Comparative analysis of SilA-laccase mediated degradation of ciprofloxacin, norfloxacin and ofloxacin and interpretation of the possible catalytic mechanism.

Fluoroquinolones (FQs) are the most commonly used antimicrobial drugs and regardless of their advantages in the healthcare sector, the pollution of these antimicrobial drugs in the environment has big concerns about human and environmental health. The presence of these antibiotic drugs even at the lowest concentrations in the environment has resulted in the emergence and spread of antibiotic resistance. Hence, it is necessary to remediate these pollutants from the environment. Previously alkaline laccase (SilA) from Streptomyces ipomoeae has been demonstrated to show degrading potentials against two of the FQs, Ciprofloxacin (CIP) and Norfloxacin (NOR); however, the molecular mechanism was not elucidated in detail. In this study, we have analyzed the possible molecular catalytic mechanism of FQ degrading SilA-laccase for the degradation of the FQs, CIP, NOR and Ofloxacin (OFL) using three-dimensional protein structure modeling, molecular docking and molecular dynamic (MD) studies. The comparative protein sequence analysis revealed the presence of tetrapeptide conserved catalytic motif, His102-X-His104-Gly105. After evaluating the active site of the enzyme in depth using CDD, COACH and S-site tools, we have identified the catalytic triad composed of three conserved amino acid residues, His102, Val103 and Tyr108 with which ligands interacted during the catalysis process. By analyzing the MD trajectories, it is revealed that the highest degradation potential of SilA is for CIP followed by NOR and OFL. Ultimately, this study provides the possible comparative catalytic mechanism for the degradation of CIP, NOR and OFL by the SilA enzyme.Communicated by Ramaswamy H. Sarma.

Study effect and mechanism of ofloxacin and levofloxacin on development of Rana nigromaculata tadpoles based on the hypothalamus-pituitary-thyroid axis.

Excessive antibiotics transferred into the aquatic environment may affect the development of amphibians. Previous studies on the aquatic ecological risk of ofloxacin generally ignored its enantiomers. The purpose of this study was to compare the effects and mechanisms of ofloxacin (OFL) and levofloxacin (LEV) on the early development of Rana nigromaculata. After 28-day exposure at environmental levels, we found that LEV exerted more severe inhibitory effects on the development of tadpoles than OFL. According to the enrichment results of differentially expressed genes in the LEV and OFL treatments, LEV and OFL had different effects on the thyroid development of tadpoles. dio2 and trh were affected by the regulation of dexofloxacin instead of LEV. At the protein level, LEV was the main component that affected thyroid development-related protein, while dexofloxacin in OFL had little effect on thyroid development. Furthermore, molecular docking results further confirmed that LEV was a major component affecting thyroid development-related proteins, including DIO and TSH. In summary, OFL and LEV regulated the thyroid axis by differential binding to DIO and TSH proteins, thereby exerting differential effects on the thyroid development of tadpoles. Our research is of great significance for comprehensive assessment of chiral antibiotics aquatic ecological risk.

Responses of cyanobacterium Microcystis aeruginosa under single and repeated ofloxacin exposure.

Antibiotics are omnipresent and pseudo-persistent in the environment. Yet, their potential ecological risks under repeated exposure, which is more environmentally relevant, are understudied. Therefore, this study used ofloxacin (OFL) as the probe chemical to investigate the toxic effects of different exposure scenarios-single dose of high concentration (4.0 µg/L) and multiple additions of low concentrations-towards the cyanobacterium Microcystis aeruginosa. Flow cytometry was employed to measure a collection of biomarkers, including endpoints related with biomass, single cell properties and physiological status. Results showed that the single dose of the highest OFL level inhibited cellular growth, chl-a content and cell size of M. aeruginosa. In contrast, OFL induced stronger chl-a autofluorescence and higher doses tended to have more remarkable effects. Repeated low OFL doses can more significantly increase the metabolic activity of M. aeruginosa than a single high dose. Viability and cytoplasmic membrane were not affected by OFL exposure. Oxidative stress was observed for the different exposure scenarios, with fluctuating responses. This study demonstrated the different physiological responses of M. aeruginosa under different OFL exposure scenarios, providing novel insights into the toxicity of antibiotics under repeated exposure.

Pseudomonas aeruginosa Can Diversify after Host Cell Invasion to Establish Multiple Intracellular Niches.

Within epithelial cells, Pseudomonas aeruginosa depends on its type III secretion system (T3SS) to escape vacuoles and replicate rapidly in the cytosol. Previously, it was assumed that intracellular subpopulations remaining T3SS-negative (and therefore in vacuoles) were destined for degradation in lysosomes, supported by data showing vacuole acidification. Here, we report in both corneal and bronchial human epithelial cells that vacuole-associated bacteria can persist, sometimes in the same cells as cytosolic bacteria. Using a combination of phase-contrast, confocal, and correlative light-electron microscopy (CLEM), we also found they can demonstrate biofilm-associated markers: cdrA and cyclic-di-GMP (c-di-GMP). Vacuolar-associated bacteria, but not their cytosolic counterparts, tolerated the cell-permeable antibiotic ofloxacin. Surprisingly, use of mutants showed that both persistence in vacuoles and ofloxacin tolerance were independent of the biofilm-associated protein CdrA or exopolysaccharides (Psl, Pel, alginate). A T3SS mutant (ΔexsA) unable to escape vacuoles phenocopied vacuole-associated subpopulations in wild-type PAO1-infected cells, with results revealing that epithelial cell death depended upon bacterial viability. Intravital confocal imaging of infected mouse corneas confirmed that P. aeruginosa formed similar intracellular subpopulations within epithelial cells in vivo. Together, these results show that P. aeruginosa differs from other pathogens by diversifying intracellularly into vacuolar and cytosolic subpopulations that both contribute to pathogenesis. Their different gene expression and behavior (e.g., rapid replication versus slow replication/persistence) suggest cooperation favoring both short- and long-term interests and another potential pathway to treatment failure. How this intracellular diversification relates to previously described "acute versus chronic" virulence gene-expression phenotypes of P. aeruginosa remains to be determined. IMPORTANCE Pseudomonas aeruginosa can cause sight- and life-threatening opportunistic infections, and its evolving antibiotic resistance is a growing concern. Most P. aeruginosa strains can invade host cells, presenting a challenge to therapies that do not penetrate host cell membranes. Previously, we showed that the P. aeruginosa type III secretion system (T3SS) plays a pivotal role in survival within epithelial cells, allowing escape from vacuoles, rapid replication in the cytoplasm, and suppression of host cell death. Here, we report the discovery of a novel T3SS-negative subpopulation of intracellular P. aeruginosa within epithelial cells that persist in vacuoles rather than the cytoplasm and that tolerate a cell-permeable antibiotic (ofloxacin) that is able to kill cytosolic bacteria. Classical biofilm-associated markers, although demonstrated by this subpopulation, are not required for vacuolar persistence or antibiotic tolerance. These findings advance our understanding of how P. aeruginosa hijacks host cells, showing that it diversifies into multiple populations with T3SS-negative members enabling persistence while rapid replication is accomplished by more vulnerable T3SS-positive siblings. Intracellular P. aeruginosa persisting and tolerating antibiotics independently of the T3SS or biofilm-associated factors could present additional challenges to development of more effective therapeutics.

Removal of oxytetracycline and ofloxacin in wastewater by microalgae-bacteria symbiosis for bioenergy production.

The development of microalgae-bacteria symbiosis for treating wastewater is flourishing owing to its high biomass productivity and exceptional ability to purify contaminants. A nature-selected microalgae-bacteria symbiosis, mainly consisting of Dictyosphaerium and Pseudomonas, was used to treat oxytetracycline (OTC), ofloxacin (OFLX), and antibiotic-containing swine wastewater. Increased antibiotic concentration gradually reduced biomass productivity and intricately changed symbiosis composition, while 1 mg/L OTC accelerated the growth of symbiosis. The symbiosis biomass productivity reached 3.4-3.5 g/L (5.7-15.3 % protein, 18.4-39.3 % carbohydrate, and 2.1-3.9 % chlorophyll) when cultured in antibiotic-containing swine wastewater. The symbiosis displayed an excellent capacity to remove 76.3-83.4 % chemical oxygen demand, 53.5-62.4 % total ammonia nitrogen, 97.5-100.0 % total phosphorus, 96.3-100.0 % OTC, and 32.8-60.1 % OFLX in swine wastewater. The microbial community analysis revealed that the existence of OTC/OFLX increased the richness and evenness of microalgae but reduced bacteria species in microalgae-bacteria, and the toxicity of OFLX to bacteria was stronger than that of OTC.

3-Phenylpropan-1-Amine Enhanced Susceptibility of Serratia marcescens to Ofloxacin by Occluding Quorum Sensing.

Serratia marcescens (S. marcescens) is an environmental bacterium that causes infections with high morbidity and mortality. Notably, infections caused by multidrug-resistant S. marcescens have become a global public health issue. Therefore, the discovery of promising compounds to reduce the virulence of pathogens and restore antibiotic activity against multidrug-resistant bacteria is critical. Quorum sensing (QS) regulates virulence factors and biofilm formation of microorganisms to increase their pathogenicity and is, therefore, an important factor in the formation of multidrug resistance. In this study, we found that 3-phenylpropan-1-amine (3-PPA) inhibited S. marcescens NJ01 biofilm formation and virulence factors, including prodigiosin, protease, lipase, hemolysin, and swimming. The combination of 3-PPA (50.0 µg/mL) and ofloxacin (0.2 µg/mL) enhanced S. marcescens NJ01 sensitivity to ofloxacin. Based on crystalline violet staining, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM), 3-PPA (50.0 µg/mL) reduced S. marcescens NJ01 biofilm formation by 48%. Quantitative real-time PCR (qRT-PCR) showed that 3-PPA regulated the expression of virulence- and biofilm-related genes fimA, fimC, bsmB, pigP, flhC, flhD, and sodB. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that 3-PPA affected intracellular metabolites of S. marcescens NJ01, leading to reduce metabolic activity. These results suggested that 3-PPA inhibits the pathogenicity of S. marcescens NJ01 by occluding QS. Thus, 3-PPA is feasible as an ofloxacin adjuvant to overcome multidrug-resistant S. marcescens and improve the treatment of intractable infections. IMPORTANCE Multidrug-resistant bacteria have become a major threat to global public health, leading to increased morbidity, mortality, and health care costs. Bacterial virulence factors and biofilms, which are regulated by quorum sensing (QS), are the primary causes of multidrug resistance. In this study, 3-PPA reduced virulence factors and eliminated biofilm formation by inhibiting QS in S. marcescens NJ01 bacteria, without affecting bacterial growth, thus restoring sensitivity to ofloxacin. Thus, the discovery of compounds that can restore antibiotic activity against bacteria is a promising strategy to mitigate multidrug resistance in pathogens.

Effects of membrane transport activity and cell metabolism on the unbound drug concentrations in the skeletal muscle and liver of drugs: A microdialysis study in rats.

The unbound concentrations of 14 commercial drugs, including five non-efflux/uptake transporter substrates-Class I, five efflux transporter substrates-class II and four influx transporter substrates-Class III, were simultaneously measured in rat liver, muscle, and blood via microanalysis. Kpuu,liver and Kpuu,muscle were calculated to evaluate the membrane transport activity and cell metabolism on the unbound drug concentrations in the skeletal muscle and liver. For Class I compounds, represented by antipyrine, unbound concentrations among liver, muscle and blood are symmetrically distributed when compound hepatic clearance is low. And when compound hepatic clearance is high, unbound concentrations among liver, muscle and blood are asymmetrically distributed, such as Propranolol. For Class II and III compounds, overall, the unbound concentrations among liver, muscle, and blood are asymmetrically distributed due to a combination of hepatic metabolism and efflux and/or influx transporter activity.

Elucidating biotransformation pathways of ofloxacin in lettuce (Lactuca sativa L).

Antibiotics can be uptaken by plants from soil desorption or directly from irrigation water, but their metabolization pathways in plants are largely unknown. In this paper, an analytical workflow based on high-resolution mass spectrometry was applied for the systematic identification of biotransformation products of ofloxacin in lettuce. The targeted metabolites were selected by comparing the mass chromatograms of exposed with control samples using an advanced spectra-processing method (Fragment Ion Search). The innovative methodology presented allowed us to identify a total of 11 metabolites, including 5 ofloxacin metabolites that are being reported for the first time in plants. Accordingly, major transformation pathways were proposed revealing insight into how ofloxacin and related chemicals are metabolized in lettuce. Furthermore, the influence of biotransformation on potential residual antimicrobial activity of identified compounds was discussed. Human exposure to antibiotics at doses below the minimum inhibitory concentrations is crucial in human risk assessment, including food ingestion; however, in the case of ofloxacin presented results reveal that plant metabolites should also be considered so as not to underestimate their risk.

MnCeOX with high efficiency and stability for activating persulfate to degrade AO7 and ofloxacin.

An efficient MnCeOx composite was successfully synthesized for activation of persulfate to degrade acid orange 7 (AO7) and ofloxacin. Pollutants degradation efficiencies with different catalytic systems were investigated. Results showed the performance of MnCeOx was better than MnOx, CeO2 and MnOx + CeO2. Thus, there was a clear synergistic effect (Se) between Mn and Ce in the composite, and the Se was 73.8% for AO7 and 39.6% for ofloxacin. In addition, AO7 removal fitted 1st order reaction while ofloxacin removal fitted 2nd order reaction in MnCeOx/persulfate system. Moreover, MnCeOx/persulfate system showed high efficiency in pH range of 5-9. Mechanism analysis showed that SO4- and OH on the surface of the catalyst were the main active species, and O2- also played an important role in pollutants degradation. Furthermore, MnCeOx showed high activity in actual water. Finally, the possible degradation pathway of ofloxacin was proposed according to the high performance liquid chromatography-mass spectrometry result. Overall, this study provides an efficient and stable catalyst to activate persulfate to degrade refractory pollutants.

How biomimetic amino modified mesoporous silica xerogel regulates loading and in vitro sustained delivery of levorotary ofloxacin.

The purpose of this study was to facilely develop biomimetic amino modified mesoporous silica xerogel (AMSX) and study how AMSX regulated loading and in vitro sustained delivery of carboxyl-containing drug levorotary ofloxacin (LOFL). Characteristics of AMSX, including morphology, porous structure, elements and crystalline state were investigated and pharmaceutical performance of AMSX for the delivery of LOFL was studied. The result showed that AMSX was accumulational spherical nanoparticles with mesoporous structure. Hydrogen bonding force was formed between carboxylic groups of LOFL and amino groups grafted on the surface of AMSX. Furthermore, a three-level three-factorial Box-Behnken experimental design was applied to optimize the amount of major agent for synthesizing AMSX with expected drug loading capacity and also to figure out how AMSX regulated in vitro delivery of LOFL. It is believed that the present work will provide novel insights for designing mesoporous silica as drug carrier and favored the development of sustained release system.

Molecular Modification of Fluoroquinolone-Biodegrading Enzymes Based on Molecular Docking and Homology Modelling.

To improve the biodegradation efficiency of fluoroquinolone antibiotics during sewage treatment, fluoroquinolone aerobic, anaerobic and facultative degrading enzymes for fluoroquinolone degradation were modified by molecular docking and homology modelling. First, amino acid residues of the binding sites of degrading enzymes for the target fluoroquinolones ciprofloxacin (CIP), norfloxacin (NOR) and ofloxacin (OFL) were analysed by the molecular docking method. The hydrophobic amino acid residues within 5 Å of the target fluoroquinolone molecules were selected as the modification sites. The hydrophobic amino acid residues at the modified sites were replaced by the hydrophilic amino acid residues, and 150 amino acid sequence modification schemes of the degrading enzymes were designed. Subsequently, a reconstruction scheme of the degrading enzyme amino acid sequence reconstruction scheme was submitted to the SWISS-MODEL server and a selected homology modelling method was used to build a new structure of the degrading enzyme. At the same time, the binding affinities between the novel degrading enzymes and the target fluoroquinolones (represented by the docking scoring function) were evaluated by the molecular docking method. It was found that the novel enzymes can simultaneously improve the binding affinities for the three target fluoroquinolones, and the degradation ability of the six modification schemes was increased by more than 50% at the same time. Among the novel enzymes, the affinity effect of the novel anaerobic enzyme (6-1) with CIP, NOR and OFL was significantly increased, with increases of 129.24%, 165.06% and 169.59%, respectively, followed by the facultative enzyme and aerobic enzyme. In addition, the designed degrading enzymes had certain selectivity for the degradation of the target quinolone. Among the novel enzymes, the binding affinities of the novel anaerobic enzyme (6-3) and CIP, the novel aerobic enzyme (3-6) and NOR, and the novel facultative enzyme (13-6) and OFL were increased by 149.71%, 178.57% and 297.12% respectively. Calculations using the Gaussian09 software revealed that the degradation reaction barrier of the novel degrading enzyme (7-1) and CIP NOR and OFL decreased by 37.65 kcal·mol-1, 6.28 kcal·mol-1 and 6.28 kcal·mol-1, respectively, which would result in efficient degradation of the target fluoroquinolone molecules. By analysing the binding affinity of the degrading enzymes before and after the modification with methanol, it was further speculated that the degradation effect of the modified aerobic degrading enzymes on organic matter was lower than that before the modification, and the increase or decrease in the degradation effect was less than 10%. The mechanism analysis found that the interaction between the modified amino acid residues of the degrading enzymes and the fluoroquinolone molecules increased. The average distance between the amino acid residues and the fluoroquinolone molecules represented a comprehensive affinity effect, and its value was positively correlated with the degradation effect of the novel degrading enzymes.

Influence of organic carbon fractions of freshwater biofilms on the sorption for phenanthrene and ofloxacin: The important role of aliphatic carbons.

Sorption to biofilms is thought to be a crucial process controlling the fate of trace organic contaminants in aquatic systems. The organic composition of biofilms is regarded as the determining factor in the sorption mechanism of biofilm organic carbon fractions; however, its role is not well known. Here, the sorption of phenanthrene and ofloxacin was modeled with classic and emerging organic contaminants, respectively, by comparatively investigating nine type of freshwater biofilms cultured in a river, lake, and reservoir in spring, summer, and autumn. The chemical features of the nine biofilms were analyzed using elemental analysis, infrared spectroscopy, X-ray photoelectron spectroscopy, and carbon-13 nuclear magnetic resonance. Results showed that the freshwater biofilms were aliphatic-rich natural amorphous solid substances with O-containing functional groups, and their surface polarity was significantly lower than their bulk polarity. All the isotherms of phenanthrene and ofloxacin sorption by the biofilms were linear. The organic carbon-normalized partition coefficient values for phenanthrene and ofloxacin on the nine biofilms ranged from 91.9 to 364.2 L g-1 and 3.2 to 43.2 L g-1, respectively. The van der Waals interaction between a majority of aliphatic carbon (73.4%-83.9%) in biofilms and the two sorbates was much stronger than π-π interactions between a minority of aromatic carbon (12.7%-21.7%) and sorbates. The surface polarity of the biofilms regulated polar interactions including the hydrogen bonding and electron donor-acceptor interactions. Both the aliphatic carbon and surface polarity in the biofilms enhanced the sorption of phenanthrene and ofloxacin. The sorption characteristics and mechanisms of polycyclic aromatic hydrocarbons and antibiotics on biofilms shown in our present and previous studies are different from those of other ubiquitous natural solid materials such as soils and sediments. This study provides insight into the importance of aliphatic carbon fractions of freshwater biofilms for the sorption of classic and emerging organic contaminants.

Photoelectrochemical aptasensing of ofloxacin based on the use of a TiO2 nanotube array co-sensitized with a nanocomposite prepared from polydopamine and Ag2S nanoparticles.

A photoelectrochemical (PEC) method is described for aptamer-based detection of ofloxacin (OFL). It is making use of a TiO2 nanotube array (NTA) that is sensitized with a structure composed of polydopamine and silver sulfide nanoparticles. The NTA were prepared by a two-step synthetic method. First, the TiO2 nanotube electrode was covered with Ag2S nanoparticles via successive ionic layer adsorption and reaction strategy. Next, they were coated with a thin film of polydopamine (PDA) by in-situ polymerization. The inorganic/organic nanocomposites exhibit distinctly enhanced visible-light PEC activity. This was exploited to fabricate a PEC aptasensor. The PDA film serves as both the sensitizer for charge separation and as a support to bind the aptamer against OFL. The aptasensor undergoes a decrease in photocurrent due to the formation of the aptamer-OFL complex. Under the optimized conditions and at a typical working potential of 0 V (vs. Hg/Hg2Cl2), the NTA has a linear response in the 5.0 pM to 100 nM OFL concentration range and a 0.75 pM detection limit (at S/N = 3). The aptasensor was successfully applied to the determination of OFL in spiked milk samples. Graphical abstract Schematic illustration for the preparation and mechanism of the photoelectrochemical aptasensor for ofloxacin. TiO2 NTs: TiO2 nanotube arrays; PDA: polydopamine; MCH: 6-mercapto-1-hexanol; OFL: ofloxacin; PEC: photoelectrochemistry; CB: conduction band; VB: valence band; LUMO: the lowest unoccupied molecular orbital; HOMO: the highest occupied molecular orbital; AA: ascorbic acid.

Phytotoxicity and degradation of antibiotic ofloxacin in duckweed (Spirodela polyrhiza) system.

The phytotoxicity and degradation of ofloxacin (OFX) in duckweed Spirodela polyrhiza based system was estimated in this study. For that, OFX was added in an environmentally relevant range (0.01-1.0 mg L-1) in medium (Hoagland nutrient) and toxicity biomarkers, i.e. changes in plant biomass, relative growth rate (RGR), photopigment (Chl-a, Chl-b and carotenoids), protein content, antioxidative enzymes (catalase, CAT; superoxide dismutase, SOD; and ascorbate peroxidases, APX) in fronds were estimated. The batch-scale setups (250 ml) was prepared in triplicate for each concentration of OFX and reared in growth chambers (Algae Tron AG 230) for 7 d. Results suggested that the high concentrations of OFX caused a reduction in biomass (4.8-41.3%), relative root growth (RGR), protein (4.16-11.28%) and photopigment contents. The fronds in OFX spiked setups showed an increased level of antioxidative enzymes: CAT (0.230-0.338 mmolH2O2 mg-1 protein), APX (0.043-0.074 mmolascorbate mg-1 protein), and SOD (0.267-0.317 U mg-1 protein) than control. At the end (7 d), the residual OFX content in the medium was also estimated, and results suggested a significant (p < 0.05) reduction (93.73-98.36%) in OFX content than control setup (54.76-75.53%) at the end of the experimentation. The trend of residual OFX suggested phytodegradation as a significant mechanism of antibiotic degradation other than hydrolysis and photodegradation processes. This study indicates that duckweed can be an effective bio-tool for the removal of environmental relevant concentration of the antibiotics from the wastewater.

Effects of ofloxacin on nitrogen removal and microbial community structure in constructed wetland.

Constructed wetlands (CWs) have emerged as a promising technology for the purification of micro-polluted water. However, their nitrogen removal performance can be significantly degraded by design, operational, and environmental factors. The present study investigates the effects of ofloxacin (OFL: 0.1, 10, and 1000 µg L-1) and plants (Cyperus alternifolius L. and Typha angustifolia L.) on nitrogen removal in a micro-polluted CW system over a duration of 12 weeks. The effects were evaluated by investigating NH4-N and NO3-N removal efficiency, nitrification genes (amoA-AOA and amoA-AOB), denitrification genes (nirK and nirS), fungal 18S rRNA gene and microorganism community structure. The results showed that in unplanted CWs, OFL increased the NH4-N removal efficiency (from 72.6% to 80.7-82.1%), the abundances of amoA-AOA, nirS, nirK and fungal 18S rRNA gene, and the bacterial diversity but decreased the abundance of both amoA-AOB and bacterial richness. In contrast, both the nitrogen removal efficiency (83.4-89.5% for NH4-N and 33.8-38.5% for NO3-N) and bacterial diversity/richness were not significantly affected by OFL in planted CWs. In planted systems, OFL increased the relative abundance of Arthrobacter, Pseudomonas, and Enterococcus, which are proven antibiotic-resistant bacteria. This study showed that CWs are able to remove nitrogen from antibiotic-contaminated micro-polluted water, which might primarily be attributed to the presence of plants that protect the microorganism community.

The role of various transporters in the placental uptake of ofloxacin in an in vitro model of human villous trophoblasts.

INTRODUCTION: Six years after the US Food and Drug Administration approval of the broad-spectrum antibiotic ofloxacin (OFLX), the chiral switching of this racemic mixture resulted in a drug composed of the L-optical isomer levofloxacin (LVFX). Since both fluoroquinolones (FQs) were introduced to the pharmaceutical market, they have been widely prescribed by physicians, with careful administration during pregnancy and breastfeeding. Therefore, the role of the influx and efflux placental transporters in the concentrations of these drugs that permeate through human placental barrier model was investigated in this study. METHODS: The contribution of major carriers on the transplacental flux of OFLX and LVFX uptake into choriocarcinoma BeWo cells was evaluated in the presence vs absence of well-known inhibitors. RESULTS: Our results reveal that neither the influx transporters such as organic cation transporters, organic anion transporters, and monocarboxylate transporters nor the efflux transporters such as P-glycoprotein or breast cancer resistance protein significantly affected the transport of OFLX. In contrast, multiple transporters revealed pronounced involvement in the transfer of the levorotatory enantiomer in and out of the in vitro placental barrier. These data suggest a non-carrier-mediated mechanism of transport of the racemic mixture, while LVFX is subjected to major influx and efflux passage through the placental brush border membranes. CONCLUSION: This study provides underlying insights to elucidate the governing factors that influence the flux of these FQs through organ barriers, in view of the controversial safety profile of these drugs in pregnant population.

Changes in resistance to and antimicrobial activity of antibiotics during in vitro human digestion.

OBJECTIVES: This study was performed to determine changes in (i) the antimicrobial activity of antibiotics (tetracycline, ofloxacin and penicillin) and (ii) the resistance of Staphylococcus aureus RN4220 (SA RN4220) and methicillin-resistant S. aureus (MRSA) to these antibiotics during in vitro human digestion. METHODS: A human gastrointestinal digestion model simulating the conditions of the mouth, stomach, small intestine and large intestine (with intestinal microbial application) was used in this study. Antimicrobial susceptibility testing was performed by the disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: Concentrations of the three antibiotics decreased during digestion. In particular, the three antibiotics were unstable under conditions of stomach to large intestine digestion, and thus a decrease in antibiotic concentration could cause a reduction in antimicrobial activity during in vitro human digestion. The resistance of SA RN4220 and MRSA to the three antibiotics increased after in vitro human digestion. SA RN4220 and MRSA showed less resistance to ofloxacin compared with tetracycline and penicillin during in vitro human digestion. CONCLUSIONS: These results may help to explain the factors affecting antimicrobial activity and resistance to antibiotics during digestion in the human alimentary canal.

Enantioselective degradation of ofloxacin and levofloxacin by the bacterial strains Labrys portucalensis F11 and Rhodococcus sp. FP1.

Fluoroquinolones are a class of antibiotics widely prescribed in both human and veterinary medicine of high environmental concern and characterized as environmental micropollutants due to their ecotoxicity and persistence and antibacterial resistance potential. Ofloxacin and levofloxacin are chiral fluoroquinolones commercialized as racemate and in enantiomerically pure form, respectively. Since the pharmacological properties and toxicity of the enantiomers may be very different, understanding the stereochemistry of these compounds should be a priority in environmental monitoring. This work presents the biodegradation of racemic ofloxacin and its (S)-enantiomer levofloxacin by the bacterial strains Labrys portucalensis F11 and Rhodococcus sp. FP1 at a laboratory-scale microcosm following the removal and the behavior of the enantiomers. Strain F11 could degrade both antibiotics almost completely when acetate was supplied regularly to the cultures. Enrichment of the (R)-enantiomer was observed in FP1 and F11 cultures supplied with ofloxacin. Racemization was observed in the biodegradation of the pure (S)-ofloxacin (levofloxacin) by strain F11, which was confirmed by liquid chromatography - exact mass spectrometry. Biodegradation of ofloxacin at 450 µg L-1 by both bacterial strains expressed good linear fits (R2 > 0.98) according to the Rayleigh equation. The enantiomeric enrichment factors were comprised between - 22.5% to - 9.1%, and - 18.7% to - 9.0% in the biodegradation of ofloxacin by strains F11 and FP1, respectively, with no significant differences for the two bacteria under the same conditions. This is the first time that enantioselective biodegradation of ofloxacin and levofloxacin by single bacteria is reported.

Probing the stereoselective interaction of ofloxacin enantiomers with corresponding monoclonal antibodies by multiple spectrometry.

Although stereoselective antibody has immense potential in chiral compounds detection and separation, the interaction traits between stereoselective antibody and the corresponding antigenic enantiomers are not yet fully exploited. In this study, the stereospecific interactions between ofloxacin isomers and corresponding monoclonal antibodies (McAb-WR1 and McAb-MS1) were investigated using time-resolved fluorescence, steady-state fluorescence, and circular dichroism (CD) spectroscopic methods. The chiral recognition discrepancies of antibodies with ofloxacin isomers were reflected through binding constant, number of binding sites, driving forces and conformational changes. The major interacting forces of McAb-WR1 and McAb-MS1 chiral interaction systems were hydrophobic force and van der Waals forces joined up with hydrogen bonds, respectively. Synchronous fluorescence spectra and CD spectra results showed that the disturbing of tyrosine and tryptophan micro-environments were so slightly that no obvious secondary structure changes were found during the chiral hapten binding. Clarification of stereospecific interaction of antibody will facilitate the application of immunoassay to analyze chiral contaminants in food and other areas.

Characterization of Ofloxacin Interaction with Mutated (A91V) Quinolone Resistance Determining Region of DNA Gyrase in Mycobacterium Leprae through Computational Simulation.

Mycobacterium leprae, the causal agent of leprosy is non-cultivable in vitro. Thus, the assessment of antibiotic activity against Mycobacterium leprae depends primarily upon the time-consuming mouse footpad system. The GyrA protein of Mycobacterium leprae is the target of the antimycobacterial drug, Ofloxacin. In recent times, the GyrA mutation (A91V) has been found to be resistant to Ofloxacin. This phenomenon has necessitated the development of new, long-acting antimycobacterial compounds. The underlying mechanism of drug resistance is not completely known. Currently, experimentally crystallized GyrA-DNA-OFLX models are not available for highlighting the binding and mechanism of Ofloxacin resistance. Hence, we employed computational approaches to characterize the Ofloxacin interaction with both the native and mutant forms of GyrA complexed with DNA. Binding energy measurements obtained from molecular docking studies highlights hydrogen bond-mediated efficient binding of Ofloxacin to Asp47 in the native GyrA-DNA complex in comparison with that of the mutant GyrA-DNA complex. Further, molecular dynamics studies highlighted the stable binding of Ofloxacin with native GyrA-DNA complex than with the mutant GyrA-DNA complex. This mechanism provided a plausible reason for the reported, reduced effect of Ofloxacin to control leprosy in individuals with the A91V mutation. Our report is the first of its kind wherein the basis for the Ofloxacin drug resistance mechanism has been explored with the help of ternary Mycobacterium leprae complex, GyrA-DNA-OFLX. These structural insights will provide useful information for designing new drugs to target the Ofloxacin-resistant DNA gyrase.