Enhancing bioremoval of textile dyes by eight fungal strains from media supplemented with gelatine wastes and sucrose

Eight Aspergillus strains were found to be successful in removing textile dyes from liquid media. These fungal strains were grown on medium containing: gelatine wastes and sucrose, as sources of nitrogen and carbon to test the possible speed up of the dyes removing while fungus biomass is building up in the media. The growth of fungal strains ranged from 10 to 110 mg biomass dry weight/100 ml medium. This growth induced high decolorization percentages, which ranged 33-95% within eight days. Two textile dyes Direct brown and Polar red were included in the study. The growth of the fungal strains as well as decolorization percentage of the dyes increased after 5, 6, and 8 days from incubation time with most tested strains. With Direct brown dye the strains number 2, 5, 31 and 37 recorded the highest percentage of decolorization (91, 92, 93 and 95 respectively) after incubation for 6 days. Fungal strains Aspergillus 5 and 31 gave the highest mycelium dry weight being 110 mg. Most of fungal strains induced 86 to 95 percentage of decolorization after 6 days incubation with Polar red dye. The possible toxicity of the remaining supernatant media after fungal biomass removal was tested by Ames test to assess the residual mutagenic agents remaining after dye removal, using three strains of Salmonella typhimurium (TA 1535, TA 1537, TA 1538). The results showed that the toxicity of the dyes, measured by Ames test could be removed by the dye absorption on the fungal biomass.     

Superoxide dismutase activity of Mycobacterium avium, M. intracellulare, and M. scrofulaceum.

Superoxide dismutase (EC 1.15.1.1) (SOD) activity has been detected in crude cell extracts of representative strains of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) group. Polyacrylamide gel electrophoresis demonstrated a single SOD activity band for each of the MAIS strains, though there were differences in mobility. All M. avium and M. intracellulare and two of five M. scrofulaceum strains demonstrated a single activity band of identical mobility (Rf = 0.83), while the SOD activity band for the three remaining M. scrofulaceum strains migrated farther (Rf = 0.85). The differences in mobility correlated with differences in sensitivity to NaN3 and H2O2. The SOD activities of the majority of the MAIS strains which displayed the slower-migrating activity band were inhibited 22 to 81% after 15 min of exposure to 5 mM H2O2, suggesting that both iron and manganese may be present in a single enzyme. The SOD activities of the three M. scrofulaceum strains which had the faster-migrating activity band were inhibited 100% after only 5 min of exposure to 5 mM H2O2 and exhibited greater sensitivity to 5 and 10 mM NaN3, characteristics of an iron-containing SOD. A concentration of 1 mM KCN did not cause inhibition of enzyme activity in any of the MAIS strains tested. Extracellular SOD activity was detected in four of six MAIS strains and was shown to be identical in mobility to the SOD activity of the crude extracts.     

Ligninolytic enzyme production in selected sub-tropical white rot fungi under different culture conditions.

Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in selected sub-tropical white rot fungal species from Zimbabwe were determined. The enzyme activities were assayed at varying concentrations of C, N and Mn2+. Manganese peroxidase and laccase activities were the only expressed activities in the fungi under the culture conditions tested. Trametes species, T. cingulata, T. elegans and T. pocas produced the highest manganese peroxidase activities in a medium containing high carbon and low nitrogen conditions. High nitrogen conditions favoured high manganese peroxidase activity in DSPM95, L. velutinus and Irpex spp. High manganese peroxidase activity was notable for T. versicolor when both carbon and nitrogen in the medium were present at high levels. Laccase production by the isolates was highest under conditions of high nitrogen and those conditions with both nitrogen and carbon at high concentration. Mn2+ concentrations between 11-25 ppm gave the highest manganese peroxidase activity compared to a concentration of 40 ppm or when there was no Mn2+ added. Laccase activity was less influenced by Mn2+ levels. While some laccase activity was produced in the absence of Mn2+, the enzyme levels were higher when Mn2+ was added to the culture medium.     

Acute and chronic alterations in calcium homeostasis in 3-nitropropionic acid-treated human NT2-N neurons

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, induced ATP depletion and both necrosis and apoptosis in human NT2-N neurons. Necrosis occurred predominantly within the first two days, and increased in a dose-dependent fashion with the concentration of 3-NP, whereas apoptosis was observed after 24 h or later at a similar rate in 0.1 mM and 5 mM 3-NP. We focused our efforts on intracellular calcium homeostasis during the first 48 h in 1 mM 3-NP, a period during which 10% of the neurons died by necrosis and 3% by apoptosis. All NT2-N neurons showed a stereotyped [Ca(2+)](i) rise, from 48+/-2 to 140+/-12 nM (mean +/-S.E.M.), during the first 2 h in 3-NP. Despite severe ATP depletion, however, [Ca(2+)](i) remained above 100 nM in only 17% and 25% of the NT2-N neurons after 24 and 48 h in 3-NP, respectively, indicating that most neurons were able to recover from acute [Ca(2+)](i) rise, and suggesting that chronic [Ca(2+)](i) dysregulation is a better indicator of subsequent necrosis. Blockade of N-methyl-D-aspartate-glutamate receptor by MK-801 substantially ameliorated 3-NP-induced ATP depletion, subsequent chronic [Ca(2+)](i) elevation, and survival. Moreover, xestospongin C, an inhibitor of endoplasmic reticulum Ca(2+) release, enhanced the capacity of NT2-N neurons to maintain [Ca(2+)](i) homeostasis and resist necrosis while subjected to sustained energy deprivation. As far as we know, this report is the first to employ human neurons to study the pathophysiology of 3-NP neurotoxicity.     

Molecular genetic analyses of mating pheromones reveal intervariety mating or hybridization in Cryptococcus neoformans

The sexual mating of the pathogenic yeast Cryptococcus neoformans is important for pathogenesis studies because the fungal virulence is linked to the alpha mating type (MAT(alpha)). We characterized C. neoformans mating pheromones (MF(alpha) 1 and MFa1) from 122 strains to understand intervariety hybridization or mating and intervariety virulence. MF(alpha) 1 in three C. neoformans varieties showed (a) specific nucleotide polymorphisms, (b) different copy numbers and chromosomal localizations, and (c) unique deduced amino acids in two geographic populations of C. neoformans var. gattii. MF(alpha) 1 of different varieties cross-hybridized in Southern hybridizations. Their phylogenetic analyses showed purifying selection (neutral evolution). These observations suggested that MAT(alpha) strains from any of the three C. neoformans varieties could mate or hybridize in nature with MATa strains of C. neoformans var. neoformans. A few serotype A/D diploid strains provided evidence for mating or hybridization, while a majority of A/D strains tested positive for haploid MF(alpha) 1 identical to that of C. neoformans var. grubii. MF(alpha) 1 sequence and copy numbers in diploids were identical to those of C. neoformans var. grubii, while their MFa1 sequences were identical to those of C. neoformans var. neoformans; thus, these strains were hybrids. The mice survival curves and histological lesions revealed A/D diploids to be highly pathogenic, with pathogenicity levels similar to that of the C. neoformans var. grubii type strain and unlike the low pathogenicity levels of C. neoformans var. neoformans strains. In contrast to MF(alpha) 1 in three varieties, MFa1 amplicons and hybridization signals could be obtained only from two C. neoformans var. neoformans reference strains and eight A/D diploids. This suggested that a yet undiscovered MFa pheromone(s) in C. neoformans var. gattii and C. neoformans var. grubii is unrelated to, highly divergent from, or rarer than that in C. neoformans var. neoformans. These observations could form the basis for future studies on the role of intervariety mating in C. neoformans biology and virulence.     

Relationship between cell surface composition, adherence, and virulence of Candida albicans.

A comparison was made of the adherence to acrylic and to human buccal epithelial cells of seven strains of Candida albicans isolated from active infections (I strains) and two strains obtained from asymptomatic carriers (C strains). After growth in defined medium containing a relatively low concentration (50 mM) of glucose as the carbon source, the adherence of I and C strains to either surface was similar and all strains were sensitive to spheroplast formation with Zymolyase 5000. Growth in medium containing a high concentration (500 mM) of sucrose or galactose enhanced the adherence of I strains up to 5- and 11-fold, respectively, and there were corresponding increases in resistance to spheroplast formation. Sucrose- or galactose-grown C strains showed only small increases in adherence and remained relatively sensitive to spheroplast formation. When inoculated intravenously into mice, I strains grown in 500 mM sucrose were up to five times more virulent than organisms grown in 50 mM glucose, while I strains grown in 500 mM galactose showed a 5- to 24-fold increase in virulence. Fifty percent lethal doses obtained for C strains were similar after growth on all three carbon sources. We conclude that I strains are able to modify their surface composition in response to high concentrations of certain sugars in the growth environment. Such modification can enhance both their ability to adhere to surfaces and their virulence. C strains lack this capability, or possess it to a lower degree, and may therefore have a lower pathogenic potential.     

Low-temperature biodegradation of high amounts of phenol by Rhodococcus spp. and basidiomycetous yeasts

Four cold-adapted microbial strains able to degrade high amounts of phenol were isolated from hydrocarbon-contaminated alpine soils. Two of the strains were bacteria identified as Rhodococcus spp., and two strains were basidiomycetous yeasts. One of the yeasts was identified as Trichosporon dulcitum, while the second yeast strain belonged to the Urediniomycetes and probably represents a novel species. This strain was not able to grow at temperatures above 20 degrees C, while the other three strains were cold-tolerant and could grow at temperatures ranging from 1-25 degrees C (T. dulcitum) or 1-30 degrees C (rhodococci). The yeast strains were characterized by a substantially lower optimum temperature for growth and biodegradation compared to the bacteria. The urediniomycete strain degraded 5 mM phenol at 1 degrees C faster than the two bacteria at 10 degrees C. The optimum temperature for phenol degradation was 10 degrees C (novel yeast species), 20 degrees C (T. dulcitum), or 30 degrees C (rhodococci). Using fed-batch cultivation in mineral medium with phenol as the sole carbon source, high amounts of phenol were degraded at 10 degrees C. Both rhodococci degraded up to 12.5 mM phenol, while the two yeast strains even utilized as much as 15 mM phenol.     

Purification and characterization of a 315 kDa keratinolytic subtilisin-like serine protease from Microsporum canis and evidence of its secretion in naturally infected cats.

A keratinolytic protease, secreted as the major component by a feline clinical isolate of Microsporum canis cultivated in a minimal medium containing cat keratin, was purified by affinity chromatography on bacitracin agarose and gel filtration. The apparent molecular mass of the enzyme was 31.5 kDa and the pI was 11.8. The enzyme was not glycosylated and its first 15 N-terminal amino acids showed numerous similarities with other fungal subtilisins. The optimum pH was around 9 while inactivation of the enzyme was reversible at pH 4, but not at pH 11. The enzyme was stable at 37 degrees C with an apparent optimum temperature around 55 degrees C. PMSF, soybean trypsin inhibitor (SBTI) and chymostatin strongly inhibited the proteinase. The highest affinity (Km of 0.37 mM) and physiological efficiency (k(cat)/Km) were obtained for the synthetic substrate N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide. These results indicate that the keratinase belongs to the subtilisin-like serine protease family. Purified rabbit immunoglobulins G prepared against the keratinase and used in an immunohistochemical test allowed the detection of the keratinase produced by the fungus invading hair structures in naturally infected cats. The in vitro keratinolytic activity of the enzyme and its production in vivo suggest that it may contribute to pathogenicity.     

Antibiosis between bacteria isolated from the vagina of women with and without signs of bacterial vaginosis.

Lactobacilli from women with and without bacterial vaginosis (BV) were tested for H2O2 production. Thirty-seven (79%) of the 47 strains of lactobacilli isolated from the women without BV produced H2O2, while only nine (23%) of the 39 strains of lactobacilli obtained from women with BV did so. Five of 20 H2O2-producing and two of 26 non-producing strains of Lactobacillus exhibited antibiosis against four of 12 strains of peptostreptococci and two of 10 strains of Mobiluncus spp. None of a further 41 different anaerobic and facultative anaerobic bacterial strains were inhibited by any of the isolates of lactobacilli tested. Some strains of Gardnerella vaginalis, Bacteriodes spp., Mobiluncus spp. and Peptostreptococcus spp. inhibited the growth of three strains of lactobacilli belonging to different species. When the pH of the culture medium was increased from 6.0 to 6.5 this led to a decrease in the number of strains inhibited and/or the size of the growth-inhibitory zones. Different concentrations of H2O2 did not inhibit any of the strains tested. The growth-inhibitory effect of lactobacilli could not be related to their bacteriocin production. Increasing the iron content of the medium by adding FeCl3 (0.01 mM-1 mM) decreased or completely abolished the antibiosis.     

Effects of 3‘, 5’-Cyclic Adenosine Monophosphate, 5-Hydroxy-tryptamine,Noradrenaline and Theophylline on the Simultaneous Release of Peroxidase and Amylase from the Guinea Pig Submanibular Gland

Noradrenaline (NA), 5-hydroxytryptamine (5-HT) and N⁶,2-0′-dibutyryl cyclic adenosine monophosphate (DBcAMP) induced a parallel discharge of peroxidase and amylase from the guinea pig submandibular gland in vitro. Theophylline alone, at a concentration of 5 mM, had only little effect on enzyme secretion, but it markedly potentiated the effects of noradrenaline, 5-HT and DBcAMP at submaximal concentrations. Adenosine-3′-monophosphoric acid (3′-AMP) and adenosine-5′-monophosphoric acid (5′-AMP) were without effect on enzyme secretion. Maximal effects of NA and DBcAMP on enzyme release were obtained at concentrations of 0.01 mM and 3 mM respectively. Dose-response curves for the stimulation of peroxidase and amylase release by 5-HT revealed that this amine was a potent secretagogue at the concentrations of 0.1 and 0.3 mM but was less effective at higher concentrations. It is concluded that peroxidase and amylase are simultaneously secreted from the guinea pig submandibular gland, and that the release of both enzymes is mediated via cyclic AMP.     

Occurrence of heavy metal-resistance in microflora from serpentine soil of Andaman

Serpentine soils collected from Saddle Hills, Chidyatapu and Rutland of Andaman Islands, India were analyzed for physico-chemical and microbiological characteristics and compared with those from adjacent non-serpentine localities. The serpentine soils contained high levels of nickel (1740.0-8033.4 mg/kg dry soil), cobalt (93.2-533.4 mg/kg dry soil) and chromium (302.9-4437.0 mg/kg dry soil), in addition to 62-152 g of iron and 37-60 g of magnesium per kg dry soil. Characteristically the serpentine soils showed low microbial density (6.2-11.3 x 10(6) colony forming unit/g soil) and activity (1.7-3.5 microg fluorescein/g dry soil/h) than non-serpentine outcrops. Serpentine microbial population was dominated by bacteria which represented 5.12 to 9.5 x 10(6) cfu/g of soil, while the fungal population ranged from 0.17 to 3.21 x 10(6) cfu/g of soil. A total of 342 (200 from serpentine and 142 from non-serpentine soils) isolates were compared for Ni, Co and Cr resistance. Serpentine microflora was in general, highly resistant than non-serpentine ones and showed a metal-resistance profile of Cr > Ni > Co. Amongst the serpentine isolates, 8 and 11 bacteria tolerated > 12.0 mM Ni and > 16.0 mM Cr respectively, while 6 fungal isolates showed a minimum inhibitory concentration (MIC) value > 8.0 mM Co. These 25 serpentine strains also showed co-resistance to Cu, Zn and Mn but were sensitive to Hg and Cd. The selected bacterial isolates were resistant to ampicillin, penicillin G and polymyxin B, whereas fungal strains showed resistance to amphotericin B, nystatin and fusidic acid.     

Biotyping of Penicillium marneffei reveals concentration-dependent growth inhibition by galactose

Thirty-two isolates of the dimorphic fungus Penicillium marneffei were studied for their biochemical properties. All isolates possessed the enzyme urease and were inhibited by 500 mg of cycloheximide per liter. No strain fermented glucose, and thus no strain fermented any of the other five sugars tested. All assimilated glucose, maltose, and cellobiose; only one of the isolates did not assimilate salicin. Totals of 65.6, 84.4, and 71.9% of the isolates assimilated trehalose, xylose, and nitrate, respectively. Twelve strains possessed the enzyme beta-galactosidase. Overall, 17 different biotypes were recognized, but no association was found between the human immunodeficiency virus status of the patients and the biotype. A novel finding of concentration-dependent growth inhibition of P. marneffei by galactose is described. Inhibition of growth occurred at a low concentration of galactose (0.015 to 0.25%) when galactose was the sole carbon source in the medium. Morphological changes of the fungal cells were observed in the presence of galactose.     

Utilization of nitrate or nitrite as single nitrogen source by Mycobacterium avium.

Twenty L-amino acids and several inorganic compounds were tested individually, as a sole nitrogen source, for ability to support the growth of Mycobacterium avium LM1 serovar 1. Of the amino acids tested, only L-glutamine provided nutritional support comparable to that of ammonium chloride at 1 mM. With either 1 mM potassium nitrate or nitrite substituted for ammonium chloride, similar numbers of CFU were produced. M. avium cells were grown in potassium nitrate or nitrite concentrations of 0.25, 0.5, 1.0, and 2.0 mM, and the medium was assayed for remaining nitrogen compound at several times during growth. Rates of utilization were of first-order kinetics, with nitrite removed more rapidly than nitrate. The rates were approximately 10 times as rapid at 0.25 mM than at 2 mM for either nitrogen source. Nine clinical isolates that included M. avium serovars 1, 4, and 8 and Mycobacterium scrofulaceum serovar 43 were tested for rate of utilization of ammonia, nitrate, or nitrite. Ammonia and nitrite were utilized with first-order kinetics by all strains. Nitrate utilization occurred but was not at the same level for all strains. Clinical tests indicate that M. avium is negative for nitrate reductase; this is because of the rapid reduction of nitrite produced from nitrate.     

Adhesion of Pseudomonas aeruginosa to respiratory mucins and expression of mucin-binding proteins are increased by limiting iron during growth.

The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cystic fibrosis. Local factors in the respiratory tract, such as osmolarity or iron concentration, might influence this colonization. In the present work, we have observed that overall levels of adhesion of two nonmucoid, nonpiliated strains of P. aeruginosa, 1244-NP and PAK-NP, to human airway mucins were higher when these strains were grown in a minimal medium of low osmolarity (M9) than when they were grown in a rich medium of higher osmolarity (tryptic soy broth [TSB]). However, increasing the NaCl concentration of M9 to increase the osmolarity did not modify the level of binding. In order to find out whether these differences were due to variations in nutrients, the influence of iron concentration was investigated: the levels of binding of the two strains increased after TSB was depleted of iron and decreased after iron was added to M9. Since the outer membranes from the two strains have been shown to contain proteins reacting with human bronchial mucins, we compared the mucin-binding proteins expressed by the two strains grown in different media. When the nonpiliated strains 1244-NP and PAK-NP were grown in the different media, previously observed mucin-binding bands were detected in the 42- to 48-kDa range but additional mucin-binding bands in the 77- to 85-kDa range were detected when these strains were grown in M9 or iron-deprived TSB. These results demonstrate that the adhesion of P. aeruginosa and the expression of mucin-binding proteins in the outer membranes of nonpiliated P. aeruginosa are affected by the iron content of the medium in which the bacteria are grown and not by the osmolarity.     

Mutagenic 1-nitropyrene in wastewater from oil-water separating tanks of gasoline stations and in used crankcase oil

Wastewater collected from oil-water separating tanks of ten gasoline stations for a year was fractionated into diethyl ether-soluble neutral, acidic, and basic fractions. Mutagenicity of these fractions was measured with Salmonella typhimurium strains TA98 and TA100 in the presence or absence of S9 mix. The neutral fractions showed high mutagenicity in the absence of S9 mix. Each neutral fraction was subjected to high-performance liquid chromatography (HPLC) and fractionated. A 1-nitropyrene(1-NP)-corresponding fraction was collected and analyzed by gas chromatography-mass spectrometry (GC-MS) and HPLC to prove that wastewater contains 1-NP and to quantitate 1-NP in wastewater. GC-MS patterns showed the following molecular and fragment ion peaks of 1-NP: 247, 217, 201, and 189. The amount of 1-NP in 36 samples of wastewater was 4.2-25,600 ng per liter of wastewater, and 1-NP accounted for 0.3-58.5% of the total mutagenicity of the neutral fractions. The other 19 samples of wastewater did not contain any detectable 1-NP. The mutagenicity of wastewater may be due to water from car washing and contamination by used crankcase oil. A Soxhlet extract of crankcase oil used in a gasoline was fractionated into three fractions as above. Mutagenicity was measured with strains TA98, TA100, TA98NR, and TA98/1,8-DNP6 in the absence or presence of S9 mix. The neutral fraction showed the highest mutagenicity with strain TA98 in the absence of S9 mix, and its mutagenicity was decreased in strains TA98NR and TA98/1,8-DNP6. The latter result indicates that the used engine-oil contained 1-NP and dinitropyrenes. Actually, the amounts of 1-NP and 1,6-diNP in used crankcase oil were 138 and 2.0 ng per ml of oil, respectively, and these concentrations accounted for 0.45 and 2.7%, respectively, of the total mutagenicity of the neutral fraction with strain TA98 in the absence of S9 mix. Moreover, the concentrations of 1-NP and 1,6-diNP in used crankcase oil of a diesel engine were 349 and 31 ng per ml of oil, respectively, accounting for 0.9 and 12%, respectively, of the total mutagenicity of the neutral fraction in the same assay system.     

New antifolate inhibitors for Mycobacterium avium

The present study extends our previous work regarding new antifolates for Mycobacterium avium (MAC) dihydrofolate reductase (DHFR). The objectives of this study were to synthesize and test new derivatives in the general class of 2,4-diamino-5-methyl-5-deazapteridines in an effort to improve solubility and selectivity for the MAC DHFR, while maintaining lack of selectivity for the human DHFR. New 6-[2', 5'-dialkoxyphenyl) methyl]-substituted DMDP analogs were synthesized as previously described. Three clinical isolates of MAC (NJ211, NJ3404, and NJ168) and M. tuberculosis H37Ra (MTB) were used to evaluate the new derivatives. A previously described colorimetric (alamarBlue(R)) microdilution broth assay was used to determine minimal inhibitory concentrations (MIC). Purified recombinant human (rDHFR), MAC rDHFR, and MTB rDHFR were used in a validated enzyme assay to obtain IC(50) values and to determine selectivity ratios (SR) for the derivatives. For the MAC strains, the MICs ranged from < 0.25 to > 16 microg/mL. The most active derivative against MAC was SRI-20920 which had MICs of 0.25, 0.25, and 8 microg/mL for the three strains, respectively. The most selective derivative was SRI-20730 with IC(50s) of 29 and 67,781 nM for MAC rDHFR and hDHFR, respectively, and a SR of 2,337. MICs for MTB ranged from 4 to >64 microg/mL and the SR, in general, ranged from 0.32 to 2.5. These results further substantiate the utility of this group of DMDP derivatives for selective activity against MAC.     

Enhanced culture of Borrelia garinii and Borrelia afzelii strains on a solid BSK-based medium in anaerobic conditions

The growth of 29 human strains from the three main pathogenic species of Borrelia burgdorferi sensu lato on a solid BSK-based medium was compared in two culture atmospheres: 3% CO(2) air and anaerobiosis. All strains grew under anaerobic conditions, whereas only 13 strains were able to grow in aerobiosis with 3% CO(2) (P<0.001). In the latter condition, 75% of the B. burgdorferi sensu stricto strains grew versus 33% of the B. garinii and B. afzelii strains. These data suggest that, especially for B. garinii and B. afzelii species, anaerobic conditions enhance growth yield and speed of low-passage Borrelia strains.     

Inhibition of fungal NADP+-isocitrate dehydrogenase by citric acid

The variations observed during earlier studies in the activity of NADP⁺-isocitrate dehydrogenase (EC. 1.1.1.42) in a strain of Aspergillus niger were found to be related to the extent of washing of mycelium. As a result the mycelium washed four times with phosphate buffer (0.05 M, pH 7.5), the enzyme activity present in 4 and 8 days old fungal mycelia increased five- and two-fold, respectively. In vivo studies showed a complete loss of enzyme activity in mycelia resuspended in HCl-KCl buffer (0.02 M, pH 2.2) containing citric acid (13 mM or more). The in vitro studies revealed 50% loss of enzyme activity in presence of 3.6 to 5.2 mM citric acid. However, in case of Aspergillus niger ATCC 1015, which produced less citric acid than the above strain, a much higher citric acid concentration (13 to 26 mM) was required to cause 50% loss of enzyme activity. These findings suggest a correlation between citric acid inhibition of NADP⁺-isocitrate dehydrogenase and the ability of A. niger to accumulate citric acid in the medium.     

Evaluating the resistance to posaconazole by E-test and CLSI broth microdilution methodologies of <Emphasis Type="Italic">Candida</Emphasis> spp. and pathogenic moulds

E-test methodology was compared with Clinical and Laboratory Standards Institute (CLSI) broth microdilution, particularly concerning the detection of resistance to posaconazole among clinical fungal isolates. The susceptibility of a large set of fungal strains (n = 300) was evaluated following 24 and 48 h in two different culture media (RPMI 1640 and Sabouraud agar). Fungal strains were highly susceptible to posaconazole; however, few less susceptible strains were found, mostly regarding Candida albicans, Candida glabrata, Acremonium sp., Cladosporium sp. and Scedosporium apiospermum. Broth microdilution and E-test methods provided similar results for posaconazole-susceptible strains, while the less susceptible fungal strains (10.3% of the strains showed MIC ≥2 μg/mL) resulted in higher discrepancies between the two methodologies, particularly concerning Candida spp. E-test susceptibility values were critically affected by the pH of the culture media. Sabouraud medium provided similar susceptibility results for moulds to those for RPMI, soon after 24 h. Posaconazole resistance was rare in this study, but routine susceptibility methods, such as the E-test, should be able to detect fungal strains with reduced susceptibility. E-test methodology still needs improvements to recognise accurately strains less susceptible to posaconazole.