norA Gene Research Articles

Characterization of aflatoxigenic Aspergillus flavus associated with aflatoxin B1 (AFB1) production in maize kernel in India

Mycotoxin-producing fungi directly impact on the yield and quality of agricultural produce. Aspergillus flavus is predominant fungi to produce aflatoxin B1 (AFB1) mycotoxin in maize. Early detection and quantification of aflatoxin B1 producing A. flavus is indispensable to mitigate maize loss. With this aim, three different detection methods (PCR, qPCR and HPLC) deployed to screen highly toxigenic and atoxigenic isolates of A. flavus. PCR analysis of four A. flavus isolates revealed possession of six toxigenic genes (aflO, aflP, aflQ, aflM, aflS and aflD), among aflO, aflP and aflQ genes are most common. In qPCR, seven isolates tested representing 5 different maize growing agroclimatic zones of India, expressed nor-1 gene. In two of the isolates, higher mean expression levels were noticed. In HPLC, aflatoxin B1 (AFB1) production capacity was tested in all isolates. About 65% of the A. flavus isolates produced aflatoxin B1 (between 43.87 and 0.88 µg/g). This study exhibited that the highly toxic isolate observed from the sample collected from Hyderabad, where maize crop is growing year round, followed by nagaon’s sample from Assam, therefore these locations will need extra care during post harvest condition to minimize the risk of aflatoxin in storage.

Inhibition of the NorA efflux pump of S. aureus by (Z)-5-(4-Fluorobenzylidene)-Imidazolidines

Searching for new alternatives to antibiotic treatments is crucial to surmount the multidrug-resistant bacteria. In this work, the antimicrobial activity of synthetic imidazolidines was evaluated as well as their modulating effect on the resistance to fluoroquinolones in a S. aureus strain (SA-1199B), which overexpresses the norA gene that encodes the NorA efflux pump. Results showed weak antimicrobial activity (512 μg mL−1) for two fluorobenzylidene derivatives against this bacterial strain, while the other benzylidene derivatives were inactive. Despite this fact, both fluorinated compounds were able to enhance the activity of norfloxacin and ciprofloxacin against SA-1199B up to 6.4– and 3.2-fold, respectively. In addition, both derivatives potentiated the action of ethidium bromide against this strain, suggesting that the modulating effect probably involves the inhibition of the NorA efflux pump, which is in concordance with the fluorimetic assays and molecular docking analyses performed in this work.

Antimicrobial activity of polypyrrole nanoparticles and aqueous extract of Moringa oleifera against Staphylococcus spp. carriers of multi-drug efflux system genes isolated from dairy farms.

Our objectives were to identify genes of the multi-drug efflux system and to evaluate the antimicrobial activities of polypyrrole nanoparticles (PPy-NPs) and aqueous extract of Moringa oleifera against Staphylococcus spp. isolated from dairy farms in Northeast Brazil. Initially, 162 Staphylococcus spp. isolates were subjected to in vitro antimicrobial sensitivity tests. Of these, 35 presented antimicrobial multi-drug resistance phenotypes. These 35 isolates were then referred for the detection of norA, norB, norC, msrA, mgrA, tet-38, and lmrS genes, all of which feature in multi-drug efflux systems. In the isolates carrying the genes, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PPy-NPs and Moringa oleifera aqueous extract were determined. In the molecular analysis of the 35 isolates norA, norC, tet-38, and msrA genes were detected and for the other genes norB, lmrS and mgrA there was no amplification. Antimicrobial activity was verified of PPy-NPs and aqueous extract of Moringa oleifera in Staphylococcus spp. carrying multi-drug efflux system genes. We concluded that there are multi-drug efflux system genes present in the Staphylococcus spp. from the agricultural environment in Northeast Brazil, and that aqueous extract of Moringa oleifera and PPy-NPs show bactericidal activity against these isolates.

Comparative activity of ozenoxacin and other quinolones in Staphylococcus aureus strains overexpressing the efflux pump-encoding genes mepA and norA

BackgroundTo evaluate the activity of ozenoxacin (OZN) in Staphylococcus aureus strains overexpressing the efflux pump-encoding genes mepA and norA. MethodsS. aureus NCTC-8325-1, S. aureus NCTC 8225-2 (overexpressing mepA), S. aureus SA 1199 and S. aureus SA 1199B (overexpressing norA) were used. The minimum inhibitory concentrations (MICs) of OZN, moxifloxacin (MOX), levofloxacin (LVX), ciprofloxacin (CIP) and norfloxacin (NOR) in the presence and absence of reserpine (20 mg/L) were determined using the microdilution method. ResultsThe MIC of OZN was lower in all evaluated strains compared with the other studied quinolones and was independent of the pump being overexpressed. MIC values of OZN ranged from 0.005 to 0.007 mg/L. Similar results were observed with MOX, with MIC values between 0.021 and 0.031 mg/L, without variations in the presence of reserpine. MIC values for LVX were between 0.167 and 1 mg/L with a slight increase in MIC observed in strains overexpressing the mepA or norA genes (from 0.250 to 0.833 mg/L and 0.167 to 1 mg/L, respectively). Overproduction of the efflux pump MepA did not affect CIP whereas it increased 8-fold the MIC of NOR. Overproduction of NorA increased ~5-fold and ~40-fold the MICs of CIP and NOR, respectively, resulting in a high-level of resistance to these antibiotics compared with OZN (0.007 mg/L). ConclusionOZN does not seem to be a substrate for the efflux pumps MepA and NorA, which are commonly found in Gram-positive bacteria and that affect other quinolones.

Structural, Vibrational and Electrochemical Analysis and Antibacterial Potential of Isomeric Chalcones Derived from Natural Acetophenone

Background: Chalcones are part of a family of small phenolic compounds that are being extensively studied for presenting a diversity of molecular structures and biological activities. In this paper, two chalcones, (E)-1-(2-hydroxy-3,4,6-trimethoxyphenyl)-3-(3-nitrophenyl)prop-2-en-1-one (1), (E)-1-(2-hydroxy-3,4,6-trimethoxyphenyl)-3-(4-nitrophenyl)prop-2-en-1-one (2), were synthesized by Claisen–Schmidt condensation. Methods: The molecular structures of these chalcones were determined by Nuclear Magnetic Resonance and characterized by infrared, Raman spectroscopy, and electrochemical analysis at room temperature. Vibrational wavenumbers were predicted using Functional Density Theory (DFT) calculations, and their normal modes were analyzed in terms of potential energy distribution (PED). Besides this, DFT calculations were performed to obtain the molecular orbitals and their quantum descriptors. The UV-Vis absorption spectrum of the synthesized chalcones was measured and compared with each other. In addition, analyses of antimicrobial activity and modulation of antibiotic resistance were carried out to assess the antibacterial potential of these chalcones. Results: The vibrational spectra of polycrystalline chalcones obtained by ATR-FTIR, FT-Raman and DFT calculations allowed a complete assignment of the vibrational modes, and revealed the quantum chemical parameters. Both chalcones did not show good responses when associated with the antibiotics Ciprofloxacin and Cephalexin against S. aureus 10 and E. coli 06 strains. However, a significant potentiating of the Gentamicin activity against S. aureus 10 and E. col 06 strains was observed for chalcone 2. On the other hand, when associated with Norfloxacin, an antagonistic effect was observed. The results found for EtBr suggest that, although the tested chalcones behave as efflux pump inhibitors, probably inhibiting other efflux pumps, they were not able to inhibit NorA. Thus, these synthetic chalcones are not recommended for use in association with Norfloxacin against strains of S. aureus 1199-B that overexpress the NorA gene. Conclusions: Spectroscopic data confirmed the structure of the chalcones, and chalcone 2 showed potential as an adjuvant in antibiotic therapy.

Effect of Vitamin K3 Inhibiting the Function of NorA Efflux Pump and Its Gene Expression on Staphylococcus aureus.

Resistance to antibiotics has made diseases that previously healed easily become more difficult to treat. Staphylococcus aureus is an important cause of hospital-acquired infections and multi-drug resistant. NorA efflux pump, present in bacteria S. aureus, is synthesized by the expression of the norA gene. Menadione, also known as vitamin K3, is one of the synthetic forms of vitamin K. Therefore, the aim of this study is to verify the menadione effect on efflux inhibition through NorA pump gene expression inhibition and assess the effects of menadione in bacterial membrane. The effect of menadione as an efflux pump inhibitor (EPI) was evaluated by the microdilution method, fluorimetry, electron microscopy, and by RT-qPCR to evaluate gene expression. In the molecular docking, association with menadione induces increased fluorescence intensity. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA. The results showed the norA gene had its expression significantly diminished in the presence of menadione. The simulation showed that several menadione molecules were able to go through the bilayer and allow the entry of water molecules into the hydrophobic regions of the bilayer. When present within membranes, menadione may have caused membrane structural changes resulting in a decline of the signaling pathways involved in norA expression. Menadione demonstrated to be an efflux pump inhibitor with dual mechanism: affecting the efflux pump by direct interaction with protein NorA and indirectly inhibiting the norA gene expression, possibly by affecting regulators present in the membrane altered by menadione.

Structural characterization, antibacterial activity and NorA efflux pump inhibition of flavonoid fisetinidol

Flavonoids belong to the extensive group of polyphenols and have several biological activities such as anti-inflammatory, anti-ulcer, anticancer, antiviral, antibacterial, and antispasmodic. These compounds can thus be explored as a tool against multidrug resistant bacteria, as nosocomial infections now constitute a major public health problem worldwide. We have carried out a structural characterization by nuclear magnetic resonance and FT-IR of fisetinidol, a flavonoid isolated from B. pentandra stems. In addition, this flavonoid was tested for antibacterial and modulatory activities, as well as inhibition of the NorA efflux pump. Fisetinidol has no intrinsic antibacterial activity. However, there is synergism in association with norfloxacin, with a two-fold decrease in MIC. When compared to chlorpromazine, a standard efflux pump inhibitor, a two-fold decrease in MIC was also verified when used against S. aureus SA-1199B, which over expresses the NorA gene encoding the NorA efflux protein. As such, it could become an alternative source to reverse the bacterial resistance if administered in association with antibiotics.

Biosynthesis of NiFe2 O4 @Ag Nanocomposite and Assessment of Its Effect on Expression of norA Gene in Staphylococcus aureus.

Activity of norA efflux pump has been known as a resistance mechanism to antibiotics like ciprofloxacin in Staphylococcus aureus. This study was carried out to assess the effect of biosynthesized NiFe2 O4 @Ag nanocomposite on expression of norA gene in Staphylococcus aureus. In this experimental study, 30 clinical samples were collected from patients hospitalized at different hospitals in Guilan Province, Iran. Then, clinical isolates of S. aureus were identified by standard microbiological tests. Antimicrobial susceptibility tests of clinical and standard strains of S. aureus were done by disk diffusion method according to CLSI guideline. Fourier transform infrared spectroscopy (FT-IR) was used to analyze the various functional groups present in the biosynthesized NiFe2 O4 @Ag nanocomposite. This analysis confirmed the formation of alga proteins coated on magnetite nanocomposite. X-ray diffraction (XRD) verified the crystalline structure of NiFe2 O4 @Ag and the deposition of silver on the surface of NiFe2 O4 . Energy dispersive X-ray mapping (EDX-map) analysis confirmed the existence of Ag, Ni, Fe and O in the final product. Scanning electron microscopy (SEM) confirmed that the nanocomposites were spherical in shape and Transmission electron microscopy (TEM) results revealed that the NiFe2 O4 @Ag had the particle size about 100 nm. Antibacterial activity of NiFe2 O4 @Ag alone and combined with ciprofloxacin was evaluated using the disk assay method, and minimum inhibitory concentration (MIC) by broth dilution method. Afterwards, the expression of norA efflux pump gene with and without of NiFe2 O4 @Ag nanocomposite and ciprofloxacin was evaluated by Real-Time PCR. Real-Time PCR results demonstrated that the expression of norA gene in the strains exposed to both NiFe2 O4 @Ag nanocomposite (1/4 MIC) and ciprofloxacin (1/8 MIC) significantly reduced in comparison to untreated strains. This study reveals that, when NiFe2 O4 @Ag nanocomposite is combined with ciprofloxacin, the inhibitory effect of ciprofloxacin increases against growth of S. aureus. Therefore, NiFe2 O4 @Ag nanocomposite can be considered as an effective factor to decrease the growth of S. aureus along with ciprofloxacin.

The effect of Propolis on inhibition of Aspergillus parasiticus growth, aflatoxin production and expression of aflatoxin biosynthesis pathway genes.

Aflatoxins are one of the most important mycotoxins, which have been classified as Group I carcinogenic compounds by the International Agency for Research on Cancer. This investigation aimed to examine the effect of Propolis on inhibition of the Aspergillus parasiticus growth, aflatoxin production and expression of aflatoxin biosynthesis pathway genes. A standard strain of Aspergillus parasiticus (ATCC 15517) was used to perform antifungal susceptibility test, using a microdilution method in accordance with the CLSI M38-A2 guidelines. The aflatoxin concentrations in the control and treated media were determined by HPLC. Also, the quantitative changes in the level of nor-1, ver-1 and omtA genes expression in aflatoxin biosynthetic pathway were analyzed using Real-Time PCR method. The results showed that the minimum inhibitory concentrations (MIC) of propolis was 100 µg/ml. The results showed that total levels of aflatoxin decreased from 386.1 ppm to 3.01 ppm at 50 µg/ml of propolis. In addition, quantitative real-time PCR analysis showed that the level of nor-1, ver-1 and omtA genes expression was significantly decreased after treatment with propolis extract. The findings reveal that propolis extract, have a significant inhibitory effect on important genes for aflatoxin biosynthesis pathway in aflatoxin production.

The antibiotics resistance mechanism and pathogenicity of cold stressed Staphylococcus aureus

Staphylococcus aureus was one of the most common foodborne pathogens around the world. S. aureus displayed strong tolerance to low temperature (4 °C and −20 °C) at which the foods are usually stocked. Here, the antibiotic resistance, biofilm-formation and virulence of S. aureus under cold stress were investigated. The results demonstrated that the MIC of S. aureus CICC10201 against quinolones and aminoglycosides enhanced 4-fold after being stressed at 4 °C and −20 °C for 4 weeks. After cold stressed for 4 weeks, the efflux pump gene norA of S. aureus CICC10201 was over-expressed >50-fold. The mutation of the target-site at position 410 of subunit-GrlA, in which the tyrosine residue mutated into phenylalanine, reduced the binding efficiency of quinolones to S. aureus CICC10201. Meanwhile, biofilm-forming ability of S. aureus W3 cells were enhanced with post-cold stress because of up-regulation of icaA and icaD gene, and the down-regulation of the icaR gene. Moreover, the mRNA expression of some virulence factors in S. aureus CICC10201 or W3 were increased significantly. It reminded that thoroughly sterilizing the foods which were stored in the refrigerator for a long time are critical to reduce food contamination caused by S. aureus infection.

Unravelling the antifungal and anti-aflatoxin B1 mechanism of chitosan nanocomposite incorporated with Foeniculum vulgare essential oil

The study reports the antifungal and aflatoxin B1 inhibitory efficacy of chemically characterized chitosan-based nanoencapsulated Foeniculum vulgare Mill. essential oil (Ne-FvEO), and its effect on sensory and nutritional properties of Sorghum bicolor. The Ne-FvEO was characterized through SEM, FTIR and XRD. The Ne-FvEO demonstrated superior antifungal (0.4 μl/ml) and aflatoxin B1 inhibitory (0.3 μl/ml) performance than the free FvEO. The biochemical studies reveal the significant alteration in ergosterol content, ions leakage, mitochondrial membrane potential, C-sources utilization and antioxidant defense system in A. flavus exposed to Ne-FvEO. The 3D modeling of the Nor-1 gene product was done using the I-TASSER server and validated by the Ramachandran plot. The in-situ result reveals that the Ne-FvEO significantly preserved the nutritional and sensory characteristics of S. bicolor seeds. Therefore, Ne-FvEO could be used as a green preservative agent to enhance the shelf-life of the food commodities.

Green Synthesis of CuFe2O4@Ag Nanocomposite Using the Chlorella vulgaris and Evaluation of its Effect on the Expression of norA Efflux Pump Gene Among Staphylococcus aureus Strains

Increasing drug resistance among Staphylococcus aureus is a global health threat and finding alternative antimicrobial agents against it has been considered. Multidrug resistance efflux pumps, including NorA, are involved with resistance to different drugs, especially fluoroquinolones, in S. aureus. Using metal nanoparticles against pathogenic bacteria is a promising approach; however, physio-chemical synthesis of nanoparticles has limitations. Biosynthesis of metal nanoparticles with antibacterial activity has gained interest, recently. In this study, biosynthesis of CuFe2O4@Ag nanocomposite using aqueous extract from microalgae Chlorella vulgaris was performed, and its antibacterial property and effect on expression of norA efflux pump gene were investigated. Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), energy dispersive X-ray mapping analysis (EDX-map), differential reflectance spectroscopy (DRS), and dynamic light scattering (DLS) were used to characterize synthesized nanocomposite. Antibacterial activity of the prepared nanocomposite individually and combined with ciprofloxacin against S. aureus strains was evaluated using the disk assay method, and minimum inhibitory concentration (MIC) of each agent was determined using the broth dilution method. Anti-biofilm activity of this nanocomposite was checked. Finally, the effect of CuFe2O4@Ag nanocomposite alone and in combination with ciprofloxacin on the expression of norA was assessed by real-time PCR. The physical analysis revealed proper synthesis of spherical and well-dispersed CuFe2O4@Ag nanocomposite with an average diameter of 20nm. Synthesized nanocomposite had synergistic antibacterial activity with ciprofloxacin. Moreover, expression of norA gene among clinical and standard strains treated with CuFe2O4@Ag nanocomposite combined with ciprofloxacin reduced by 59% and 65%, respectively. Thus, CuFe2O4@Ag nanocomposite synthesized in this study can be considered as a promising candidate to be used to inhibit staphylococcal efflux pump genes and increasing the antibiotic efficacy.

Small Molecular Contaminant and Microorganism Can Be Simultaneously Detected Based on Nanobody-Phage: Using Carcinogen Aflatoxin and Its Main Fungal Aspergillus Section Flavi spp. in Stored Maize for Demonstration.

Simultaneous detection technology has become a hot topic in analytical chemistry; however, very few reports on how to simultaneously detect small molecular contaminants and microorganisms have been in place. Aflatoxins are a group of highly toxic and carcinogenic compounds, which are produced mainly by Aspergillus flavus and Aspergillus parasiticus from section Flavi responsible for aflatoxin accumulation in stored cereals. Both aflatoxins and Aspergillus section Flavi were used to demonstrate the duplex real-time RCR method of simultaneously detecting small molecular contaminants and microorganisms. The detection of aflatoxins and Aspergillus section Flavi was carried out depending on the anti-idiotypic nanobody-phage V2–5 and aflatoxin-synthesis related gene nor-1 (=aflD), respectively. The quantitative standard curves for simultaneous detection of aflatoxins and Aspergillus section Flavi were constructed, with detection limits of 0.02 ng/ml and 8 × 102 spores/g, respectively. Naturally contaminated maize samples (n = 25) were analyzed for a further validation. The results were in good agreement between the new developed method and the referential methods (high-performance liquid chromatography and the conventional plating counts).

Antimicrobial activity and inhibition of the NorA efflux pump of Staphylococcus aureus by extract and isolated compounds from Arrabidaea brachypoda

Arrabidaea brachypoda is a native shrub of the Brazilian Cerrado widely used in the folk medicine for treatment of renal diseases and articular pains. This study aimed to, first, evaluate the antimicrobial activity of both extracts and isolated molecules Brachydins BR-A and BR-B obtained from the flowers of A. brachypoda against Staphylococcus aureus, Escherchia coli and Candida albicans species. A second objective was to investigate if these natural products were able to potentiate the Norfloxacin activity against the strain Staphylococcus aureus SA1199-B that overexpress the norA gene encoding the NorA efflux pump. Extracts and isolated compounds were analyzed by HPLC-PDA and LC-ESI-MS respectively. Minimal inhibitory concentrations of Norfloxacin or Ethidium Bromide (EtBr) were determined in the presence or absence of ethanolic extract, dichloromethane fraction, as well as BR-A or BR-B by microdilution method. Only BR-B showed activity against Candida albicans. Addition of ethanolic extract, dichloromethane fraction or BR-B to the growth media at sub-inhibitory concentrations enhanced the activity of both Norfloxacin and EtBr against S. aureus SA1199-B, indicating that these natural products and its isolated compound BR-B were able to modulate the fluoroquinolone-resistance possibly by inhibition of NorA. Moreover, BR-B inhibited the EtBr efflux in the SA1199-B strain confirming that it is a NorA inhibitor. Isolated BR-B was able to inhibit an important mechanism of multidrug-resistance very prevalent in S. aureus strains, thus its use in combination with Norfloxacin could be considered as an alternative for the treatment of infections caused by S. aureus strains overexpressing norA.

Polyphasic Assessment of Aflatoxin Production Potential in Selected Aspergilli.

This study investigated the aflatoxin production potentials of selected fungi using a polyphasic approach. Internally transcribed spacer region of the fungi was amplified using the polymerase chain reaction. Forty-five Aspergillus strains were further assessed for aflatoxin production using the conventional methods such as growth on yeast extract sucrose, β-cyclodextrin neutral red desiccated coconut agar (β-CNRDCA); expression of the aflatoxin regulatory genes and the use of both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A large proportion (82.22%) of the isolates harbored the Nor-1 gene while 55.56%, 68.89%, and 80% possessed the ver-1, omt-A, and aflR genes, respectively. All 100% the isolates harbored the aflJ gene. Twenty-three isolates were positive for aflatoxin production based on the yeast extract sucrose medium (YES) test; ammonium vapor test (51%), yellow pigment production (75.5%), and β-CNRDCA tests; and blue/green fluorescence (57.7%). Based on TLC detection 42.2% produced aflatoxins while in the HPLC, total aflatoxin (AFTOT) production concentrations ranged from 6.77–71,453 µg/g. Detectable aflatoxin B1 (AFB1) concentrations obtained from the HPLC ranged between 3.76 and 70,288 µg/g; 6.77 and 242.50 µg/g for aflatoxin B2 (AFB2); 1.87 and 745.30 µg/g for aflatoxin G1 (AFG1); and 1.67 and 768.52 µg/g for aflatoxin G2 (AFG2). AFTOT contamination levels were higher than European Union tolerable limits (4 µg/kg). The regression coefficient was one (R2 = 1) while significant differences exist in the aflatoxin concentrations of Aspergillus (p ≤ 0.05). This study reports the potentials of Aspergillus oryzae previously known as a non-aflatoxin producer to produce AFG1, AFG2, AFB1, and AFB2 toxins. Aspergillus species in feedlots of animals reared for food are capable of producing aflatoxins which could pose hazards to health.

Evaluation of multidrug efflux pump expression in clinical isolates of Staphylococcus.aureus

BackgroundOne of the mechanisms of Staphylococcus aureus resistance to ciprofloxacin is the presence of efflux pumps. The aim of this study was to determine the frequency and expression of norA, norB and norC efflux pump genes in ciprofloxacin-resistant Staphylococcus aureus clinical isolates. Materials and methodsIn this study, 40 isolates of S. aureus were obtained from blood and urine samples. Antibiotic resistance pattern by disc diffusion method and the minimum inhibitory concentration (MIC) was evaluated. Identification and expression of nor efflux family genes (norA, norB, and norC) in ciprofloxacin-resistant isolates of S. aureus were determined using Real-Time PCR method. ResultsThe resistant to ciprofloxacin, Gentamicin, Cotrimoxazol, Erythromycin and Amoxicillin was 42.5%, 12.5%, 12.5%, 50% and 87.5%, respectively. All of ciprofloxacin-resistant isolates were positive for of norA, norB, and norC genes. The results of Real-Time PCR showed that all ciprofloxacin-resistant isolates are active in the case of efflux pumps. Isolates showed different expressions of the norA, norB and norC genes. Expression levels of norA and norB increased by 6.8 and 7.1 fold, respectively. ConclusionThe results indicated that nor efflux family genes were expressed in all ciprofloxacin-resistant isolates of S. aureus. Moreover, there is an association between the expression of the efflux pump genes and resistance to ciprofloxacin in S. aureus isolates.

<p>A novel mechanism of action of ketoconazole: inhibition of the <em>NorA</em> efflux pump system and biofilm formation in multidrug-resistant <em>Staphylococcus aureus</em></p>

Background: The rapid emergence of antimicrobial resistance among Gram-positive organisms, especially staphylococci, has become a serious clinical challenge. Efflux machinery and biofilm formation are considered two of the main causes of antimicrobial resistance and therapy failure.Aim: Our study aims to evaluate the antibiofilm and efflux pump inhibitory activity of the antifungal ketoconazole against multidrug-resistant (MDR) Staphylococcus aureus.Methods: Ketoconazole was tested for its effect on the following: minimum inhibitory concentrations (MICs) of ciprofloxacin, norfloxacin, levofloxacin, and ethidium bromide (EtBr) by the broth microdilution method, the efflux of EtBr by NorA-positive MDR S. aureus, and the relative expression of NorA, NorB, and NorC efflux pump genes. Docking studies of ketoconazole were performed using 1PW4 (glycerol-3-phosphate transporter from Escherichia coli which was the representative structure from the major facilitator superfamily).Results: Ketoconazole significantly decreased the MICs of levofloxacin, ciprofloxacin, norfloxacin, and EtBr (a substrate for efflux pump) by 8 to 1024-fold (P<0.01) and decreased the efflux of EtBr. Furthermore, a time-kill assay revealed that combinations of levofloxacin with ketoconazole or carbonyl cyanide m-chlorophenylhydrazone showed no growth for the tested strains after 24 h in comparison to the effect of levofloxacin alone. Docking studies and the ability of ketoconazole to diminish the relative expression of NorA gene in comparison to control (untreated strains) confirmed its action as an efflux pump inhibitor. Conclusion: The findings showed that the antifungal ketoconazole has no antibacterial activity but can potentiate the activity of the fluroquinolones against MDR S. aureus via inhibiting efflux pump and biofilm formation in vitro.

Determination of aflatoxins by HPLC and the identification of biosynthetic nor-1 gene of aflatoxins in poultry products by PCR assay

Aflatoxins residues in different poultry products were determined using HPLC technique and the expression of biosynthetic nor-1 gene of these aflatoxins by PCR assay. 90 different chicken products were analyzed for presence of aflatoxins (AFB1, AFB2, AFG1 and AFG2) using highly accurate, robust, selective, sensitive and precise HPLC-FLD assay. Incidence of aflatoxins in examined samples showed that the level of AFB1, AFB2 and AFG2 were 10 % for each and of AFG1 was 16.7%. The highest aflatoxins levels of analyzed samples were found in the liver. Expression of nor-1 gene of aflatoxins in polluted poultry products by PCR assay. A Real time PCR (RT-PCR) technique was applied to detect the gene expression of structural nor-1, which catalyzed the first step in the biosynthetic pathway of aflatoxins. The obtained results showed a very strong relationship between the presences of aflatoxins biosynthetic genes as analyzed by multiplex PCR and aflatoxins determination by HPLC/FLD.

Essential Oil, Extracts, and Sesquiterpenes Obtained From the Heartwood of Pilgerodendron uviferum Act as Potential Inhibitors of the Staphylococcus aureus NorA Multidrug Efflux Pump.

Staphylococcus aureus is a serious human pathogen that is highly adaptive to environmental conditions and rapidly develops antibiotic resistance. The use of efflux pumps to reduce antibiotic concentrations at the intracellular level is one of the main mechanisms by which bacteria develop antibiotic resistance. The management of efflux pumps, specifically NorA, which is expressed by S. aureus strains, is a valuable strategy for restoring susceptibility in strains resistant to antibacterial agents. In recent years, many studies have focused on searching for natural substances that can reverse efflux pump-mediated resistance in S. aureus. Extracts and compounds obtained from plants can be efficient efflux pump inhibitors (EPIs) and represent a potentially patient-friendly strategy for controlling S. aureus. In the present study, we evaluated the ability of essential oils, petroleum ether extracts, dichloromethane extract (DCME) and six compounds isolated from the heartwood of Pilgerodendron uviferum (Cupressaceae) and two synthetic derivatives to inhibit efflux in NorA pumps in the following three S. aureus strains: K2378, which overexpressed the norA gene (norA++), K1902 (norA-deleted, ΔnorA) and the parental strain, NCTC 8325-4. Efflux activity was evaluated using a fluorometric method that measured the accumulation of the universal efflux pump substrate ethidium bromide (EtBr). Only DCME and the compounds 15-copaenol and epi-cubenol inhibited EtBr efflux by K2378. Even the lowest concentration of 15-copaenol exhibited a stronger inhibitory effect than carbonyl cyanide m-chlorophenyl hydrazone on EtBr efflux by K2378. 15-copaenal only showed inhibition of EtBr efflux in K2378 cells at 125 μg/mL, but not superior to the control inhibitor and 15-copaenyl acetate exerted no intrinsic EPI activity against K2378. Fractional inhibitory concentration index (FICI) values obtained in the checkerboard assays, indicated that all combinations between DCME, epi-cubenol and 15-copaenol, and tested antibiotics showed a synergistic effect in wild type, norA++ and ΔnorA strains. Moreover, those were not toxic for the HeLa cell line at concentrations in which the synergistic effect and inhibitory activity of efflux pumps was determined. Other extracts and compounds obtained from P. uviferum did not display EtBr efflux-inhibiting activity against the evaluated S. aureus strains.

Biosynthesis of Fe3O4@Ag Nanocomposite and Evaluation of Its Performance on Expression of norA and norB Efflux Pump Genes in Ciprofloxacin-Resistant Staphylococcus aureus.

At present, the universal health problem with Staphylococcus aureus is the emergence of multidrug-resistant strains due to the overuse of antibiotics. Drug extrusion through efflux pumps is one of the bacterial mechanisms to neutralize the bactericidal effect of antibiotics. The antibacterial activity of silver nanoparticle as well as Fe3O4 nanoparticle had been previously studied and widely described. Today, the development of green methods for nanomaterial synthesis is an important aspect of research in the field of nanotechnology. Here, we report the biosynthesis and characterization of Fe3O4@Ag nanocomposite by Spirulina platensis cyanobacterium and it impacts on the expression of efflux pump genes in ciprofloxacin-resistant S. aureus (CRSA). The physical properties of biosynthesized nanocomposite measured and confirmed by ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and scanning and transmission electron microscopy. The minimum inhibitory concentration (MIC) of ciprofloxacin in CRSA strains was determined in the presence of Fe3O4@Ag nanoparticles by broth microdilution method. The effect of Fe3O4@Ag nanocomposite on the expression of norA and norB genes was evaluated by real-time PCR. The physical analysis confirmed well-dispersed, highly stable, and mostly spherical Fe3O4/Ag NPs with the average size of 30-68nm. The results of antibacterial tests showed the synergistic effects of nanocomposite and antibiotics in MIC reduction. Additionally, in the presence of Fe3O4@Ag nanocomposite, the expression of norA and norB genes was decreased more than twofold compared to control. In conclusion, the Fe3O4/Ag nanocomposite can use as an effective inhibitor of antibiotic resistance in medicine.