Understanding the response of Desulfovibrio desulfuricans ATCC 27774 to the electron acceptors nitrate and sulfate - biosynthetic costs modulate substrate selection.

Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that obtain their energy from dissimilatory sulfate reduction. Some SRB species have high respiratory versatility due to the possible use of alternative electron acceptors. A good example is Desulfovibrio desulfuricans ATCC 27774, which grows in the presence of nitrate (end product: ammonium) with higher rates and yields to those observed in sulfate containing medium (end product: sulfide). In this work, the mechanisms supporting the respiratory versatility of D. desulfuricans were unraveled through the analysis of the proteome of the bacterium under different experimental conditions. The most remarkable difference in the two-dimensional gel electrophoresis maps is the high number of spots exclusively represented in the nitrate medium. Most of the proteins with increase abundance are involved in the energy metabolism and the biosynthesis of amino acids (or proteins), especially those participating in ammonium assimilation processes. qPCR analysis performed during different stages of the bacterium's growth showed that the genes involved in nitrate and nitrite reduction (napA and nrfA, respectively) have different expressions profiles: while napA did not vary significantly, nrfA was highly expressed at a 6h time point. Nitrite levels measured along the growth curve revealed a peak at 3h. Thus, the initial consumption of nitrate and concomitant production of nitrite must induce nrfA expression. The activation of alternative mechanisms for energy production, aside several N-assimilation metabolisms and detoxification processes, solves potential survival problems in adapting to different environments and contributes to higher bacterial growth rates.

Systematic review and meta-analysis of the nitrate reductase assay for drug susceptibility testing of Mycobacterium tuberculosis and the detection limits in liquid medium.

Recently, the need for rapid, reliable, and low-cost drug susceptibility testing (DST) methods has increased due to the emergence of multidrug-resistant Mycobacterium tuberculosis. Colorimetric methods of DST provide results more quickly than standard culture methods and are inexpensive than molecular methods. Thus, colorimetric methods, such as the nitrate reductase assay (NRA), are being recommended. We searched Medline PubMed for reports on the NRA for DST of M. tuberculosis written in English and published within the last five years. We selected 20 reports on six major anti-TB drugs and conducted a meta-analysis using Meta-Disc software. The pooled sensitivities for isoniazid, rifampicin, streptomycin, ethambutol, ofloxacin, and kanamycin were 95.4%, 96.4%, 91.5%, 93.1%, 99.3%, and 88.4%, and the pooled specificities were 98.5%, 99.2%, 92.9%, 97.8%, 97.4%, and 99.4%, respectively. The area under the summary receiver operator curve for all drugs was 0.9723-0.9952. The time to results (TTR) for the direct and indirect NRAs was 7-28days and 6-15days, respectively. Quality assessments were conducted using the quality of diagnostic accuracy studies tool (QUADAS-2) items, and most reports showed good performance. However, ethambutol, streptomycin, and kanamycin showed relatively low sensitivity. We performed a quantitative NRA in liquid media at various inoculum concentrations. The TTR at 4.94×106, 1.67×104, and 2.27×102CFU/mL was 4, 14, and 14days, respectively. The minimum absorbance and nitrite concentration for positive samples were 0.8 and 168µM, respectively. We propose a quantitative standard to determine sample positivity to address the problems with the current standard NRA which is much less expensive than the conventional assay conducted on solid medium.

A Low Cost/Low Power Open Source Sensor System for Automated Tuberculosis Drug Susceptibility Testing.

In this research an open source, low power sensor node was developed to check the growth of mycobacteria in a culture bottle with a nitrate reductase assay method for a drug susceptibility test. The sensor system reports the temperature and color sensor output frequency change of the culture bottle when the device is triggered. After the culture process is finished, a nitrite ion detecting solution based on a commercial nitrite ion detection kit is injected into the culture bottle by a syringe pump to check bacterial growth by the formation of a pigment by the reaction between the solution and the color sensor. Sensor status and NRA results are broadcasted via a Bluetooth low energy beacon. An Android application was developed to collect the broadcasted data, classify the status of cultured samples from multiple devices, and visualize the data for the end users, circumventing the need to examine each culture bottle manually during a long culture period. The authors expect that usage of the developed sensor will decrease the cost and required labor for handling large amounts of patient samples in local health centers in developing countries. All 3D-printerable hardware parts, a circuit diagram, and software are available online.

Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples.

AIMS AND OBJECTIVES: Current methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB) are either costly or slow. As the prevalence of multidrug-resistant (MDR) strains increases, the need for fast, reliable, and inexpensive methods is obvious. This study evaluated a rapid colorimetric nitrate reductase assay (NRA) for direct DST of MTB directly from clinical sputum samples. METHODS: A total of 111 sputa with positive microscopy results for acid-fast bacilli (AFB) with more than 10 AFB per high-power field were used in the study. The samples were decontaminated using the modified Petroff method. The NRA results were compared with the reference indirect proportion method. RESULTS: The sensitivity and the specificity of the direct NRA were 90% and 97.3%, 92.6% and 98.2%, 52.9% and 100%, and 28.6% and 100% for rifampin, isoniazid, streptomycin, and ethambutol, respectively. The results were in most cases available in 28days (84.3%). CONCLUSIONS: The direct NRA could be used as a rapid, inexpensive, and accurate method to determine rifampin and isoniazid susceptibility directly from sputum. The technique might become a valid alternative to traditional methods, especially in low-income countries.

Nitric oxide generated by the rice blast fungus Magnaporthe oryzae drives plant infection.

Plant-derived nitric oxide (NO) triggers defence, priming the onset of the hypersensitive response and restricting pathogen ingress during incompatibility. However, little is known about the role of pathogen-produced NO during pre-infection development and infection. We sought evidence for NO production by the rice blast fungus during early infection. NO production was measured using fluorescence of DAR-4M and the role of NO assessed using NO scavengers. The synthesis of NO was investigated by targeted knockout of genes potentially involved in NO synthesis, including nitric oxide synthase-like genes (NOL2 and NOL3) and nitrate (NIA1) and nitrite reductase (NII1), generating single and double Δnia1Δnii1, Δnia1Δnol3, and Δnol2Δnol3 mutants. We demonstrate that Magnaporthe oryzae generates NO during germination and in early development. Removal of NO delays germling development and reduces disease lesion numbers. NO is not generated by the candidate proteins tested, nor by other arginine-dependent NO systems, by polyamine oxidase activity or non-enzymatically by low pH. Furthermore, we show that, while NIA1 and NII1 are essential for nitrate assimilation, NIA1, NII1, NOL2 and NOL3 are all dispensable for pathogenicity. Development of M. oryzae and initiation of infection are critically dependent on fungal NO synthesis, but its mode of generation remains obscure.

Prevalence of antibiotic resistance in coagulase-negative staphylococci from spontaneously fermented meat products and safety assessment for new starters.

To provide new meat starter strains lacking antibiotic (AB) resistances, we explored the AB susceptibility in 116 coagulase-negative Staphylococcus (CNS) isolates from traditionally fermented sausages (n=40) manufactured with meat from conventional animal breeding, and from meat products (n=76) made from meat of animals raised in natural habitats under low- or no-antibiotic pressure. Less than 50% of these CNS isolates showed phenotypic resistances to at least one antibiotic (AB) by using microdilution assay. Resistances to penicillins and tetracycline were most often observed and could be traced back to blaZ and tet(K) genes. Prevalence of AB resistances was species-dependent and mainly found in isolates of Staphylococcus warneri (78%), Staphylococcus capitis (75%) and Staphylococcus epidermidis (67%), but only sporadically detected in Staphylococcus carnosus (27%) and Staphylococcus equorum (18%). AB resistances were more often observed in S. xylosus isolates originating from natural habitats compared to traditionally fermented sausages made from conventional meat. A selection of 101 isolates belonging to S. xylosus (n=63), S. carnosus (n=21) and S. equorum (n=17) were subsequently grouped by pulsed-field gel electrophoresis (PFGE) into strain clusters. No S. carnosus and only five S. xylosus strains were lacking AB resistances and exhibited a PFGE genotype different from commercial starters. These strains, together with 17 S. equorum strains, were further studied for safety and technological characteristics. The ability to produce biogenic amines was not detected in any strain. PCR amplifications for enterotoxin encoding genes seg-sej were detected in one, and for δ-hemolysin encoding gene hld in four S. equorum strains, but phenotypic hemolytic activity was visible for three S. xylosus and 15 S. equorum strains. Catalase and nitrate reductase activity was observed in all isolates tested; particularly S. equorum showed high nitrate reduction. In conclusion, we were able to select four new meat starter strains (two S. xylosus and two S. equorum strains) out of 116 investigated CNS, fulfilling all safety criteria including the absence of AB resistances, production of biogenic amines and genes encoding virulence factors but exhibiting high nitrate reductase and catalase activity as suitable technological characteristics. Thus, S. equorum isolates, often the dominant species in spontaneously fermented meat products, provided a prospective meat starter species exhibiting high nitrate reduction and low prevalence of AB resistances.

NO loading: Efficiency assessment of five commonly used application methods of sodium nitroprusside in Medicago truncatula plants.

Nitric oxide (NO) is a bioactive, diffusible molecule involved in a multitude of physiological and developmental processes in plants, which has been reported to display both antioxidant and pro-oxidant properties in plants. Several reports exist highlighting the protective action of sodium nitroprusside (SNP), an NO donor, which demonstrate its important role as a signal molecule in plants responsible for the expression regulation of antioxidant and other defense enzymes. However, the mode of application of this compound varies greatly between studies. The present study provides a comprehensive efficiency comparison of the most commonly used application methods using 2.5mM SNP on mature (40 day) Medicago truncatula plants. Measurement of NO content in both leaves and roots suggests that vacuum infiltration is the most efficient method for NO donation in leaf tissue, whereas hydroponic application resulted in highest NO content in roots. NO content correlated with activity levels of nitrate reductase (NR; EC 1.7.99.4), a key enzyme involved in the generation of NO in plants and which is known to be regulated by NO itself.

Fractionation and availability of heavy metals in tannery sludge-amended soil and toxicity assessment on the fully-grown Phaseolus vulgaris cultivars.

This study was conducted to assess the effect of tannery sludge on the bush bean (Phaseolus vulgaris) cultivars fully-grown on a culture sandy soil, as tannery sludge is valuable to improve soil fertility but long term studies evaluating the effect on fully grown plants are scarce. Tannery sludge amendments (0, 0.77, 1.54, 3.08 and 6.16 g tannery sludge kg(-1) soil) were characterized and the main heavy metals identified (Cr, Mn, Fe, K, and Zn) later on sequentially and singly extracted, for soil fractionation and availability determination, respectively. Metals showed different fractionation and availability patterns, being the most toxic metal (Cr) found to primarily bind to the carbonate fraction in soil, while almost 10% of the total Cr was available for plant uptake. In the green house experiments, bush bean cultivars exposed to increasing tannery sludge amendments were evaluated at different plant stages. Metal accumulation and physiological parameters (chlorophyll, carotenoids, nitrate reductase activity and dry weight) were determined. Toxicity was primarily due to Cr, stimulating or affecting the response of physiological parameters and suppressing seed formation at the highest tannery sludge ratio. Metals were mainly accumulated in the roots of bush beans, diminishing in the upper part of the plants with minimal translocation to seeds, supposing little risk for human consumption. Additionally, important correlations, antagonistic and synergistic relationships were observed between the extracted metals and metal accumulation in plant tissues.

Field assessment of the direct nitrate reductase assay for rapid detection of multidrug-resistant tuberculosis in Honduras.

SETTING: The national TB reference laboratory and four health care units connected to the national laboratory network in Honduras, Central America. OBJECTIVE: To evaluate the performance of the direct nitrate reductase assay (NRA) for rapid, low-cost detection of multidrug-resistant tuberculosis (MDR-TB) in a resource-limited setting. DESIGN: Consecutive smear-positive samples (n = 185) were prospectively analysed with NRA and compared to the proportion method on Löwenstein Jensen medium (PM-LJ) to detect resistance to isoniazid (INH) and rifampicin (RMP). RESULTS: The NRA sensitivity, specificity, positive and negative predictive values for INH and RMP were respectively 100%, 99%, 91%, 100% and 80%, 100%, 100%, 99%. Good agreement was observed between NRA and PM-LJ (κ > 0.8). CONCLUSION: The direct NRA is a reliable alternative for rapid and low-cost identification of MDR-TB cases in resource-limited settings.

Direct nitrate reductase assay versus microscopic observation drug susceptibility test for rapid detection of MDR-TB in Uganda.

The most common method for detection of drug resistant (DR) TB in resource-limited settings (RLSs) is indirect susceptibility testing on Lowenstein-Jensen medium (LJ) which is very time consuming with results available only after 2-3 months. Effective therapy of DR TB is therefore markedly delayed and patients can transmit resistant strains. Rapid and accurate tests suitable for RLSs in the diagnosis of DR TB are thus highly needed. In this study we compared two direct techniques--Nitrate Reductase Assay (NRA) and Microscopic Observation Drug Susceptibility (MODS) for rapid detection of MDR-TB in a high burden RLS. The sensitivity, specificity, and proportion of interpretable results were studied. Smear positive sputum was collected from 245 consecutive re-treatment TB patients attending a TB clinic in Kampala, Uganda. Samples were processed at the national reference laboratory and tested for susceptibility to rifampicin and isoniazid with direct NRA, direct MODS and the indirect LJ proportion method as reference. A total of 229 specimens were confirmed as M. tuberculosis, of these interpretable results were obtained in 217 (95%) with either the NRA or MODS. Sensitivity, specificity and kappa agreement for MDR-TB diagnosis was 97%, 98% and 0.93 with the NRA; and 87%, 95% and 0.78 with the MODS, respectively. The median time to results was 10, 7 and 64 days with NRA, MODS and the reference technique, respectively. The cost of laboratory supplies per sample was low, around 5 USD, for the rapid tests. The direct NRA and MODS offered rapid detection of resistance almost eight weeks earlier than with the reference method. In the study settings, the direct NRA was highly sensitive and specific. We consider it to have a strong potential for timely detection of MDR-TB in RLS.

Evaluation of nitrate reductase assay for direct detection of drug resistance in Mycobacterium tuberculosis: rapid and inexpensive method for low-resource settings.

The aim of this study was to evaluate a nitrate reductase assay (NRA) for the direct detection of multidrug resistance (MDR) in Mycobacterium tuberculosis from 100 smear-positive sputum samples. The NRA results were compared with the reference proportion method for 100 sputum specimens for which comparable results were available. NRA results were obtained at day 7 for 61 specimens, results for 26 specimens were obtained at day 10, and the results for 13 specimens were obtained at day 14. Thus, 87% of NRA results were obtained in 10 days. NRA is a rapid, accurate, and cost-effective method for the detection of MDR in M. tuberculosis isolates as compared to the proportion method, which is time consuming. Therefore, NRA constitutes a useful tool for detection of tuberculosis drug resistance in low-resource countries with limited laboratory facilities due to its low-cost, ease of performance and lack of requirement of sophisticated equipment.

Determination of the susceptibility of Mycobacterium tuberculosis to pyrazinamide in liquid and solid media assessed by a colorimetric nitrate reductase assay.

OBJECTIVES: The aim of this study was to develop and evaluate two colorimetric nitrate reductase-based antibiotic susceptibility (CONRAS) tests, the CONRAS-liquid test and the CONRAS-LJ test, to enable susceptibility testing of Mycobacterium tuberculosis to pyrazinamide. To enhance the growth of M. tuberculosis in 7H9 broth with acid pH (6.0), the effect of three potential growth-promoting substances (reconstitution fluid, fastidious organism supplement and epidermal growth factor) was evaluated. METHODS: Seventy-five M. tuberculosis strains were tested for susceptibility to pyrazinamide in the CONRAS-liquid test performed in Middlebrook 7H9 broth, and 77 M. tuberculosis strains were tested for susceptibility to pyrazinamide in the CONRAS-LJ test performed on Löwenstein-Jensen medium. The BACTEC 460TB system, sequencing of the pncA gene and Wayne's assay were used as reference tests. Growth of 10 M. tuberculosis strains in conventional 7H9 broth and in acid 7H9 broth with and without growth-promoting substances added was evaluated by the nitrate reductase assay. RESULTS: By using the BACTEC 460TB system as the reference test, the sensitivity and specificity of the CONRAS-liquid test (100 mg/L pyrazinamide) tested on 75 M. tuberculosis strains were 87.5% and 83.7%, respectively. The corresponding sensitivity and specificity of the CONRAS-LJ test (1200 mg/L pyrazinamide) tested on 77 M. tuberculosis strains were 100% and 84.1%, respectively. The mean turn around time of the CONRAS-liquid test was significantly shorter than that of the CONRAS-LJ test (mean, 9.4 and 14.5 days, respectively; P < 0.001). In addition, no effect of growth-promoting substances on the growth of M. tuberculosis in a broth with acid pH was seen. CONCLUSIONS: The CONRAS tests were rapid, cheap and easy to perform and interpret. Both tests should be evaluated on extended strain batteries in multicentre studies before they can be considered for use in susceptibility testing of M. tuberculosis to pyrazinamide in resource-limited settings.

Direct drug susceptibility testing of Mycobacterium tuberculosis to primary anti-tubercular drugs by nitrate reductase assay.

OBJECTIVES: Traditional drug susceptibility testing for Mycobacterium tuberculosis takes weeks and/or expensive. In this study, we evaluated nitrate reductase assay for drug susceptibility testing which is faster than the visual detection of colonies. MATERIALS AND METHODS: 32 clinical specimens (direct microscopy positive for AFB with 1+, 2+ or 3+ grading) were decontaminated and the sediment was inoculated onto the L-J medium with INH or Rifampicin incorporated with Potassium nitrate and the same medium without antibiotics at 1;10 dilution as control. After 2 weeks, the control was first tested for color change with addition of nitrate reductase reagents. If found positive, the media with antibiotics were tested and compared. Futher incubation was done if the control was found to be negative. The results obtained was compared with standard direct proportion method for drug susceptibility testing. RESULTS: Resistance of isolates as shown by both methods for INH and Rifampicin was 37.5% and 31.3% respectively. The results showed that NRA and proportion method do not differ significantly ( P < 0.05 for both drugs). Thus an excellent agreement between the results of NRA and proportion method was found for two primary anti-tubercular drugs, 87.5% for INH and 97% for Rifampicin. CONCLUSION: Nitrate reductase assay is a rapid and inexpensive method for susceptibility testing of M. tuberculosis for primary anti-tubercular drugs and could be an alternative to existing methods, particularly in resource poor settings.

Fluoroquinolone resistance in Mycobacterium tuberculosis: an assessment of MGIT 960, MODS and nitrate reductase assay and fluoroquinolone cross-resistance.

OBJECTIVES: The aim of this study was to assess the sensitivity, specificity and time to results of mycobacterial growth indicator tube (MGIT) 960, microscopic observation drug susceptibility (MODS) assay and nitrate reductase assay (NRA) compared with the gold standard agar proportion method (PM), and to determine whether there is cross-resistance between older-generation fluoroquinolones and moxifloxacin. METHODS: Mycobacterium tuberculosis isolates from culture-confirmed tuberculosis patients from 2002 to 2007 were tested for ofloxacin (2 mg/L) resistance by PM and MGIT 960. All isolates from 2005 and 2006 were also tested by MODS and NRA. Ofloxacin-resistant isolates by PM were further tested by all four methods using ciprofloxacin, levofloxacin and moxifloxacin. For each ofloxacin-resistant isolate, two ofloxacin-susceptible isolates were tested against all three fluoroquinolones using all four methods. RESULTS: Of the 797 M. tuberculosis isolates, 19 (2.4%) were ofloxacin-resistant by PM. MGIT 960 had 100% sensitivity (95% CI, 83%-100%) and specificity (95% CI, 99.5%-100%). Of the 797 isolates, 239 were from 2005 to 2006 and 6 of these (2.5%) were resistant by PM. MODS had 100% sensitivity (95% CI, 61%-100%) and specificity (95% CI, 98%-100%). NRA had 100% sensitivity (95% CI, 61%-100%) and 98.7% specificity (95% CI, 96%-99.6%). The median time to results was shorter using MGIT 960 (8 days), MODS (6 days) or NRA (9 days) compared with PM (21 days) (P < 0.001). All 19 ofloxacin-resistant isolates were resistant to ciprofloxacin, levofloxacin and moxifloxacin by PM. CONCLUSIONS: MGIT 960, MODS and NRA are sensitive and specific and more rapid than PM for identifying fluoroquinolone resistance in M. tuberculosis. Ofloxacin resistance was associated with cross-resistance to ciprofloxacin, levofloxacin and moxifloxacin.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis in Cotonou (Benin) using two low-cost colorimetric methods: resazurin and nitrate reductase assays.

We have evaluated two simple, rapid and low-cost colorimetric methods for the detection of multidrug-resistant Mycobacterium tuberculosis. A total of 151 M. tuberculosis strains were tested for resistance to rifampicin (RMP) and isoniazid by resazurin microplate assay (REMA) and nitrate reductase assay (NRA) in comparison with the conventional proportion method (PM) on Löwenstein-Jensen medium. A complete agreement was found between NRA and PM, while one false RMP-susceptible result was found by REMA. REMA and NRA tests are rapid and inexpensive, and could be good alternatives to the conventional PM in low-resource countries.

Microbial succession of nitrate-reducing bacteria in the rhizosphere of Poa alpina across a glacier foreland in the Central Alps.

Changes in community structure and activity of the dissimilatory nitrate-reducing community were investigated across a glacier foreland in the Central Alps to gain insight into the successional pattern of this functional group and the driving environmental factors. Bulk soil and rhizosphere soil of Poa alpina was sampled in five replicates in August during the flowering stage and in September after the first snowfalls along a gradient from 25 to 129 years after deglaciation and at a reference site outside the glacier foreland (>2000 years deglaciated). In a laboratory-based assay, nitrate reductase activity was determined colorimetrically after 24 h of anaerobic incubation. In selected rhizosphere soil samples, the community structure of nitrate-reducing microorganisms was analysed by restriction fragment length polymorphism (RFLP) analysis using degenerate primers for the narG gene encoding the active site of the membrane-bound nitrate reductase. Clone libraries of the early (25 years) and late (129 years) succession were constructed and representative clones sequenced. The activity of the nitrate-reducing community increased significantly with age mainly due to higher carbon and nitrate availability in the late succession. The community structure, however, only showed a small shift over the 100 years of soil formation with pH explaining a major part (19%) of the observed variance. Clone library analysis of the early and late succession pointed to a trend of declining diversity with progressing age. Presumably, the pressure of competition on the nitrate reducers was relatively low in the early successional stage due to minor densities of microorganisms compared with the late stage; hence, a higher diversity could persist in this sparse environment. These results suggest that the nitrate reductase activity is regulated by environmental factors other than those shaping the genetic structure of the nitrate-reducing community.