adult female Wistar albino rats into four equal groups: control, quercetin treated, ciprofloxacin treated, and ciprofloxacin + quercetin treated. At the end of the experiment, liver samples were processed for light microscopic examination and biochemical measurements. Sections were prepared and stained with hematoxylin and eosin, and a histopathologic damage score was calculated. The sections from the control group appeared normal. Hemorrhage, inflammatory cell infiltration and intracellular vacuolization were observed in the ciprofloxacin group. The histopathological findings were reduced in the group treated with quercetin. Significant differences were found between the control and ciprofloxacin groups, and between the ciprofloxacin and ciprofloxacin + quercetin groups. Quercetin administration reduced liver injury caused by ciprofloxacin in rats. We suggest that quercetin may be useful for preventing ciprofloxacin induced liver damage.