norA Gene Research Articles

Selection of non-toxigenic strains of Aspergillus flavus for biocontrol of aflatoxins in maize in Thailand

Aflatoxin production in maize and peanuts remains a major public health problem, especially in developing countries. One promising method for combating aflatoxin formation is biocontrol using competitive exclusion, a management strategy currently being studied in maize crops in Thailand. It is important that the strains of Aspergillus flavus used in biocontrol be non-toxigenic and be incapable of reversion to toxigenicity. In the current study, 80 non-toxigenic strains of A. flavus, randomly selected from commercially produced dried maize samples from several sources in Thailand, were screened for their potential as biocontrol strains by examining the 24 aflatoxin biosynthesis genes, using a PCR assay. Assessment of the presence or absence of PCR products provides an indication of the function of pathway genes. Of the 80 strains, 78 showed no PCR products from one or more genes in the aflatoxin biosynthesis pathway. Twenty-three isolates showed only one failure, in the aflD (nor-1) gene, but most isolates failed to produce a PCR product for two or more genes. Nineteen isolates (24%) failed to show a PCR product in 10 or more genes. Altogether, 45 PCR product patterns were observed, usually common to only one or two isolates, indicating great diversity in the aflatoxin biosynthesis pathway in A. flavus isolates taken from dried Thai maize. Although the absence of a particular PCR product is not conclusive evidence that the particular gene is non-functional, the absence of several such PCR products provides reasonable evidence that the isolate in question will not revert to toxigenicity in the field.

Molecular differentiation between aflatoxinogenic and non-aflatoxinogenic strains of Aspergillus flavus and Aspergillus parasiticus

Three types of media and a multiplex PCR procedure with a set of four primers were used to differentiate between aflatoxinogenic and non-aflatoxinogenic strains of Aspergillus flavus and Aspergillus parasiticus. Four sets of primers were the aflR, nor-1, ver-1, and omt-A genes of the aflatoxin biosynthetic pathway. Multiplex PCR showed that the four aflatoxinogenic strains gave a quadruplet pattern, indicating the presence of all the genes involved in the aflatoxin biosynthetic pathway which encode for the products. Non-aflatoxinogenic strains gave varying results with two, three, or four banding patterns. A banding pattern in seven non-aflatoxinogenic strains resulted in non-differentiation between these and aflatoxinogenic strains.

Association of norB overexpression and fluoroquinolone resistance in clinical isolates of Staphylococcus aureus from Korea

Although the prevalence of fluoroquinolone resistance among methicillin-resistant Staphylococcus aureus (MRSA) is known to be higher than in methicillin-susceptible S. aureus (MSSA), the reasons have never been identified. We randomly selected 115 isolates of S. aureus collected from 10 different hospitals in Korea between June 2009 and May 2011. To investigate the difference in fluoroquinolone resistance mechanisms between MRSA and MSSA, we evaluated gyrA and parC mutations and the relative expression of the multidrug efflux pump genes norA, norB and norC. All 62 ciprofloxacin-resistant S. aureus had either gyrA or parC mutations. The S84L mutation of gyrA (59/62, 95.2%) and the S80F mutation of parC (61/62, 98.4%) were the most common. Fifty-eight (93.6%) strains had both the S84L mutation of gyrA and the S80F mutation of parC. Among the 115 isolates, norB overexpression was the most common, occurring in 49 (42.6%) strains. There were only two (1.7%) strains with norA overexpression and none with norC overexpression. Strains overexpressing norB were more common among ciprofloxacin-resistant S. aureus (33/62, 53.2%) than ciprofloxacin-susceptible S. aureus (16/53, 30.2%) (P = 0.013). When we analysed 62 ciprofloxacin-resistant S. aureus strains, those overexpressing norB were more common in ciprofloxacin-resistant MRSA (28/46, 60.9%) than in ciprofloxacin-resistant MSSA (5/16, 31.3%) (P = 0.041). Increased expression of norB can be a factor that contributes to ciprofloxacin resistance in MRSA strains.

The potential effects of Zataria multiflora Boiss essential oil on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes of toxigenic Aspergillus parasiticus

This study aims at evaluating the effects of Zataria multiflora (Z. multiflora) essential oil (EO) on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes. Total RNAs of Aspergillus parasiticus (A.parasiticus) ATCC56775 grown in yeast extract sucrose (YES) broth medium treated with Z. multiflora EO were subjected to reverse transcription- polymerase chain reaction (RT-PCR). Specific primers of nor-1, ver-1, omt-A and aflR genes were used. In parallel mycelial dry weight of samples were measured and all the media were assayed by high-pressure liquid chromatography (HPLC) for aflatoxinB1 (AFB1), aflatoxinB2 (AFB2), aflatoxinG1 (AFG1), aflatoxinG2 (AFG2) and aflatoxin total (AFTotal) production. The results showed that mycelial dry weight and aflatoxin production reduce in the presence of Z. multiflora EO (100 ppm) on day 5 of growth. It was found that the expression of nor-1, ver-1, omt-A and aflR genes was correlated with the ability of fungus to produce aflatoxins on day 5 in YES medium. RT-PCR showed that in the presence of Z.multiflora EO (100 ppm) nor-1, ver-1 and omtA genes expression was reduced. It seems that toxin production inhibitory effects of Z. multiflora EO on day 5 may be at the transcription level and this herb may cause reduction in aflatoxin biosynthesis pathway genes activity.

Testis Selective Expression of NOR1 Gene in Rat and Down Regulation of NOR1 Protein in Human Testicular Germ Cell Tumors

Testis Selective Expression of NOR1 Gene in Rat and Down Regulation of NOR1 Protein in Human Testicular Germ Cell Tumors

Overexpression of oxidored-nitro domain containing protein 1 inhibits human nasopharyngeal carcinoma and cervical cancer cell proliferation and induces apoptosis: Involvement of mitochondrial apoptotic pathways

Oxidored-nitro domain containing protein1 (NOR1) is a novel member of the nitroreductase family that was first isolated as a tumor suppressor gene from human nasopharyngeal carcinoma (NPC). However, the role of NOR1 gene dysfunction in human cancers has not been addressed. We analyzed the expression of NOR1 in various human cancer and benign tissue specimens and found significant downregulation in nine types of cancer compared with corresponding non-tumor tissues. The recombinant expression vector pCDNA3.1-myc-his-NOR1 was constructed and transfected into human NPC 6-10B nasopharyngeal cancer and HeLa cervical cancer (CCA) cells. We found that stable NOR1 overexpression resulted in suppression of 6-10B and HeLa cell proliferation and led to S phase cell cycle arrest. In addition, NOR1 upregulation enhanced apoptosis in pCDNA3.1-myc-his-NOR1 stably transfected cells, and it also altered the expression of proteins involved in the mitochondria-dependent apoptotic pathway. Furthermore, we also found that the NOR1 protein is a cytoplasmic protein that is partially localized in the mitochondria and endoplasmic reticulum. Therefore, NOR1 is an important tumor suppressor gene associated with NPC and CCA and may play antitumor roles by inhibiting proliferation, preventing colony formation, and promoting the apoptosis of tumor cells via the mitochondrial-dependent apoptotic pathway. However, the precise mechanism behind the NOR1 antitumor effects needs to be investigated further.

Inhibition of Grb2-mediated activation of MAPK signal transduction suppresses NOR1/CB1954-induced cytotoxicity in the HepG2 cell line

The nitroreductase oxidored-nitro domain containing protein 1 (NOR1) gene may be involved in the chemical carcinogenesis of hepatic cancer and nasopharyngeal carcinoma (NPC). We have previously demonstrated that NOR1 overexpression is capable of converting the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) into a toxic form by reducing the 4-nitro group of CB1954. Toxic CB1954 is able to enhance cell killing in the NPC cell line CNE1; however, the underlying mechanisms remain unknown. Using cDNA microarrays and quantitative real-time PCR, we previously discovered that NOR1 increases the expression of growth factor receptor-bound protein 2 (Grb2) mRNA by 4.8-fold in the human hepatocellular carcinoma cell line HepG2. In the present study, we revealed that NOR1 increased Grb2 protein expression by 3-fold in HepG2 cells. Additionally, we demonstrated that NOR1 enhanced CB1954-induced cell killing in HepG2 cells, and cell cytotoxicity was inhibited with the tyrosine kinase inhibitor genistein, or by stable transfection of Grb2 small hairpin RNA (shRNA) pU6+27-shGrb2 to silence the expression of Grb2. Western blot analysis revealed that Grb2 downregulation may reduce the activity of the mitogen-activated protein kinase (MAPK). Inhibiting the activation of MAPK using the methyl ethyl ketone (MEK) inhibtor PD98059 suppressed CB1954-induced cell killing. These results suggested that the NOR1 gene enhances CB1954-mediated cell cytotoxicity through the upregulation of Grb2 expression and the activation of MAPK signal transduction in the HepG2 cell line.

Target gene mutations among methicillin-resistant Staphylococcus aureus and methicillin-susceptible S. aureus with elevated MICs of enrofloxacin obtained from diseased food-producing animals or food of animal origin

Sir, Fluoroquinolones are broad-spectrum antimicrobial agents that are effective against Gram-positive and Gram-negative bacteria. Enrofloxacin, the first fluoroquinolone approved for veterinary use, is still commonly used in veterinary practice to treat infections in food-producing, pet and companion animals, caused by a wide variety of bacterial pathogens including methicillinresistant Staphylococcus aureus (MRSA) and methicillinsusceptible S. aureus (MSSA). However, CLSI-approved clinical breakpoints for enrofloxacin applicable to Staphylococcus spp. are restricted to staphylococci from cats and dogs (susceptible, ≤0.5 mg/L; intermediate, 1–2 mg/L; resistant, ≥4 mg/L). No CLSI-approved clinical breakpoints applicable to staphylococci from pigs, cattle or poultry are currently available. DNA gyrase and DNA topoisomerase IV are the target enzymes of fluoroquinolones in S. aureus, and resistance in staphylococci is mainly due to mutations in the quinolone resistance-determining regions (QRDRs) of the genes gyrA, gyrB, grlA and grlB, which code for the corresponding subunits of these two enzymes. Another mechanism involved in fluoroquinolone resistance of S. aureus is overexpression of the norA gene, which encodes a multidrug efflux pump, by mutations in the norA promoter region. The aim of the present study was to gain insight into target gene mutations and norA regulator mutations present among MRSA and MSSA from diseased pigs, cattle and poultry, as well as food of poultry origin. For this, all MRSA and MSSA isolates that exhibited enrofloxacin MICs of ≥1 mg/L, from five different strain collections, were comparatively analysed. This cut-off was chosen based on the breakpoint for enrofloxacin-non-susceptible canine and feline Staphylococcus spp. isolates. The test population included 8/54 MRSA from diseased pigs and 9/32 MRSA from poultry meat and poultry meat products characterized in two previous studies, 2/11 MRSA from diseased cattle and 1/2 MRSA from diseased poultry collected in the GERM-Vet programme 2008–09, 1/2 MRSA and 12/24 MSSA from diseased poultry collected in the GERM-Vet programme 2006–07, and 3/27 MSSA from diseased pigs collected in the BfT-GermVet study 2006–08. In total, 36 isolates with enrofloxacin MICs of 1 to ≥32 mg/L were identified (Table 1). If not already done in previous studies, isolates were subjected to spa typing (http ://spaserver.ridom.de) as well as to two clonal complex (CC) 398-specific PCRs as described previously. Isolates that were negative in these PCRs were subjected to multilocus sequence typing (MLST; http://saureus.mlst.net). Of the 36 isolates, 24 were assigned to sequence type (ST) 398 or CC398, depending on whether MLST or the CC398-specific PCRs had been conducted. Five isolates had ST9, another five isolates had ST5 and one isolate had ST1791 (both ST5 and ST1791 belong to CC5). One avian isolate had ST2269, which is a novel ST (allelic profile 1-4-1-4-243-250-10) that has been identified for the first time during the course of this study. Nine different spa types (t002, t011, t034, t214, t899, t1197, t1419, t1430 and t2346) were detected (Table 1). The QRDRs of the gyrA, gyrB, grlA and grlB genes and the promoter region of the norA gene of all 36 S. aureus isolates were amplified by PCR using previously described primers. Mutations were identified by sequencing the PCR products, and the results are summarized in Table 1 in relation to the enrofloxacin MICs. Regardless of their origin and their methicillinresistance status, all isolates with enrofloxacin MICs of ≥4 mg/L exhibited mutations in both grlA at codon 80 (resulting in a Ser to Phe exchange) and gyrA at codon 84 (resulting in exchange of Ser to Leu or Ala) (Table 1). The Ser84Ala exchange in GyrA observed in two MRSA isolates has not been reported before in staphylococci. In contrast, isolates with enrofloxacin MICs of 1 or 2 mg/L also exhibited a mutation in grlA at codon 80 (resulting in the exchange of Ser to Phe, Tyr or Leu), but lacked the amino acid substitutions at position 84 in GyrA. Another interesting observation was the presence of the Glu422Asp exchange in GrlB in all isolates assigned to CC398/ ST398, independent of enrofloxacin MIC, the year of isolation,

Identification and expression of nor efflux family genes in Staphylococcus epidermidis that act against gatifloxacin

NorA, NorB, and NorC are efflux proteins in the Nor family that regulate the secretion of fluoroquinolones, and MgrA/NorR is a transcription factor of the Nor family. Overexpression of Nor family proteins provides fluoroquinolone resistance in Staphylococcus aureus. However, in coagulase-negative staphylococci (CNS), members of the Nor family had not been identified. In this work, the presence of Nor family proteins in Staphylococcus spp. and the expression of Nor family in gatifloxacin resistant S. epidermidis strains obtained from ocular infections (OI) were identified and analyzed.S. epidermidis strains from OIs (n = 44) and healthy skin (HS; n = 52) were isolated. The nor family genes were identified in CNS using PCR, sequencing and phylogenetic approaches. Nor family expression was determined by RT-PCR. NorA efflux activity was determined using the automated ethidium bromide method.In-silico analysis showed that norA, mgrA/norR, and “norB-like” and “norC-like” (norB/norC) genes are present in CNS. The nor family genes were distributed and constitutively expressed in all S. epidermidis strains studied. In one gatifloxacin resistant strain isolated from the endophthalmitis, treatment with gatifloxacin induced overexpression of the norA gene and resulted in high activity of NorA efflux.These results indicate that the Nor family of proteins is present in CNS, and the NorA efflux mechanism for gatifloxacin response occurs in at least one strain of S. epidermidis, contributing to gatifloxacin resistance.

Influence of sub-lethal antioxidant doses, water potential and temperature on growth, sclerotia, aflatoxins and aflD (=nor-1) expression by Aspergillus flavus RCP08108

Effects of interacting conditions of sub-lethal levels of antioxidants, water potential (Ψ) and temperature were evaluated on growth, sclerotial characteristics, aflatoxin B1 (AFB1) production and aflD (=nor-1) gene expression by Aspergillus flavus strain RCP08108. These studies were carried out on peanut meal extract agar osmotically modified to −2.8,−7.1, −9.9 and −16.0MPa and incubated at 28 and 20°C. The food grade antioxidants added were butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) at (1+1mM−M1) and (5+5mM−M2). To relate the aflD expression after toxigenic A. flavus grew under interacting stress conditions, real-time PCR was used. Antioxidant mixtures caused a higher and significant (p<0.001) reduction in growth rate. The major impact on size and volume sclerotia was produced by Ψ; followed by antioxidant mixtures. High AFB1 levels were observed in response to the M1 applied at −7.1MPa. Induction of the aflD gene was observed in response to the M1 treatment at −2.8, −7.1 and −9.9MPa; but significant decreases of AFB1 production and aflD transcripts were observed; when the fungus grew in the presence of the M2 treatment.These results showed that it is necessary to apply food-grade antioxidants into the peanut storage system at levels higher than 5mM. This is an important tool to avoid sub-lethal antioxidant doses that can lead to fungal growth, increase resistance structures, and stimulate aflD gene expression and AFB1 accumulation in this substrate.

A Novel Non Radioactive PCR-DNA Probe for the Detection of Aflatoxin Producing &amp;lt;i&amp;gt;Aspergillus&amp;lt;/i&amp;gt; Species from Major Food Crops Grown in India

In the present study, a novel non radioactive digoxigenen labelled PCR-DNA probe was developed targeting nor-1 gene to assess the contamination of aflatoxigenic Aspergillus species in food grain samples from Southern parts of India. The sensitivity of developed PCR-DNA probe was determined to be 10 pg of genomic DNA and 1 pg of purified PCR product. The specificity of the DNA probe was validated by testing against an array of Aspergillus, Fusarium and Penicillium spp. A total of 89 Aspergillus isolates were recovered from 152 grain samples of maize, paddy, and groundnut. Among them, maize had the highest (90%) incidence of toxigenic Aspergillus species. When developed PCR-DNA probe was evaluated onto pure cultures of toxigenic and nontoxigenic Aspergillus species, 60 samples were positive for the nor-1 gene probe. DNA probe results unequivocally matched with the HPLC analysis. In conclusion, the novel PCR-DNA probe developed in this study may find its application in rapid detection of aflatoxin-producing Aspergillus isolates from contaminated cereal grains.

Construction of Streptomyces nogalater Lv65 strains with enhanced nogalamicin biosynthesis using regulatory genes

Influence of cloned regulatory genes on nogalamycin biosynthesis by Streptomyces nogalater LV65 strain has been studied. Gene snorA from the S. nogalater genome was cloned in multicopy replicative plasmid pSOKA and integrative plasmid pR3A. Introduction of these plasmids into S. nogalater wild type cells resulted in enhanced nogalamicin biosynthesis. A similar effect was observed at heterologous expression of gene (p)ppGpp-synthetase gene relA cloned from Streptomyces coelicolor A3(2). Heterologous expression of genes absA2 from Streptomyces ghanaensis ATCC14672 and lndYR from genome Streptomyces globisporus 1912 decreased synthesis of antibiotic. The study results indicate the presence of homologs of these genes in chromosome of S. nogalater, their possible participation in regulation of nogalamicin biosynthesis, and provide us with a possibility for genetic design of the strains with higher synthesis of this antibiotic.

Increased expression of NOR-1 mRNA in the skeletal muscles of cold-exposed neonatal chicks

Nuclear receptor subfamily 4, group A (NR4A) subgroup orphan receptors are rapidly induced by various physiological stimuli and have been suggested to regulate oxidative metabolism and muscle mass in mammalian skeletal muscle. The results showed that the NR4A subgroup orphan receptor, NOR-1 (NR4A3), was acutely increased in skeletal muscles of neonatal chicks in response to short-term cold exposure. The increased NOR-1 gene expression was concomitant with cold-induced changes in gene expression of both myostatin and proliferator-activated receptor-gamma coactivator-1 (PGC-1α), and the increase in skeletal muscle mass. These observations suggest that NOR-1 might play a role in controlling skeletal muscle growth in cold-exposed neonatal chicks.

NOR1 is an HSF1- and NRF1-regulated putative tumor suppressor inactivated by promoter hypermethylation in nasopharyngeal carcinoma

Promoter hypermethylation-mediated silencing of tumor suppressor genes (TSGs) is a hallmark of oncogenesis. Oxidored-nitro domain-containing protein 1 (NOR1) is a candidate TSG that is downregulated in nasopharyngeal carcinoma (NPC). In the present study, we identified a functional NOR1 promoter that is regulated by heat shock factor 1 and nuclear respiratory factor 1. The promoter is located within a CpG island. Hypermethylation of this CpG island was found in NPC tissue samples and cancer cell lines, whereas no aberrant promoter methylation was detected in non-cancerous nasopharyngeal tissue samples or normal nasopharyngeal epithelial cells. Treatment of NPC 6-10B cells and leukemia HL60 cells with 5'-aza-2'-deoxycytidine increased endogenous levels of NOR1 messenger RNA. Ectopic expression of NOR1 in NPC HNE1 cells inhibited tumor cell colony formation and viability. These findings suggest that promoter hypermethylation may participate in transcriptional inactivation of the NOR1 gene in NPC. Frequent epigenetic inactivation of the NOR1 gene in NPC suggests that it may be a critical tumor suppressor involved in the development of NPC.

Distribution and expression of alternative splice isoforms of NOR1 in human tissues and cell lines.

To analyze the distribution and subcellular localization of NOR1 alternative splice isoforms in human tissues and cell lines. NOR1 open reading frame(ORF) was amplified from human fetal brain cDNA library and subcloned into pCMV/myc vector. The level of NOR1 mRNA in human tissues was determined by real-time RT-PCR. Region spanning exon 2 was amplified from cDNA or genomic DNA by specific primers and sequenced. The expression plasmids of NOR1 were transfected into cells and immunofluoresence assay was performed to determine the subcellular localization of NOR1 protein isoforms in human cells. The expression of NOR1 mRNA was high in human adult testis, moderate in human fetus nasopharynx, trachea, brain, and kidney tissues, and weak or undetectable in other tissues. Two splice variants of human NOR1 gene resulted from alternative splicing at exon 2 were identified. Both of isoform 1 and isoform 2 mRNA were detected in human fetus brain. Isoform 2 was the sole isoform in other tissues but brain. Only isoform 2 mRNA was detected in cell lines used in this study, though no genomic deletion of exon 2 could be found in all these cell lines. Immunofluoresence assay showed both isoform 1 and isoform 2 proteins were distributed in cytoplasm. Alternative splice isoforms of tumor suppressor gene NOR1 are identified. NOR1 isoform 1 and isoform 2 are both detected in fetus brain. NOR1 isoform 2 lacking of exon 2 is the sole isoform in multiple tissues except for brain. The exon 2 encoded peptide does not affect the subcellular location of NOR1 protein.

Optimization of prokaryotic expression condition and purification of anti-cancer protein NOR1 in E.coli

To optimize the induction condition of human NOR1 gene expression in E.coli. and purify NOR1 recombinant proteins. A full-length cDNA of human NOR1 was inserted into the corresponding region of pET28b expression vector to yield recombinant prokaryotic expression vector pET28b-NOR1. The prokaryotic expression vector pET28b-NOR1 was introduced into the bacterial host E.coli Rosettablue(DE3). Recombinant NOR1 protein was induced at different conditions. Induction condition was optimized to obtain high yield of recombinant protein. At last, the recombinant NOR1 protein was purified by Ni-IDE chromatography resin. Recombinant NOR1 protein was induced by IPTG in a dose-dependent manner. Increase of kanamycin concentration and induction temperature resulted in high yield of recombinant protein. The most recombinant protein was found in inclusion bodies. The recombinant His-NOR1 protein was purified with Ni-IDE chromatography resin under denature condition. IPTG, kanamycin concentration and temperature can affect the expression of recombinant NOR1 protein in pET28b system. High yield of recombinant NOR1 protein is achieved by inducing 1 mmol/L IPTG and 200 μg/mL kanamycin at 37 degree. Recombinant His-NOR1 protein with high purity is purified.

Control of Aflatoxin Production of Aspergillus flavus and Aspergillus parasiticus Using RNA Silencing Technology by Targeting aflD (nor-1) Gene

Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B1 (AFB1), and aflatoxin G1 (AFG1) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB1 production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB1 production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB1 production by A. flavus EGP9 and AFG1 production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG1 production by A. parasiticus SSWT 2999. Changes in AFB1 production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.

Isolation of the drug-resistant Staphylococcus aureus strains from burn wound flora and analysis of norA genetic mutation

：Objective To investigatethe genetic mutation of the norA gene and its promotor from the wild-type drug-resistanceStaphylococeus aureus(S.aureus)strains. Methods A total of 10 antibiotic-resistantS.aureus strains were isolated and screened from the burn wound for the sequencing andanalysis of the nora gene and its promoter. Results There isolated 87 S.aureus strainsfrom the burn wound flora,which were completely sensitive to vacomycin,highly sensitive toQuinupristin and Nitrofurantoin,but highly resistant to the other antibiotics,even upto91.7% of MRSA.There found the same point mutation(G→A) located at 1 349 sites of the norA gene coding regionin all the S.aureus strains,saying that the amino acid was changed from Gly(glycin)toAsp(agpartic acid) in 291 sites.The resetpine reverse test showed that the MICs value ofthree antibiotics was lowered at various degrees in all 10strains.Conclusion NorA genemutation is one of the mechanisms for antibiotic-resistance of S.aureus. Key words: Burns; Staphylococcus aureus; Drug resistance; norAgene; Point mutation

Temporal monitoring of the nor-1 (aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different water activity levels

A relative quantification system (RQ-PCR) was used to monitor the correlations between the activity of the nor-1 (=aflD) gene of Aspergillus flavus using real-time PCR in relation to phenotypic aflatoxin B(1) (AFB(1) ) production and populations of A. flavus in stored peanuts at three water activity levels (a(w) , 0·95, 0·90 and 0·85) for 6 weeks. Real-time PCR was used to amplify the nor-1 gene (target gene), and benA56 (β-tubulin gene) used as a control gene. Expression of three structural genes, nor-1 (=aflD), ver-1 (=aflM), and omtA (=aflP), and the regulatory gene aflR of the aflatoxin biosynthetic pathway were also assayed. There were significant differences between nor-1 gene expression at the three a(w) levels; higher expression at 0·90 a(w) in weeks 1-3, when compared to 0·95. In contrast, in the driest treatment (0·85 a(w) ) none or very low nor-1 expression occurred. The populations of A. flavus colony-forming units (CFUs g(-1) ) increased over time with the highest at 0·95 a(w) . Highest AFB(1) production was at 0·90 and 0·95 a(w) from weeks 3-6. A(w) had a significant effect on aflR transcription at 0·95 a(w) over the 6-week period, while at 0·90 a(w) , only in the last 2 weeks. Correlations between different factors showed that log AFB(1) × log CFUs, log AFB(1) × a(w) , and log CFUs × a(w) were statistically significant, while log CFUs × RQ-PCR and RQ-PCR × a(w) were not. The AflR gene may not have an important role in the regulation of nor-1 expression in food matrices (e.g. peanuts). Determination of correlations between nor-1 expression and aflatoxin production by A. flavus in raw peanuts under different a(w) levels could be helpful to predict potential risk of aflatoxin production during storage of this hygroscopic food product and minimize contamination with the AFB(1) .

Detection and quantification of Aspergillus section Flavi spp. in stored peanuts by real-time PCR of nor-1 gene, and effects of storage conditions on aflatoxin production

Aspergillus flavus and A. parasiticus are the main species from section Flavi responsible for aflatoxin accumulation in stored peanuts. A real-time PCR (RT-PCR) system directed against the nor-1 gene of the aflatoxin biosynthetic pathway as target sequence was applied to monitor and quantify Aspergillus section Flavi population in peanuts. Kernels were conditioned at four water activity ( a W) levels and stored during a 4-month period. The quantification of fungal genomic DNA in naturally contaminated peanut samples was performed using TaqMan fluorescent probe technology. Sensitivity tests demonstrated that DNA amounts accounting for a single conidium of A. parasiticus RCP08300 can be detected. A standard curve relating nor-1 copy numbers to colony forming units (cfu) was constructed. Counts of species of Aspergillus section Flavi from unknown samples obtained by molecular and conventional count (CC) methodologies were compared. A correlation between cfu data obtained by RT-PCR and CC methods was observed ( r = 0.613; p < 0.0001); and the former always showed values higher by 0.5–1 log units. A decrease of fungal density was observed throughout the storage period, regardless of the quantification methodology applied. Total aflatoxin levels ranging from 1.1 to 200.4 ng/g were registered in peanuts conditioned at the higher a W values (0.94–0.84 a W). The RT-PCR assay developed appears to be a promising tool in the prediction of potential aflatoxigenic risk in stored peanuts, even in case of low-level infections, and suitable for rapid, automated and high throughput analysis.