Concentrations Of Nalidixic Acid Research Articles

Subinhibitory concentrations of nalidixic acid alter bacterial physiology and induce anthropogenic resistance in a commensal strain of Escherichia coli invitro.

The human gut houses a complex group of bacterial genera, including both opportunistic pathogens and commensal micro-organisms. These are regularly exposed to antibiotics, and their subinhibitory concentrations play a pivotal role in shaping the microbial responses. This study was aimed to investigate the effects exerted by sub-MICs of nalidixic acid (NA) on the growth rate, bacterial motility, biofilm formation and expression of outer membrane proteins (OMPs) in a commensal strain of E.coli. The NA-sensitive strain was sequentially passaged under sub-MICs of NA. E-test was used to determine the MIC values of NA. Results indicated significant changes in the growth profile of commensal E.coli upon exposure to NA at sub-MICs. Differential expression of OMPs was observed in cells treated with sub-MICs of NA. Bacterial motility was reduced under 1/2 MIC of NA. Interestingly, successive passaging under 1/2 MIC of NA led to the emergence of resistant E.coli with an increased MIC value of 64 µg ml-1 in just 24 days. The NA-resistant variant was confirmed by comparing its 16S rRNA sequence to that of the sensitive commensal strain. Mutations in the Quinolone Resistance-Determining Regions (QRDRs) of chromosomal gyrA, and Topoisomerase IV-encoding parC genes were detected in NA-resistant E.coli. Our results demonstrate how antibiotics play an important role as signalling molecules or elicitors in driving the pathogenicity of commensal bacteria invitro.

Quinolone resistance among Salmonella Kentucky and Typhimurium isolates in Tunisia: first report of Salmonella Typhimurium ST34 in Africa and qnrB19 in Tunisia.

Characterization of quinolone-resistant Salmonella Kentucky and Typhimurium isolates in Tunisia from various sources, detection of some plasmid-mediated quinolone resistance genes and the genetic relatedness. A total of 1404 isolates of S. Kentucky (n=1059)/S. Typhimurium (n=345) from various sources from all over Tunisia were tested for quinolone resistance by disk diffusion method. Minimum inhibitory concentrations of nalidixic acid, ciprofloxacin and ofloxacin were determined. Quinolone-resistant isolates were screened for plasmid-mediated quinolone-resistance genes (qnrA,qnrB,qnrS, aac(6')-Ib-cr and qepA) by polymerase chain reaction (PCR). Mutations in the quinolone-resistance-determining regions of the gyrA and parC genes were detected by PCR and DNA sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were accomplished for isolates harbouring plasmid-mediated quinolone-resistance genes. According to our selection criteria (NAL=resistance phenotype; CIP=resistant with diameter 0, or intermediate), only 63 S. Kentucky/41 S. Typhimurium isolates were investigated: 49% (5/104) were multidrug resistant. Two S. Typhimurium isolates harboured qnrB19 with different PFGE profiles. A mutation was detected in the gyrA gene for each of these two isolates. MLST revealed the presence of ST313 and ST34, an endemic sequence type. Our study highlights the presence of quinolone multidrug-resistant Salmonella in humans and animals in Tunisia. This is the first report of S. Typhimurium ST34 in Africa and qnrB19 in Tunisia. This is the first report that describes not only the current epidemiological situation of the quinolone resistance in S. Kentucky and Typhimurium isolated from various sources and regions in Tunisia, but also, the genetic resistance determinants associated with phenotypic antibiotic resistance and the molecular mechanisms of their quinolone-resistance. Also, we provide the first report of S. Typhimurium ST34 in Africa, and the first report of qnrB19 in Salmonella in Tunisia.

Removal of nalidixic acid from aqueous solutions using a cathode containing three-dimensional graphene

Herein three-dimensional graphene (3DG) was synthesized by solution combustion method. A nanostructured 3DG modified carbon paper as the cathode was employed in the homogeneous electro-Fenton method for degradation of nalidixic acid from aqueous solutions and as an anode electrode, graphite was used. The physic-chemical properties of 3DG powder and modified carbon paper were studied by the SEM, XRD, AFM, FTIR, CV and BET analyses. The ability of the resultant 3DG modified carbon paper cathode for hydrogen peroxide production was investigated in solution fed with air. Results showed that the value of hydrogen peroxide that was electrogenerated by using 3DG electrode is 4 times more than bare carbon paper and graphene oxide modified electrode. The consequence of running parameters including current intensity (mA), initial solution pH, initial concentration of nalidixic acid (mg L−1) and process time were investigated comprehensively in a series of batch experiments which were optimized by response surface method (RSM) and the optimized amounts of each parameter were respectively 300 mA, 3.5, 15 mg L−1 and 300 min. The pharmaceutical removal efficiency was 90% under these optimum conditions. The total organic carbon (TOC) measurements showed 87.3% mineralization of 15 mg L−1 drug at 7 h. The gas chromatography (GC) linked to a mass spectrometry (MS) was used to recognize the generated intermediates through the nalidixic acid degradation.

Novel Antibiotic Testing Approaches Reveal Reduced Antibiotic Efficacy Against Shiga Toxin-Producing Escherichia coli O157:H7 Under Simulated Microgravity.

As a foodborne and environmental pathogen, Shiga toxin-producing Escherichia coli O157:H7 could pose a health threat to immunocompromised astronauts during a space mission. In this study, novel approaches, including real-time testing and direct evaluation of resistance mechanisms, were used to evaluate antibiotic efficacy against E. coli O157:H7 under low-shear modeled microgravity (LSMMG) produced using a rotary cell culture system. When compared with normal gravity (NG), bacterial growth was increased under LSMMG in the presence of sub-inhibitory nalidixic acid concentrations and there was an accompanying up-regulation of stress-related genes. LSMMG also induced transcriptional changes of the virulence genes stx1 and stx2, highlighting the potential risk of inappropriate antibiotic use during a spaceflight. The degree of bacterial cell damage induced by the antibiotics was reduced under LSMMG, suggesting low induction of reactive oxygen species. Efflux pumps were also shown to play an important role in these responses. Increased cell filamentation was observed under LSMMG upon ampicillin treatment, possibly reflecting a protective mechanism against exposure to antibiotics. These observations indicate that, in the presence of antibiotics, the survival of E. coli O157:H7 is greater under LSMMG than under NG, indicating that antibiotic therapies may need to be adjusted during space missions.

Suppression of Reactive Oxygen Species Accumulation Accounts for Paradoxical Bacterial Survival at High Quinolone Concentration.

When bacterial cells are exposed to increasing concentrations of quinolone-class antibacterials, survival drops, reaches a minimum, and then recovers, sometimes to 100%. Despite decades of study, events underlying this paradoxical high-concentration survival remain obscure. Since reactive oxygen species (ROS) have been implicated in antimicrobial lethality, conditions generating paradoxical survival were examined for diminished ROS accumulation. Escherichia coli cultures were treated with various concentrations of nalidixic acid, followed by measurements of survival, rate of protein synthesis, and ROS accumulation. The last measurement used a dye (carboxy-H2DCFDA) that fluoresces in the presence of ROS; fluorescence was assessed by microscopy (individual cells) and flow cytometry (batch cultures). High, nonlethal concentrations of nalidixic acid induced lower levels of ROS than moderate, lethal concentrations. Sublethal doses of exogenous hydrogen peroxide became lethal and eliminated the nalidixic acid-associated paradoxical survival. Thus, quinolone-mediated lesions needed for ROS-executed killing persist at high, nonlethal quinolone concentrations, thereby implicating ROS as a key factor in cell death. Chloramphenicol suppressed nalidixic acid-induced ROS accumulation and blocked lethality, further supporting a role for ROS in killing. Nalidixic acid also inhibited protein synthesis, with extensive inhibition at high concentrations correlating with lower ROS accumulation and paradoxical survival. A catalase deficiency, which elevated ROS levels, overcame the inhibitory effect of chloramphenicol on nalidixic acid-mediated killing, emphasizing the importance of ROS. The data collectively indicate that ROS play a dominant role in the lethal action of narrow-spectrum quinolone-class compounds; a drop in ROS levels accounted for the quinolone tolerance observed at very high concentrations.

A flow injection chemiluminescence method for determination of nalidixic acid based on KMnO4–morin sensitized with CdS quantum dots

A simple and sensitive flow injection chemiluminescence (CL) method was developed for determination of nalidixic acid by application of CdS quantum dots (QDs) in KMnO4–morin CL system in acidic medium. Optical and structural features of l-cysteine capped CdS quantum dots which were synthesized via hydrothermal approach were investigated using X-ray diffraction (XRD), scanning electron microscopy (SEM), photoluminescence (PL), and ultraviolet–visible (UV–Vis) spectroscopy. Moreover, the potential mechanism of the proposed CL method was described using the results of the kinetic curves of CL systems, the spectra of CL, PL and UV–Vis analyses. The CL intensity of the KMnO4–morin–CdS QDs system was considerably increased in the presence of nalidixic acid. Under the optimum condition, the enhanced CL intensity was linearly proportional to the concentration of nalidixic acid in the range of 0.0013 to 21.0mgL−1, with a detection limit of (3σ) 0.003mgL−1. Also, the proposed CL method was utilized for determination of nalidixic acid in environmental water samples, and commercial pharmaceutical formulation to approve its applicability. Furthermore, corona discharge ionization ion mobility spectrometry (CD-IMS) method was utilized for determination of nalidixic acid and the results of real sample analysis by two proposed methods were compared. Comparison the analytical features of these methods represented that the proposed CL method is preferable to CD-IMS method for determination of nalidixic acid due to its high sensitivity and precision.

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.

Drug Resistance Reversal Potential of Ursolic Acid Derivatives against Nalidixic Acid- and Multidrug-resistant Escherichia coli.

As a part of our drug discovery program, ursolic acid was chemically transformed into six semi-synthetic derivatives, which were evaluated for their antibacterial and drug resistance reversal potential in combination with conventional antibiotic nalidixic acid against the nalidixic acid-sensitive and nalidixic acid-resistant strains of Escherichia coli. Although ursolic acid and its all semi-synthetic derivatives did not show antibacterial activity of their own, but in combination, they significantly reduced the minimum inhibitory concentration of nalidixic acid up to eightfold. The 3-O-acetyl-urs-12-en-28-isopropyl ester (UA-4) and 3-O-acetyl-urs-12-en-28-n-butyl ester (UA-5) derivatives of ursolic acid reduced the minimum inhibitory concentration of nalidixic acid by eightfold against nalidixic acid-resistant and four and eightfold against nalidixic acid-sensitive, respectively. The UA-4 and UA-5 were further evaluated for their synergy potential with another antibiotic tetracycline against the multidrug-resistant clinical isolate of Escherichia coli-KG4. The results showed that both these derivatives in combination with tetracycline reduced the cell viability in concentration-dependent manner by significantly inhibiting efflux pump. This was further supported by the in silico binding affinity of UA-4 and UA-5 with efflux pump proteins. These ursolic acid derivatives may find their potential use as synergistic agents in the treatment of multidrug-resistant Gram-negative infections.

Enhanced resistance to fluoroquinolones in laboratory-grown mutants & clinical isolates of Shigella due to synergism between efflux pump expression & mutations in quinolone resistance determining region.

There is a worldwide emergence of fluoroquinolone resistance in Shigella species. To understand the molecular mechanisms associated with fluoroquinolone resistance, naturally occurring fluoroquinolone-resistant strains and laboratory-induced spontaneous mutants of Shigella spp. were used and the relative contributions of acrAB-tolC efflux pumps, gyrase and topoisomerase target gene mutations towards fluoroquinolone resistance were determined. Eight Shigella flexneri and six S. dysenteriae clinical isolates were studied. Three consecutive mutants resistant to ciprofloxacin for S. flexneri SFM1 (≥ 0.25 µg/ml), SFM2 (≥ 4 µg/ml) and SFM3 (≥ 32 µg/ml) were selected in 15 steps from susceptible isolates by serial exposure to increasing concentrations of nalidixic acid and ciprofloxacin. Similarly, two mutants for S. dysenteriae SDM1 (≥ 0.25 µg/ml) and SDM2 (≥ 4 µg/ml) were selected in eight steps. After PCR amplification sequence analyses of gyrase and topoisomerase target genes were performed. Expression of efflux genes acrA, acrB, acrR and tolC was measured using real-time PCR. Mutations were observed in gyrA Ser [83]→Leu, Asp [87]→Asn/Gly, Val [196]→Ala and in parC Phe [93]→Val, Ser [80]→Ile, Asp [101]→Glu and Asp [110]→Glu. Overall, acrA and acrB overexpression was associated with fluoroquinolone resistance ( p0 <0.05); while tolC and acrR expression levels did not. Fluoroquinolone resistance in Shigella spp. is the end product of either a single or a combination of mutations in QRDRs and/ or efflux activity. Novel polymorphisms were observed at Val [196]→Ala in gyrA in clinical isolates and Phe [93]→Val, Asp [101]→Glu, Asp [110]→Glu and in parC in majority of laboratory-grown mutants.

Antimicrobial resistance in Salmonella enterica Serovar Typhi isolates from Bangladesh, Indonesia, Taiwan, and Vietnam.

We characterized Salmonella enterica serovar Typhi isolates from Bangladesh, Indonesia, Taiwan, and Vietnam to investigate their genetic relatedness and antimicrobial resistance. The isolates from Bangladesh and Vietnam were genetically closely related but were distant from those from Indonesia and Taiwan. All but a few isolates from Indonesia and Taiwan were susceptible to all antimicrobials tested. The majority of isolates from Bangladesh and Vietnam were multidrug resistant (MDR) and belonged to the widespread haplotype H58 clone. IncHI1 plasmids were detected in all MDR S. Typhi isolates from Vietnam but in only 15% of MDR isolates from Bangladesh. Resistance genes in the majority of MDR S. Typhi isolates from Bangladesh should reside in the chromosome. Among the isolates from Bangladesh, 82% and 40% were resistant to various concentrations of nalidixic acid and ciprofloxacin, respectively. Several resistance mechanisms, including alterations in gyrase A, the presence of QnrS, and enhanced efflux pumps, were involved in the reduced susceptibility and resistance to fluoroquinolones. Intensive surveillance is necessary to monitor the spread of chromosome-mediated MDR and fluoroquinolone-resistant S. Typhi emerging in Bangladesh.

Lactococcus garvieae carries a chromosomally encoded pentapeptide repeat protein that confers reduced susceptibility to quinolones in Escherichia coli producing a cytotoxic effect

This study characterises a chromosomal gene of Lactococcus garvieae encoding a pentapeptide repeat protein designated as LgaQnr. This gene has been implicated in reduced susceptibility to quinolones in this bacterium, which is of relevance to both veterinary and human medicine. All of the L. garvieae isolates analysed were positive for the lgaqnr gene. The expression of lgaqnr in Escherichia coli reduced the susceptibility to quinolones, producing an adverse effect. The reduced susceptibility to ciprofloxacin was 16-fold in E. coli ATCC 25922 and 32-fold in E. coli DH10B, compared to the control strains. The minimum inhibitory concentration of nalidixic acid was also increased 4 or 5-fold. The effect of the expression of lgaqnr in E. coli was investigated by electron microscopy and was observed to affect the structure of the cell and the inner membrane of the recombinant cells.

ALTERATIONS OF DNA TOPOISOMERASES OF RIEMERELLA ANATIPESTIFER WITH IN VITRO-REDUCED SUSCEPTIBILITY TO QUINOLONES

To elucidate on a potential association between mutations of topoisomerases and quinolone resistance in Riemerella anatipestifer, mutants of reduced quinolones susceptibility were generated from the four clinical isolates by serial passages in the presence of increased concentrations of nalidixic acid, ofloxacin, enrofloxacin and ciprofloxacin. Eleven alterations at eight positions (81, 82, 83, 84, 87, 117, 119, and 181) in GyrA and five different alterations at four (431, 452, 470, and 471) positions in GyrB were induced within mutants, resulting in various levels of resistance to different quinolones. The most frequent alteration was at Ser83 in GyrA, which concurred with that of previous clinical studies. To our best knowledge, alterations at Ala183 in GyrA and at Arg452 as well as Glu471 in GyrB were firstly reported. The study demonstrated that alterations of GyrA and GyrB are the prior mechanisms by which R. anatipestifer develops its resistance to quinolones. Expanding the quinolone-resistance determining region for R. anatipestifer is suggested as well.

Development of an alternative culture medium for the selective enumeration of Lactobacillus casei in fermented milk

Monitoring the populations of probiotic strains of the species Lactobacillus casei in food is required by food industries in order to assure that a minimum concentration of these organisms will be ingested by consumers. In this context, Petrifilm™ AC plates can be used along with selective culture media to allow the enumeration of specific groups of lactic acid bacteria. The present study aimed to assess chemical substances as selective agents for Lb. casei in order to propose a selective culture medium to be used with Petrifilm™ AC plates as an alternative protocol for the enumeration of probiotic strains of this species in fermented milk. Twenty-six probiotic and starter cultures (including six strains of Lb. casei) were plated on de Man Rogosa and Sharpe (MRS) agar with distinct concentrations of nalidixic acid, bile, lithium chloride, metronidazole, sodium propionate, and vancomycin. Vancomycin at 10 mg/L demonstrated selective activity for Lb. casei. In addition, 2,3,5-triphenyltetrazolium chlorine was identified as a compound that did not inhibit Lb. casei, and Petrifilm™ AC plates used with MRS and vancomycin at 10 mg/L (MRS-V) demonstrated more colonies of this organism when incubated under anaerobic conditions than aerobic conditions. Acidophilus milk and yoghurt were prepared, added to Lb. casei strains, and stored at 4 °C. Lb. casei populations were monitored using MRS-V and MRTLV by conventional plating and associated with Petrifilm™ AC plates. All correlation indices between counts obtained by conventional plating and Petrifilm™ AC were significant (p < 0.05), but the best performance was observed for growth on MRS-V. The obtained data indicate the efficiency of using MRS-V associated with Petrifilm™ AC plates for the enumeration of Lb. casei strains in fermented milk. However, the selective potential of this culture medium must be evaluated considering the specific strains of Lb. casei and the starter cultures inoculated in the fermented milk that requires monitoring.

Isolation and characterization of smallqnrS1-carrying plasmids from imported seafood isolates ofSalmonella entericathat are highly similar to plasmids of clinical isolates

Dissemination of plasmid-mediated quinolone resistance among pathogenic bacteria is a concern for public health because of decreased sensitivity to fluoroquinolones and increased potentials to develop high fluoroquinolone resistance. Two qnrS1-positive isolates of Salmonella enterica Corvallis (468) and Typhimurium (484) from imported seafood (Thailand and Vietnam) were tested for quinolone sensitivity using disk agar diffusion and the Sensititre system. The presence of qnr genes, qnr-carrying plasmids, and mutations in the quinolone resistance determining regions were also determined. Minimal inhibitory concentrations of nalidixic acid for isolates 468 and 484 were 8 and 16 μg mL(-1) , respectively, and those of ciprofloxacin were 1 and 2 μg mL(-1), respectively. Disk agar diffusion indicated that isolate 468 was moderately resistant to moxifloxacin, and isolate 484 was resistant to moxifloxacin and moderately resistant to norfloxacin. Isolates 468 and 484 carried a mutation on parC, but not on gyrA, gyrB, or parE. Sequences of qnrS1-carrying plasmids from isolates 468 and 484, sized 10,039 and 10,047 bp, were nearly identical (> 99% similarity) to each other and to published sequences of plasmids from clinical isolates of Salmonella Typhimurium isolated in the United Kingdom and Taiwan, indicating a dissemination of qnrS1-carrying plasmids among different serovars of Salmonella from geographically separated sources. This is the first complete sequence of a qnrS1-carrying plasmid from imported seafood isolate of S. enterica.

Effect of nalidixic acid on the morphology and protein expression of Pseudomonas aeruginosa

ObjectiveTo determine the effect of nalidixic acid on the morphology and protein expression of Pseudomonas aeruginosa (P. aeruginosa). MethodsNalidixic acid solution of 1 600 μg/mL was prepared. The minimum inhibitory concentration (MIC) for P. aeruginosa was determined with tube dilution test. The effect of nalidixic acid on the morphology of P. aeruginosa was studied using light microscope and scanning electron microscope. Changes in protein profile were studied using SDS-PAGE. ResultsThe MIC of nalidixic acid was 700 μg/mL against P. aeruginosa. The exposure of P. aeruginosa to different concentrations of nalidixic acid resulted in deformation of most of the growing cells. At the concentration of 600 μg/mL most of the cells turned into elongated and adhere to each other while some of the cells were bulged. The intensity of protein bands were changed when they exposed to nalidixic acid. ConclusionsThe present findings suggest that the morphology and protein expression of P. aeruginosa is greatly affected by nalidixic acid.

Removal of Organics and Degradation Products from Industrial Wastewater by a Membrane Bioreactor Integrated with Ozone or UV/H2O2 Treatment

The treatment of a pharmaceutical wastewater resulting from the production of an antibacterial drug (nalidixic acid) was investigated employing a membrane bioreactor (MBR) integrated with either ozonation or UV/H(2)O(2) process. This was achieved by placing chemical oxidation in the recirculation stream of the MBR. A conventional configuration with chemical oxidation as polishing for the MBR effluent was also tested as a reference. The synergistic effect of MBR when integrated with chemical oxidation was assessed by monitoring (i) the main wastewater characteristics, (ii) the concentration of nalidixic acid, (iii) the 48 organics identified in the raw wastewater and (iv) the 55 degradation products identified during wastewater treatment. Results showed that MBR integration with ozonation or UV/H(2)O(2) did not cause relevant drawbacks to both biological and filtration processes, with COD removal rates in the range 85-95%. Nalidixic acid passed undegraded through the MBR and was completely removed in the chemical oxidation step. Although the polishing configuration appeared to give better performances than the integrated system in removing 15 out of 48 secondary organics while similar removals were obtained for 19 other compounds. The benefit of the integrated system was however evident for the removal of the degradation products. Indeed, the integrated system allowed higher removals for 34 out of 55 degradation products while for only 4 compounds the polishing configuration gave better performance. Overall, results showed the effectiveness of the integrated treatment with both ozone and UV/H(2)O(2).

Salmonella antibiotic-mutant strains reduce fecal shedding and organ invasion in broiler chicks

We investigated the exposure to antibiotics in the production of antibiotic-mutant strains of Salmonella. Ten isolates of poultry origin were assayed for antibiotic susceptibilities. One strain of Salmonella Enteritidis, one of Salmonella Heidelberg, and one of Salmonella Typhimurium were selected to induce antimicrobial resistance. Each strain was exposed to high concentrations of streptomycin, rifampicin, and nalidixic acid, respectively. Parent and antibiotic-mutant strains were assayed for antibiotic susceptibilities using a commercial microdilution test and the disk susceptibility test. The strains were assessed for virulence genes and evaluated for fecal shedding, cecal colonization, organ invasion, and mean Salmonella counts after inoculation in 1-day-old chicks. The study revealed that exposure to high concentrations of streptomycin produced the antibiotic-mutant strain SE/LABOR/USP/08 and the exposure to rifampicin produced the antibiotic-mutant SH/LABOR/USP/08. These strains showed significantly reduced fecal shedding (P≤0.05) and organ invasion, persisting less than the parental strains and showing no clinical signs in inoculated chicks. High concentrations of nalidixic acid produced the antibiotic-mutant strain ST/LABOR/USP/08, which did not show any differences compared with the parent strain. Likewise, SE/LABOR/USP/08 did not show the expression of plasmid-encoded fimbriae (pefA) and plasmid virulence protein (spvC), suggesting that after exposure to streptomycin, the parent isolate lost the original gene expression, reducing fecal shedding and organ invasion in inoculated chicks.

Mathematical Modeling of a Bioluminescent E. Coli Based Biosensor

In this work we present a mathematical model for the bioreporter activity of an E. coli based bioluminescent bioreporter. This bioreporter is based on a genetically modified E. coli which harbors the recA promoter, a member of the bacterial SOS response, fused to the bacterial luminescence (lux) genes. This bioreporter responds to the presence of DNA damaging agents such as heavy metals, H2O2 and Nalidixic Acid (NA) that activate the SOS response. In our mathematical model we implemented basic physiological mechanisms such as: the penetration of the NA into the biosensor; gyrase enzyme inhibition by the NA; gyrase level regulation; creation of chromosomal DNA damage; DNA repair and release of ssDNA into the cytoplasm; SOS induction and chromosomal DNA repair; activation of lux genes by the fused recA promoter carried on a plasmidal DNA; transcription and translation of the luminescence responsible enzymes; luminescence cycle; energy molecules level regulation and the regulation of the O2 consumption. The mathematical model was defined using a set of ordinary differential equations (ODE) and solved numerically. We simulated the system for different concentrations of NA in water for specific biosensors concentration, and under limited O2 conditions. The simulated results were compared to experimental data and satisfactory matching was obtained. This manuscript presents a proof of concept showing that real biosensors can be modeled and simulated. This sets the ground to the next stage of implementing a comprehensive physiological model using experimentally extracted parameters. Following the completion of the next stage, it will be possible to construct a “Computer Aided Design” tool for the simulation of the genetically engineered biosensors. We define a term “bioCAD” for a Biological System Computer Aided Design. The specific bioCAD that is described here is aimed towards whole cell biosensors which are under investigation today for functional sensing. Usage of the bioCAD will improve the biosensors design process and boost their performance. It will also reduce Non Recurring Engineering (NRE) cost and time. Finally, using a parameterized solution will allow fair and quick evaluation of whole cell biosensors for various applications.

Lon Protease Is Essential for Paradoxical Survival of Escherichia coli Exposed to High Concentrations of Quinolone

A deficiency of the Escherichia coli Lon protease blocked paradoxical survival occurring at very high nalidixic acid concentrations. The absence of Lon also blocked a parallel increase in cell lysate viscosity likely to reflect DNA size. Thus, Lon may participate in repairing quinolone-mediated DNA lesions formed at high drug concentrations.

Antibiotic-Mediated Inhibition of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection: A Novel Quinolone Function Which Potentiates the Antiviral Cytokine Response in MARC-145 Cells and Pig Macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically significant agent for which there currently are no effective treatments. Development of antiviral agents for PRRSV as well as many other viruses has been limited by toxicity of known antiviral compounds. In contrast, antibiotics for non-virus microbial infections have been widely useful, in part because of their acceptable toxicity in animals. We report here the discovery that the quinolone-containing compound Plasmocin™, as well as the quinolones nalidixic acid and ciprofloxacin, have potent anti-PRRSV activity in vitro. PRRSV replication was inhibited by these antibiotics in both cultured MARC-145 cells and cultured primary alveolar porcine macrophages (PAMs). Furthermore, sub-optimal concentrations of nalidixic acid synergized with antiviral cytokines (AK-2 or IFN-γ) to quantitatively and qualitatively inhibit PRRSV replication in MARC-145 cells or PAMs. The antiviral activity of Plasmocin and nalidixic acid correlated with reduced actin expression in MARC-145 cells. Replication of the related lactate dehydrogenase-elevating virus (LDV) was also inhibited in primary mouse macrophages by Plasmocin. These results are significant to the development of antiviral strategies with potentially reduced toxicity, and provide a model system to better understand regulation of arterivirus replication.