norA Gene Research Articles

A novel α-mangostin derivative synergistic to antibiotics against MRSA with unique mechanisms.

Methicillin-resistant Staphylococcus aureus (MRSA) remains a leading cause of hospital-acquired infections, often linked to complicated treatments, increased mortality risk, and significant cost burdens. Several antibacterial agents have been developed to address MRSA resistance. In this study, potential agents to combat MRSA resistance were explored, with the antibacterial activity of synthesized α-mangostin (α-MG) derivatives being evaluated alongside investigations into their cellular mechanisms against MRSA2. α-MG-4, featuring an allyl group at C3 of the lead compound α-MG, restored the sensitivity of MRSA2 to penicillin, enrofloxacin, and gentamicin, while also demonstrating improved safety profiles. Although α-MG-4 alone was ineffective against MRSA2, it exhibited an optimal synergistic ratio in vitro when combined with these antibiotics. This significant synergistic antibacterial effect was further confirmed in vivo using a mouse skin abscess model. Additionally, the synergistic mechanisms revealed that α-MG-4 was associated with changes in membrane permeability and inhibition of the MepA and NorA genes, which encode the efflux pumps of MRSA2. α-MG-4 also inhibited PBP2a expression, potentially by occupying a crucial binding site in a dose-dependent manner.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA)'s resistance to multiple antibiotics poses significant health and safety concerns. A novel α-mangostin (α-MG) derivative, α-MG-4, was first identified as a xanthone-based PBP2a inhibitor that reverses MRSA2 resistance to penicillin. The synergistic antibacterial effects of α-MG-4 were linked to increased cell membrane permeability and the inhibition of genes involved in efflux pump function.

Antimicrobial Susceptibility and Genomic Profiles of Multidrug-Resistant Staphylococcus aureus from Nasopharynx of Asymptomatic Children in Dhaka, Bangladesh.

Children carrying Staphylococcus aureus in their nasopharynx are at a higher risk of contracting systemic infection. Due to lack of sufficient information regarding such carriage, this study was conducted to explore the prevalence, antibiotic susceptibility, and genomic profiles of S. aureus isolated from nasopharyngeal samples of 163 randomly selected asymptomatic Bangladeshi children aged from 5-<15 years. Antibiotic susceptibility pattern and genomic analysis of the samples were conducted using standard microbiological methods and genomic tools. The carriage was confirmed in 44 (27%) children who were mostly well nourished without respiratory symptoms in the last 3 months. Higher carriage was observed among the younger age group (5-<10 years) who completed vaccines for pneumonia (p = 0.002) and influenza (p = 0.004). Among the isolates, 84.1% were multidrug-resistant and 47.5% (n = 40) were methicillin-resistant S. aureus (MRSA). All the isolates (100%) were resistant to cefixime with higher resistance to ampicillin (95.5%) and penicillin (90.9%). Among the three investigated isolates, two were ST80 (ID-1 and ID-52) and one was a novel strain (ID-19) with the presence of aph-Stph, blaI, blaZ, dha1, fosB, lmrS, mepA, norA, and tet38 genes. The current research demonstrates a high incidence of multidrug-resistant S. aureus and reports the first instance of ST80 in asymptomatic children in Bangladesh.

Thymoquinone’ potent impairment of multidrug-resistant Staphylococcus aureus NorA efflux pump activity

The drug efflux pump is a crucial mechanism implicated in resistance to multiple antimicrobials. Thymoquinone (TQ) has evidently demonstrated multiple activities, antibacterial being the most effective. Knowledge about TQ activity against multidrug-resistant Staphylococcus aureus is very scarce. Therefore, the present study was conducted to investigate TQ resistance modulation in ciprofloxacin (CIP) and doxycycline (DO) multidrug-resistant S. aureus. Forty-seven samples were collected from different sources, and S. aureus was isolated and identified. Then, S. aureus resistance profiles to antimicrobials, N. sativa essential oil, and TQ; the correlation between TQ-MIC readings and disc diffusion; cartwheel and ethidium bromide (EtBr) accumulation assays; and norA gene expression were all described within silico molecular docking for TQ interactions with norA efflux pump protein. TQ-MICs ranged from 5–320 µg/ml. TQ down-regulated norA gene expression, resulting in a drop in efflux pump activity of 77.5–90.6% in the examined strains, comparable to that observed with verapamil. Exposure of S. aureus strains to CIP and DO raises the initial basal efflux pumping expression to 34.2 and 22.9 times, respectively. This induced efflux pumping overexpression was substantially reduced by 97.7% when TQ was combined with CIP or DO. There was a significant reduction of MICs of CIP and DO MICs by 2–15 and 2–4 folds, respectively, after treatment with 0.5XMIC-TQ in resistance modulation assays. These results refer to TQ ligand inhibitory interactions with NorA protein in molecular docking. Interpretations of inhibition zone diameters (IZDs) of disc diffusion and TQ-MICs exhibit independence of MICs from IZDs, as indicated by invalid linear regression analysis. TQ significantly reduced efflux pumping S. aureus induced by CIP and DO, but further investigations are needed to improve TQ-pharmacokinetics to restore CIP and DO activity and suppress fluoroquinolone and doxycycline-resistant S. aureus selection in clinical and animal settings.

SUMOylation of nuclear receptor Nor1/NR4A3 coordinates microtubule cytoskeletal dynamics and stability in neuronal cells

BackgroundNor1/NR4A3 is a member of the NR4A subfamily of nuclear receptors that play essential roles in regulating gene expression related to development, cell homeostasis and neurological functions. However, Nor1 is still considered an orphan receptor, as its natural ligand remains unclear for mediating transcriptional activation. Yet other activation signals may modulate Nor1 activity, although their precise role in the development and maintenance of the nervous system remains elusive.MethodsWe used transcriptional reporter assays, gene expression profiling, protein turnover measurement, and cell growth assays to assess the functional relevance of Nor1 and SUMO-defective variants in neuronal cells. SUMO1 and SUMO2 conjugation to Nor1 were assessed by immunoprecipitation. Tubulin stability was determined by acetylation and polymerization assays, and live-cell fluorescent microscopy.ResultsHere, we demonstrate that Nor1 undergoes SUMO1 conjugation at Lys-89 within a canonical ψKxE SUMOylation motif, contributing to the complex pattern of Nor1 SUMOylation, which also includes Lys-137. Disruption of Lys-89, thereby preventing SUMO1 conjugation, led to reduced Nor1 transcriptional competence and protein stability, as well as the downregulation of genes involved in cell growth and metabolism, such as ENO3, EN1, and CFLAR, and in microtubule cytoskeleton dynamics, including MAP2 and MAPT, which resulted in reduced survival of neuronal cells. Interestingly, Lys-89 SUMOylation was potentiated in response to nocodazole, a microtubule depolymerizing drug, although this was insufficient to rescue cells from microtubule disruption despite enhanced Nor1 gene expression. Instead, Lys-89 deSUMOylation reduced the expression of microtubule-severing genes like KATNA1, SPAST, and FIGN, and enhanced α-tubulin cellular levels, acetylation, and microfilament organization, promoting microtubule stability and resistance to nocodazole. These effects contrasted with Lys-137 SUMOylation, suggesting distinct regulatory mechanisms based on specific Nor1 input SUMOylation signals.ConclusionsOur study provides novel insights into Nor1 transcriptional signaling competence and identifies a hierarchical mechanism whereby selective Nor1 SUMOylation may govern neuronal cytoskeleton network dynamics and resistance against microtubule disturbances, a condition strongly associated with neurodegenerative diseases.

Potential fungicidal and antiaflatoxigenic effects of cinnamon essential oils on Aspergillus flavus inhabiting the stored wheat grains

Wheat is one of the essential crops for the human and animal nutrition, however, contamination with aflatoxigenic fungi, due to the improper storage conditions and high humidity, was the main global threats. So, preventing the growth of aflatoxigenic fungi in stored wheat grains, by using different essential oils was the main objective of this work. Aspergillus flavus EFBL-MU12 PP087400, EFBL-MU23 PP087401 and EFBL-MU36 PP087403 isolates were the most potent aflatoxins producers inhabiting wheat grains. The effect of storage conditions of wheat grains “humidity, temperature, incubation period, and pH” on growth of A. flavus, was assessed by the response surface methodology using Plackett-Burman design and FCCD. The highest yield of aflatoxins EFBL-MU12 B1 and B2 by A. flavus grown on wheat grains were 145.3 and 7.6 μg/kg, respectively, at incubation temperature 35°C, 16% moisture contents, initial pH 5.0, and incubated for 14 days. The tested oils had a powerful antifungal activity for the growth and aflatoxins production by A. flavus in a concentration-dependent manner. Among these oils, cinnamon oil had the highest fungicidal activity for A. flavus at 0.125%, with about 85-90 % reduction to the aflatoxins B1 and B2, conidial pigmentation and chitin contents on wheat grains. From the SEM analysis, cinnamon oils had the most deleterious effect on A. flavus with morphological aberrations to the conidial heads, vegetative mycelia, alteration in conidiophores identity, hyphae shrank, and winding. To emphasize the effect of the essential oils on the aflatoxins producing potency of A. flavus, the molecular expression of the aflatoxins biosynthetic genes was estimated by RT-qPCR. The molecular expression of nor-1, afLR, pKsA and afLJ genes was suppressed by 94-96%, due to cinnamon oil at 0.062% compared to the control. Conclusively, from the results, cinnamon oils followed by the peppermint oils displayed the most fungicidal activity for the growth and aflatoxins production by A. flavus grown on wheat grains.

Yolk-shell nanospheres based silane-functionalized carbon dots: Designed synthesis and application in highly selective determination of aflatoxin biosynthesis-related genes nor-1 in cereal crops

This study presented advanced monitoring approaches for the aflatoxin gene cluster in Aspergillus flavus and Aspergillus parasiticus, which are effective tools for controlling aflatoxin proliferation in the food chain. A novel approach involved a ratiometric fluorescence probe based on yolk-shell nanospheres (bCDs@SiO2@gQDs). The probe's fluorescence was activated by DNA hybridization with the modified bCDs@SiO2@gQDs and gold nanoparticles (AuNPs), functioning through donor–acceptor pairs. The exceptional properties of the gQDs and the high FRET efficiency of this system established a functionalized platform for DNA labeling. When the nor-1 gene was present, FRET between the gQDs and AuNPs triggered fluorescence quenching, enabling nor-1 gene detection. The yolk-shell nanostructure of the fluorescent nanoprobes provided an internal reference, reducing experimental errors due to background noise, instrumentation factors, and probe concentration. This allowed for accurate and highly sensitive quantitative determination of the nor-1 gene. The ratiometric fluorescence assay demonstrated a broad linear range (0.1–100 nM) and a low detection limit (12.83 pM) for the nor-1 gene. Tests on non-contaminated maize samples spiked with various concentrations of the nor-1 gene achieved recovery rates between 95.43 % and 101.46 %. This biosensor represents a significant advancement in food safety, offering a simple, rapid, and sensitive method for monitoring agricultural products.

Impact of clonal lineages on susceptibility of Staphylococcus lugdunensis to chlorhexidine digluconate and chloride benzalkonium.

Little is known about susceptibility of Staphylococcus lugdunensis to antiseptics. The objective of this study was to evaluate, at the molecular and phenotypic level, the susceptibility of 49 clinical S. lugdunensis strains (belonging to the seven clonal complexes [CCs] defined by multilocus sequence typing) to two antiseptics frequently used in healthcare settings (chlorhexidine digluconate [CHX] and chloride benzalkonium [BAC]). The minimum inhibitory concentrations (MICs), by broth microdilution method, varied for BAC from 0.25mg/L to 8mg/L (MIC50 = 1mg/L, MIC90 = 2mg/L) and for CHX from 0.5mg/L to 2mg/L (MIC50 = 1mg/L, MIC90 = 2mg/L). The BAC and CHX minimum bactericidal concentrations (MBCs) varied from 2mg/L to 8mg/L (MBC50 = 4mg/L, MBC90 = 8mg/L) and from 2mg/L to 4mg/L (MBC50 and MBC90 = 4mg/L), respectively. A reduced susceptibility to CHX (MIC = 2mg/L) was observed for 12.2% of the strains and that to BAC (MIC ≥ 4mg/L) for 4.1%. The norA resistance gene was detected in all the 49 isolates, whereas the qacA gene was rarely encountered (two strains; 4.1%). The qacC, qacG, qacH, and qacJ genes were not detected. The two strains harboring the qacA gene had reduced susceptibility to both antiseptics and belonged to CC3. The norA gene was detected in all the strains, suggesting that it could belong to the core genome of S. lugdunensis. S. lugdunensis is highly susceptible to both antiseptics tested. Reduced susceptibility to BAC and CHX was a rare phenomenon. Of note, a tendency to higher MICs of BAC was detected for CC3 isolates. These results should be confirmed on a larger collection of strains.

Inhibition of the norA gene expression and the NorA efflux pump by the tannic acid

The NorA efflux pump of Staphylococcus aureus is known to play a major role in the development of resistance against quinolone drugs by reducing their concentration inside target pathogens. The objective of this study was to evaluate the ability of tannic acid to inhibit the gene expression of the NorA efflux pump in Staphylococcus aureus and to evaluate the in silico effect on the pump. Efflux pump inhibition was evaluated by fluorimetry. The checkerboard method evaluates the effect of the test substance in combination with an antimicrobial at different concentrations. To gene expression evaluation NorA the assay was performed using: a sub-inhibitory concentration preparation (MIC/4) of the antibiotic; a sub-inhibitory concentration preparation (MIC/4) of the antibiotic associated with tannic acid at a sub-inhibitory concentration (MIC/4). In this study, docking simulations were performed by the SWISSDOCK webserver. The ability of tannic acid to inhibit the NorA efflux pump can be related to both the ability to inhibit the gene expression of this protein, acting on signaling pathways involving the ArlRS membrane sensor. As well as acting directly through direct interaction with the NorA protein, as seen in the approach and in silico and in vitro per checkerboard method and fluorimetry of bromide accumulated in the cell.

Efflux pump inhibitory potential of indole derivatives as an arsenal against norA over-expressing Staphylococcus aureus.

NorA, an extensively studied efflux pump in Staphylococcus aureus, has been connected to fluoroquinolone, antiseptic, and disinfection resistance. Several studies have also emphasized how efflux pumps, including NorA, function as the first line of defense of S. aureus against antibiotics. In this study, we have screened some chemically synthesized indole derivatives for their activity as efflux pump inhibitors (EPIs). The derivative SMJ-5 was found to be a potent NorA efflux pump inhibitor among the screened indole derivatives, owing to increased ethidium bromide and norfloxacin accumulation in norA over-expressing S. aureus. The combination of SMJ-5 and ciprofloxacin demonstrated the eradication of S. aureus biofilm and prolonged the post-antibiotic effect more than ciprofloxacin alone. SMJ-5 was able to inhibit staphyloxanthin virulence. In in vitro time-kill trials and in vivo efficacy investigations, the combination enhanced the bactericidal activity of ciprofloxacin against S. aureus. Additionally, reverse transcription PCR results revealed that SMJ-5 also inhibits the NorA efflux pump indirectly at the transcriptional level. IMPORTANCE The NorA efflux pump is the most effective resistance mechanism in S. aureus. The clinical importance of NorA efflux pumps is demonstrated by the expression of pump genes in S. aureus strains in response to fluoroquinolones and biocides. Along with the repercussions of decreased fluoroquinolone sensitivity, increasing expression of efflux pump genes by their substrate necessitates the importance of efflux pump inhibitors. Reserpine and verapamil are clinically used to treat ailments and have proven NorA inhibitors, but, unfortunately, the concentration needed for these drugs to inhibit the pump is not safe in clinical settings. In the current study, we have screened some indole derivatives, and among them, SMJ-5 was reported to potentiate norfloxacin and ciprofloxacin at their sub-inhibitory concentration by inhibiting the norA gene transcriptionally. Here we highlight the promising points of this study, which could serve as a model to design a therapeutic EPI candidate against norA over-expressing S. aureus.

Docking Complexes of Active Phytochemicals with VK-deficient Genes

Background:: Vitamin K (VK) deficiency occurs when the body does not have enough vitamin K to produce proteins that are essential for blood clotting and bone health. Vitamin K is a cofactor that plays a major role in various comorbidities. Over the years, efforts have been made to identify the interaction between natural compounds, such as K vitamers, that could play a sig-nificant role in regulation of the blood coagulation. We intended to obtain insights into the poten-tial therapeutic implications of phytochemicals for treating VK deficiency-related diseases by in-vestigating the interactions between phytochemicals and VK-deficient genes. Methods:: On active phytochemical docking complexes with VK-deficient genes, there is no spe-cific information available as of yet. In this computationally aided docking study, we were inter-ested in finding the pathogenic blood coagulation-related genes that are linked to VK deficiency. Based on literature reviews and databases, bioactive phytochemicals and other ligands were con-sidered. To provide precise predictions of ligand-protein interactions, docking parameters and scoring algorithms were thoroughly optimized. We have performed molecular docking studies and observed the way the complexes interact. Results:: Specific binding interactions between active phytochemicals and VK pathogenic muta-tions have been identified by the docking study. Hydrogen bonds, van der Waals interactions, and hydrophobic contacts, which are indications of high binding affinities, have been observed in the ligand-protein complexes. Few phytochemicals have demonstrated the ability to interact with the targets of VK-deficient genes, indicating their capacity to modify pathways relevant to VK defi-ciency. The results of the docking study have explained the three pathogenic genes, viz. VWF, F8, and CFTR, wherein VWF and F8 play important roles in blood coagulation and people with cyst-ic fibrosis, to have a deficiency in vitamin K. Thirty-five compounds from different plant and natural sources were screened through molecular docking, out of which two compounds have been considered as controls, including curcumin and warfarin (R-warfarin and S-warfarin), which are the most common anticoagulants readily available in the market. They act by inhibiting vita-min K epoxide reductase (VKOR), which is needed for the gamma-carboxylation of vitamin K-dependent factors. Conclusion:: A focus on other compounds, like theaflavin, ellagic acid, myricetin, and catechin was also made in this study as they show more binding affinity with the three pathogenic proteins. Based on the results, the complexes have been found to possess great potential and thus may be considered for further interaction studies. The potential for active phytochemicals to generate docking complexes with VK-deficient genes is highlighted in this computational analysis. Health disorders related to VK insufficiency may be significantly impacted by these interactions. To val-idate the expected interactions and determine the therapeutic potential of the identified phyto-chemicals, more experimental research, including in vitro and in vivo experiments, is needed.

Identification of norA, qacAB and smr genes and determination of antibiotic and biocide resistance patterns in methicillin-resistant Staphylococcus aureus isolated from Isfahan-Iran

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections, which have become a serious problem due to antibiotic resistance. In addition to antibiotic resistance, biocide resistance is also increasing due to the high use of these substances. The aim of this study was to investigate the pattern of antibiotic resistance and the prevalence of biocide resistance genes norA, qacA/B and smr, and to determine the minimum inhibitory concentration of chlorhexidine digluconate (CHG) and benzalkonium chloride (BKC) in MRSA isolates taken from teaching hospitals in Isfahan city, Iran. Materials and Methods: This study was conducted on 63 MRSA isolates collected from teaching hospitals in Isfahan city, Iran. MRSA isolates were identified by performing phenotypic testing and then confirmed by using PCR. The resistance pattern was determined by using disc diffusion method. Broth microdilution method was performed to determine the minimum inhibitory concentration (MIC) of CHG and BKC according to the Clinical and Laboratory Standards Institute (CLSI). The presence of biocide resistance genes was determined by using PCR and the Time kill assay was performed to investigate the lethality of biocides. Results: MRSA isolates had MIC values of 0.5–4 μg/ml for BKC and 0.125–4 μg/ml for CHG. norA was the most common biocide resistance gene among 63 isolates (88.9%), followed by qacAB and smr with 28.6% and 22.2%, respectively. Among the isolates, the highest percentage of resistance belonged to penicillin, erythromycin and doxycycline, respectively. Conclusion: Due to the high prevalence of MRSA isolates in hospitals and the role of this pathogen in nosocomial infections, continuous monitoring of antimicrobial resistance for appropriate antibiotic administration and continuous evaluation of biocide resistance due to the high prevalence of resistance genes and appropriate use of biocides for disinfection, allows us to minimize further prevalence of resistant strains in hospital environments.

A graphene-oxide-based fluorometric assay for norA gene transcription in MRSA using Nb.BbvCI-assisted target recycling and T7 exonuclease-triggered cascade dual recycling signal amplification

We describe a graphene oxide (GO)-based bioassay for the fluorometric determination of norA gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This approach is based on Nb.BbvCI-assisted target recycling (NATR) and T7 exonuclease (T7 Exo)-triggered cascade dual-recycling signal amplification (TTCDRSA). The system included GO, a capture probe (CP), an assistant probe (AP), two carboxyfluorescein (FAM)-labeled hairpins (HP1 and HP2), endonuclease Nb.BbvcI, and exonuclease T7. In the presence of a target, AP, together with the target RNA, can hybridise with CP via partial complementarity to one another and open its hairpin structure to form a triple complex that is recognised by Nb.BbvCI. Once the CP is cleaved, the released AP and target RNA can walk on the carboxylated graphene oxide (CGO) surface to bind with another CP which induces the next round of cleavage, accumulating many trigger probes (TPs). The TPs then activate TTCDRSA with the assistance of T7 Exo, HP1, and HP2 to produce large amounts of free FAMs. These free FAMs are repelled by GO and exhibit enhanced fluorescence signals at excitation/emission wavelengths of 480/514 nm. The limit of detection (LOD) of the bioassay was calculated to be 0.37 fM, and the linear range of the method ranged from 1 fM to 1 nM. More importantly, the bioassay also exhibited high sensitivity and selectivity for target RNA detection in real samples, which may open a new promising avenue for monitoring drug efflux and studying the mechanisms of drug actions.

The Effects of Antiperspirant Aluminum Chlorohydrate on the Development of Antibiotic Resistance in Staphylococcus epidermidis.

This study investigates the effects of the antiperspirant aluminum chlorohydrate on the development of antibiotic resistance in commensal Staphylococcus epidermidis isolates. The isolates were exposed to aluminum chlorohydrate for 30 days. The bacteria that developed resistance to oxacillin and ciprofloxacin were isolated, and the expression levels of some antibiotic resistance genes were determined using quantitative reverse transcriptase PCR. Before and after exposure, the minimum inhibitory concentration (MIC) values of the bacteria were determined using the microdilution method. A time-dependent increase was observed in the number of bacteria that developed resistance and increased MIC values. Consistent with the ciprofloxacin resistance observed after exposure, an increase in norA, norB/C, gyrA, gyrB, parC, and parE gene expression was observed. In addition to aluminum chlorohydrate exposure, oxacillin resistance was observed in all test bacteria in the group only subcultured in the medium, suggesting that phenotypic resistance cannot be correlated with chemical exposure in light of these data. The increase in mecA gene expression in selected test bacteria that acquired resistance to oxacillin after exposure compared with control groups suggests that the observed resistance may have been related to aluminum chlorohydrate exposure. To our knowledge, this is the first time in the literature that the effects of aluminum chlorohydrate as an antiperspirant on the development of antibiotic resistance in Staphylococcus epidermidis have been reported.

Evaluation of Gold Nanoparticles on the Expression of NorA and NorB Efflux Pumps in Ciprofloxacin-resistant Staphylococcus aureus Isolated from Burn Patients

Background: In the development of multidrug resistance, efflux pumps effectively pump drug compounds out of cells, which results in reduced membrane permeability to drug compounds. This study evaluated the effect of gold nanoparticles on the inhibition of norA and norB efflux pumps in ciprofloxacin-resistant Staphylococcus aureus isolated from burn patients in Qom province, Iran. Methods: In this cross-sectional study, S. aureus strains were isolated from burn patients in Qom hospital, Iran. After gold nanoparticles were synthesized using chemical reduction and characterized by spectrophotometry, transmission electron microscopy (TEM), and dynamic light scattering (DLS), ciprofloxacin resistance of S. aureus was screened by the disc diffusion method. The Minimum Inhibitory Concentration (MIC) of ciprofloxacin (CCCP), ciprofloxacin + gold nanoparticles (CCCP + gold nanoparticles), and ciprofloxacin + CCCP was determined. Moreover, norA and norB genes were evaluated by PCR using special primers. Real-time PCR was then performed for norA and norB genes. Results: Of 88 S. aureus strains tested, 50 (56.81%) were resistant to ciprofloxacin. From the 50 ciprofloxacin-resistant S. aureus strains, 12 isolates had active pumps. Real-time PCR of 12 ciprofloxacin- resistant S. aureus and S. aureus ATCC 25923 before and after exposure to ciprofloxacin, gold nanoparticles, and gold nanoparticles with sub-MIC ciprofloxacin revealed significant differences in expression of norA and norB genes before exposure to the treatments compared to after exposure (p &lt;0.05). Conclusion: Gold nanoparticles with ciprofloxacin could be used to prevent the expression of pump genes involved in resistance to fluoroquinolone compounds.

Deciphering the antibiofilm potential of 2-Phenylethyl methyl ether (PEME), a bioactive compound of Kewda essential oil against Staphylococcus aureus

Opportunistic pathogenic bacteria and their pathogenicity linked with biofilm infections become a severe issue as they resist the actions of multiple antimicrobial drugs. Naturally derived drugs having antibiofilm properties are more effective than chemically synthesized drugs. The plant derived essential oils are a rich source of phytoconstituents with widespread pharmacological values. In the present investigation, a major phytoconstituent, 2-Phenyl Ethyl Methyl Ether (PEME) of Kewda essential oil extracted from the flowers of Pandanus odorifer was explored for its prospective antimicrobial and anti-biofilm properties against ESKAPE pathogenic bacterial strains, Staphylococcus aureus and MTCC 740. The minimum inhibitory concentration (MIC) of PEME was found to be 50 mM against the tested bacterial strains. A gradual decrease in biofilm production was observed when PEME was treated with the sub-MIC concentration. The reduction in biofilm formation was noticeable from qualitative assay i.e., Congo Red Agar Assay (CRA) and further quantified by crystal violet staining assay. The decline in exopolysaccharides production was quantified, with the highest inhibition against MTCC 740 with a decrease of 71.76 ± 4.56% compared to untreated control. From the microscopic analysis (light and fluorescence microscopic method), PEME exhibited inhibitory effect on biofilm formation on the polystyrene surface. The in silico studies stated that PEME could invariably bind to biofilm associated target proteins. Further, transcriptomic data analysis suggested the role of PEME in the down-regulation of specific genes, agrA, sarA, norA and mepR, which are critically associated with bacterial virulence, biofilm dynamics and drug resistance patterns in S. aureus. Further, qRT-PCR analysis validated the role of PEME on biofilm inhibition by relative downregulation of agrA, sarA, norA and mepR genes. Further, advanced in silico methodologies could be employed in future investigations to validate its candidature as promising anti-biofilm agent.

Molecular characterization of aflatoxigenic and non-aflatoxigenic Aspergillus flavus strains isolated from peanut sold in northern Togo

The contamination of agricultural products and aflatoxins synthesis can be controlled and reduced by the application of a biological control product based on atoxinogenic Aspergillus flavus strains. The purpose of this work was to select atoxinogenic Aspergillus flavus strains that can be used in the biological control of aflatoxins. Thirty-four (34) strains of Aspergillus flavus were used and the Nor-1, Ver-1, Omt-A and AflR genes involved in the aflatoxin biosynthesis pathway were sought. The growth speed and ability of atoxigenic strains to inhibit the growth of toxigenic strains were also determined. The results showed that 50% of the strains have at least one of the genes sought and the percentages of inhibition (PI) of the atoxinogenic strains varied significantly from 16.98% to 62.50% at the threshold of 5% according to the Tukey test (p-value = 0.0000136). The mycelial growth speed (VCM) of the atoxinogenic strains varied from 7.5 to 10 mm/day with an average VCM of 8.4 ± 0.05 mm/day. Strains AKA-10; AGA-8; AMAN-35; ABA-28 and AGA-46 were found to be more effective in inhibiting mycelial growth of toxigenic strains.

The Evaluation of Efflux Pump Genes norA, norB and norC Related to Fluoroquinolones Resistance in Staphylococcus aureus Strains Isolated from Blood Infection

&#x0D; &#x0D; &#x0D; &#x0D; Background &amp; Objective: Recently, ciprofloxacin resistance in Staphylococcus aureus strains, due to efflux pumps, has become a significant challenge. Therefore, this study was performed to evaluate the frequency of norA, norB, and norC efflux pump genes and their roles in resistance to ciprofloxacin in clinical isolates of S. aureus.&#x0D; &#x0D; &#x0D; &#x0D; &#x0D; Materials &amp; Methods: A total of one hundred clinical blood samples were collected from patient in Qom hospitals and S. aureus isolates were identified by standard microbiological tests. Antimicrobial susceptibility patterns were determined by the disk diffusion method using CLSI guidelines. Subsequently, the presence of norA, norB, and norC efflux pump genes in ciprofloxacin isolates was detected using the PCR method.&#x0D; &#x0D; &#x0D; &#x0D; &#x0D; Results: Among one hundred clinical samples, 36 S. aureus isolates were recovered and the results of antibiotic susceptibility tests showed that twenty of them were resistant to ciprofloxacin. 15 isolates were resistant to norfloxacin and one isolate was resistant to ofloxacin. Moreover, the norA, norB, and norC genes were found in 58%, 30%, and 41% of ciprofloxacin-resistant isolates, respectively.&#x0D; &#x0D; &#x0D; &#x0D; &#x0D; Conclusion: Based on the results of this study, norA, norB, and norC efflux pumps may play a significant role in the development of resistance to ciprofloxacin in clinical isolates of S. aureus. Detecting these genes may prove useful in suggesting an effective treatment model for infections caused by S. aureus.&#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D;

Association of Antibacterial Susceptibility Profile with the Prevalence of Genes Encoding Efflux Proteins in the Bangladeshi Clinical Isolates of Staphylococcus aureus

Expelling antibiotic molecules out of the cell wall through multiple efflux pumps is one of the potential mechanisms of developing resistance against a wide number of antibiotics in Staphylococcus aureus. The aim of this study was to investigate the association between the antibiotic susceptibility profile and the prevalence of different efflux pump genes i.e., norA, norB, norC, mepA, sepA, mdeA, qacA/B, and smr in the clinical isolates of S. aureus. Sixty clinical isolates were collected from a tertiary level hospital in Bangladesh. The disc diffusion method using ten antibiotics of different classes was used to discern the susceptibility profile. polymerase chain reaction (PCR) was employed to observe the resistance patterns and to detect the presence of plasmid and chromosomal encoded genes. Among the clinical isolates, 60% (36 out of 60) of the samples were Methicillin-resistant Staphylococcus aureus (MRSA), whereas 55% (33 out of 60) of the bacterial samples were found to be multi-drug resistant. The bacteria showed higher resistance to vancomycin (73.33%), followed by ciprofloxacin (60%), cefixime (53.33%), azithromycin (43.33%), and amoxicillin (31.67%). The prevalence of the chromosomally-encoded efflux genes norA (91.67%), norB (90%), norC (93.33%), mepA (93.33%), sepA (98.33%), and mdeA (93.33%) were extremely high with a minor portion of them carrying the plasmid-encoded genes qacA/B (20%) and smr (8.33%). Several genetic combinations of efflux pump genes were revealed, among which norA + norB + norC + mepA + sepA + mdeA was the most widely distributed combination among MRSA and MSSA bacteria that conferred resistance against ciprofloxacin and probably vancomycin. Based on the present study, it is evident that the presence of multiple efflux genes potentiated the drug extrusion activity and may play a pivotal role in the development of multidrug resistance in S. aureus.

Identification of Quinolones/Fluoroquinolones Resistance Genes from Staphylococci Strains Isolated at the University Hospital of Brazzaville, Republic of the Congo

Staphylococci strains, like the majority of bacterial strains, have developed the resistance to several antibiotics, including Quinolones and Fluoroquin-olones In the Republic of the Congo, cases of resistance leading to treat-ment failures have been observed during the treatment of staphylococcal infections with antibiotics in hospitals. The objective of this study was to identify the Quinolone/Fluoroquinolone resistance genes from staphylo-cocci strains isolated in hospitals. A total of 51 strains of Staphylococci were isolated, including 16 (31.37%) community strains, and 35 (68.62%) clinical strains. 46 strains of Staphylococcus aureus (S. aureus) and 5 SCNs were identified. A total of 34 DNA fragments from different strains resistant to Quinolones/Fluoroquinolones, including 21 (61.67%) DNA fragments from clinical S. aureus and 13 (38.23%) from community SCN strains were analyzed by the molecular method (genotypic detection) by PCR. The genotypic results made it possible to identify the gyrA, grLA and norA genes and to show that these genes are involved in the resistance of the strains to the various antibiotics used. The grLA gene was the most identified gene with a frequency of 75%. The gyrA and grLA genes have been identified in Staphylococcus aureus and Coagulase Negative Staphy-lococci. The norA gene, on the other hand, has only been identified in Staphylococcus aureus. Two mechanisms are essentially involved in the resistance of Staphylococci to quinolones/Fluoroquinolones, the mecha-nism of resistance by efflux, which takes place thanks to a transmembrane protein coded by the norA gene and by point mutations (substitution and deletion of acids or nucleotides) observed within the protein and nucleic sequences of the chromosomal gyrA and grLA genes.

Inhibitory activities of vitamins K2 against clinical isolates of quinolone-resistant and methicillin-resistant Staphylococcus aureus (QR-MRSA) with different multi-locus sequence types (MLST), SCCmec, and spa types

BackgroundThe inhibitory activities of vitamins K2 against clinical isolates of quinolone-resistant and methicillin-resistant Staphylococcus aureus (QR-MRSA) are unclear. The main aim is to better understand of inhibitory activities of vitamins K2, multi-locus sequence typing (MLST), SCCmec, and spa typing in clinical isolates of QR-MRSA on those mutation and gene expressions.Materials and methodsAfter collecting S. aureus clinical isolates and detecting QR-MRSA, the genes encoding norA, grlA, grlB, gyrA, and gyrB were sequenced. After treating isolates by vitamin K2, isolates were prepared to measure norA, grlA, grlB, gyrA, and gyrB gene expression. The quantitative-real-time PCR was used to measure the expression of efflux pump genes.ResultsQR-MRSA, MDR, and XDR strains were reported in 59.4%, 73.9%, and 37.6% of isolates, respectability. SCCmecIV (36.5%) and SCCmecV (26.8%) had the highest frequency. Thirty-nine spa types were identified, t021, t044, and t267 types most prevalent in QR-MRSA isolates. ST22 and ST30 dominated the invasive, drug-resistant isolates and QR-MRSA. In 24 h incubated isolates, the most noticeable change of gene expression with vitamin K2 was that the norA, gyrA, and grlB genes were highly repressed. However, the down-regulation of grlA at 24 h after being treated by vitamin K2 was more than another gene. Further, a significant decrease was observed in QR-MRSA-treated isolates compared to un-treated isolates. In other words, norA, grlA, grlB, gyrA, and gyrB genes were less suppressed by QR-MRSA (p ≤ 0.01, p ≤ 0.05).ConclusionVitamin K2 has significant inhibitory effects on the genes responsible for resistance to fluoroquinolone antibiotics. However, a subminimum inhibitory concentration (sub-MIC) level of vitamin K2 was delayed but did not completely inhibit norA, grlA, grlB, gyrA, and gyrB genes in MRSA strains.