Production of laccase from Pleurotus florida using agro‐wastes and efficient decolorization of Reactive blue 198

Pleurotus florida NCIM 1243 produced laccase as the dominant lignolytic enzyme during the dye decolorization. Banana peel was the best substrate for extracellular laccase production under solid state fermentation when compared to mandarin peel and cantaloupe peel. The maximum activity of laccase (5.4 U/g) was detected on the 10 day. The ratio of banana peel: mandarin peel: cantaloupe peel (5:2:3) showed increased production of laccase (6.8 U/g). P. florida produced two extracellular laccase isoenzymes (L1 and L2). The half life of laccase at 60 °C was 2 h and at 4 h it retained 25% residual activity. P. florida laccase showed high thermostability and an interesting difference was noticed in the behavior of laccase isoenzymes at different temperature. The L1 isoenzyme of laccase showed remarked thermostability at 60 °C in the native PAGE when compared to L2 isoenzyme. The optimum pH, temperature and enzyme concentration for maximum decolorization was found to be 4.5, 60 °C and 1.2 U/ml, respectively. Partially purified laccase enzyme showed excellent decolorization activity to Reactive blue 198. The maximum decolorization (96%) was observed at lower dye concentrations (50–100 ppm) which decreased markedly when the dye concentration was increased beyond 150 ppm. The thermostable laccase of P. florida could be effectively used to decolorize the synthetic dyes in the textile effluent and other biotechnological applications. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Astrazon Red dye decolorization by growing cells and pellets of Funalia trogii

The dye decolorization activity of fungal pellets has been compared with another method based on the decolorization of dye by growing cells. The pellet method was more advantageous than the growing cell method. The growing cells of F. trogii decolorized 21% of the dye in distilled water medium and 16% in stock basal medium in 24 h. On the other hand, Funalia trogii pellets rapidly decolorized the Astrazon Red dye, mono-azo textile dye, in 24 h, without any visual sorption of any dye to the pellets. The effect of various supplements on longevity of decolorization by free pellets was also tested. Glucose and cheese whey supplementation improved dye decolorization performance of the pellets and remained high and stable for 10 days. We also tested the dye decolorization ability of pellets immobilized on activated carbon. These pellets showed the stable dye decolorization activity during the repeated batch experiments. The study revealed that dye decolorization by pellets is more effective method than the growing cell method.     

Isolation and characterization of microorganisms capable of decolorizing various triphenylmethane dyes

Various soil and sludge samples collected from the vicinity of textile dyeing industries and waste disposal sites were used for enrichment of microbial population in the presence of triphenylmethane (TPM) dye Acid Violet-17 (AV-17). Twenty-five (25) isolates were screened for their ability to decolorize AV-17 dye added at a rate of 10 mgl(-1) in mineral salts medium (MSM) agar plates. Five bacterial isolates belonging to Bacillus sp., Alcaligenes sp. and Aeromonas sp. were selected on the basis of their higher decolorization ability and were used to develop a bacterial consortium. The consortium was able to efficiently decolorize various TPM dyes viz. Acid Violet-17 (86%), Acid Blue-15 (85%), Crystal Violet (82%), Malachite Green (82%) and Brilliant Green (85%). The consortium will be further used for designing efficient and cost effective treatment system for effluents of textile processing industries (TPI).     

Decolorization of synthetic dyes by the deuteromycete Pestalotiopsis guepinii CLPS no. 786 strain

The ability of Pestalotiopsis guepinii CLPS no. 786 to decolorize 13 dyes of varied structure was analyzed and compared with that of the basidiomycete Phanerochaete chrysosporium . Nine dyes representing anthrapyridone, azo, N-heterocyclic and triphenylmethane chromophores, but none with the O-heterocyclic group, were decolorized by P. guepinii when added to an agar medium. Studies with liquid cultures revealed that P. guepinii transformed both crystal violet and methylene blue B dyes, and adsorbed different chromophores onto its mycelium.     

Optimization of laccase production by Pleurotus ostreatus IMI 395545 using the Taguchi DOE methodology

Production of laccase using a submerged culture of Pleurotus orstreatus IMI 395545 was optimized by the Taguchi orthogonal array (OA) design of experiments (DOE) methodology. This approach facilitates the study of the interactions of a large number of variables spanned by factors and their settings, with a small number of experiments, leading to considerable savings in time and cost for process optimization. This methodology optimizes the number of impact factors and enables to calculate their interaction in the production of industrial enzymes. Eight factors, viz. glucose, yeast extract, malt extract, inoculum, mineral solution, inducer (1 mM CuSO₄) and amino acid (l‐asparagine) at three levels and pH at two levels, with an OA layout of L18 (2¹ × 3⁷) were selected for the proposed experimental design. The laccase yield obtained from the 18 sets of fermentation experiments performed with the selected factors and levels was further processed with Qualitek‐4 software. The optimized conditions shared an enhanced laccase expression of 86.8% (from 485.0 to 906.3 U). The combination of factors was further validated for laccase production and reactive blue 221 decolorization. The results revealed an enhanced laccase yield of 32.6% and dye decolorization up to 84.6%. This methodology allows the complete evaluation of main and interaction factors. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Immune-stimulatory potential of hot water extracts of selected edible mushrooms

Polysaccharides isolated from mushrooms have recently attracted attention due to its potential immune-stimulatory activity. The aim of this study was to validate the in vitro immune-stimulatory activities of various mushroom extracts. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that Pleurotus eryngii, with the highest β-glucan (18.94%) content, displayed highest viability on macrophage cells of 62.59% at 200?μg/ml concentration. Pleurotus cystidiosus, with 18.16% β-glucan, content showed highest activation of NF-kB (0.7?µg/ml) at a concentration of 100?µg/ml. Termitomyces heimii, with the lowest percentage of β-glucan (0.51%), exhibited highest phagocytosis index of 9.38 at 12.5?µg/ml. The brown strain of Agaricus bisporus with 1.54% of β-glucan stimulates the highest nitric oxide (NO) production of 12.39?µM nitrite oxide at 100?µg/ml. This study revealed that hot water extracts of mushrooms have different β-glucan contents and produced varying immune-stimulatory activities. Among these, Pleurotus spp. demonstrated the highest percentage of β-glucan content and viability of macrophage cells. Pleurotus spp. are deemed immune-stimulatory by increasing phagocytic activity, NO production, and triggered the activation of NF-kB.     

Wild boars as reservoirs of extended‐spectrum beta‐lactamase (ESBL) producing Escherichia coli of different phylogenetic groups

ESBL‐producing E. coli isolates have been isolated from eight of seventy seven faecal samples (10.4%) of wild boars in Portugal. The ESBL types identified by PCR and sequencing were bla_CTX‐M‐1 (6 isolates) and bla_CTX‐M‐1 + bla_TEM1‐b (2 isolates). Further resistance genes detected included tet (A) or tet (B) (in three tetracycline‐resistant isolates), aad A (in three streptomycin‐resistant isolates), cml A (in one chloramphenicol‐resistant isolate), sul 1 and/or sul 2 and/or sul 3 (in all sulfonamide‐resistant isolates). The intI 1 gene encoding class 1 integrase was detected in all ESBL‐producing E. coli isolates. One isolate also carried the intI 2 gene, encoding class 2 integrase. The ESBL‐producing E. coli isolates could be assigned to phylogenetic groups B1 (3 isolates), B2 (3 isolates) or A (2 isolates). Amino acid change in GyrA protein (Ser83Leu or Asp87Tyr) was detected in three nalidixic acid‐resistant and ciprofloxacin‐susceptible isolates. Two amino acid changes in GyrA (Ser83Leu + Asp87Asn) and one in ParC (Ser80Ile) were identified in two nalidixic acid‐ and ciprofloxacin‐resistant isolates. As evidenced by this study wild boars could be a reservoir of antimicrobial resistance genes. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Production of laccase from Pleurotus florida NCIM 1243 using Plackett–Burman Design and Response Surface Methodology

Statistically‐based experimental designs were applied to optimize the fermentation for the production of laccase by Pleurotus florida NCIM 1243. Eleven components were screened for their significant effect on laccase production using Plackett–Burman factorial design. Glucose (carbon source), asparagine (nitrogen source), CuSO₄(inducer) and incubation period were found to have highest positive influence on the laccase production. The combined effect of these factors on laccase production was studied using central composite design of Response surface methodology. The optimal point of variables for maximum laccase production using Response surface methodology are glucose (15.21 g/l), asparagine (6.40 g/l), CuSO₄ (91.78 μM) and incubation period (178.55 h), respectively. The maximum enzyme activity predicted by the model was 5.0 U/ml which was in perfect agreement with the actual experimental value (4.8 U/ml). Further, partially purified laccase from the optimized cultural condition was used for the decolorization of reactive dyes, Reactive Blue 198 and Reactive Red 35. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Decolorization of diazo-dye Reactive Blue 172 by Pseudomonas aeruginosa NBAR12

A novel bacterial strain capable of decolorizing textile dyes was isolated from dye contaminated soil obtained from industrial estate of Ahmedabad, Gujarat, India. The bacterial isolate Pseudomonas aeruginosa NBAR12 was capable of decolorizing 12 different dyes tested with decolorization efficiency varying in the range of 80 to 95%. Maximum extent as well as rate of Reactive Blue 172 (RB 172) decolorization was observed when glucose (2 g x l(-1)) and yeast extract (2.5 g x l(-1)) were supplemented in the medium. The optimum dye pH and temperature for dye decolorization was found to be 7 and 40 degrees C, respectively. The decolorizing activity was found to increase with increasing the dye concentration from 50 to 400 mg x l(-1). The dye decolorization was strongly inhibited at 500 mg dye l(-1) in the medium. High performance thin layer chromatography analysis indicated that dye decolorization occurred due to the breakdown of dye molecules into colorless end products.     

Isolation of Enterobacter aerogenes carrying blaTEM‐1 and blaKPC‐3 genes recovered from a hospital Intensive Care Unit

Enterobacter aerogenes has recently emerged as an important hospital pathogen. In this study, we showed the emergence of E. aerogenes isolates carrying the bla_KPC gene in patients colonized by carbapenem‐resistant Klebsiella pneumoniae strains. Two multiresistant E. aerogenes isolates were recovered from bronchial aspirates of two patients hospitalized in the Intensive Care Unit at the “Santa Maria della Scaletta” Hospital, Imola. The antimicrobial susceptibility test showed the high resistance to carbapenems and double‐disk synergy test confirmed the phenotype of KPC and AmpC production. Other investigation revealed that ESBL and bla_KPC genes were carried on the conjugative pKpQIL plasmid. This is a relevant report in Italy that describes a nosocomial infection due to the production of KPC beta‐lactamases by an E. aerogenes isolate in patients previously colonized by K. pneumoniae carbapenem‐resistant. In conclusion, it's necessary a continuous monitoring of multidrug‐resistant strains for the detection of any KPC‐producing bacteria that could expand the circulation of carbapenem‐resistant pathogens.     

Microflora involved in textile dye waste removal

Textile dyes are heavily used in factories for coloring different cloth materials. This work was designed to identify microorganisms capable of removing textile dyes, either by biodegradation or by biosorption. We expected to isolate microorganisms adapted to high dye concentrations from sites near textile industry complex. An experiment was conducted to study the efficiency of the isolates in removing textile dyes. The tested dyes were used as carbon and nitrogen sources for isolation of soil and/or water microorganisms capable of removing textile dyes wastes from factories effluent. The results indicated the low efficiency of both bacteria and actinomycetes in clean-up the effluent from the waste dyes in 10-21 days. On the other hand six fungal isolates were obtained by plating factory effluent on Martin's medium and media containing dyes as the sole source of carbon and nitrogen for growth. These isolates fell in two genera, Aspergillus and Trichoderma. Results of these studies revealed the potential capacity of these fungi to decolorize the tested dyes in comparatively short time (2-24 hours) indicating strong efficiency of dye bioremediation by the fungal isolates. Since the process involved is mostly fast interaction between the fungal mycelium and the dye in the media, the possible mechanism could be based on a biosorption of such chemicals on the intact fungal biomass, rather than direct biodegradation of the compounds.     

Detection of antibiotic resistant E. coli and Enterococcus spp. in stool of healthy growing children in Portugal

From stool specimens of 118 healthy children's (1–14 years) in Portugal 92 E. coli and 101 Enterococcu s spp. strains have been isolated. Almost half (40.2%) of the E. coli isolates were resistant to ampicillin, 25.0% were resistant to tetracycline and 26.1% were resistant to streptomycin. Resistance genes detected by specific PCR included bla_TEM and/or bla_SHV and/or bla_CTX‐M (33 of 37 ampicillin and/or cefotaxime resistant isolates), tet (A) and/or tet (B) (16 of 23 tetracycline‐resistant isolates), aad A (19 of 24 streptomycin‐resistant isolates), cml A (in the two chloramphenicol‐resistant isolates), aac (3)‐II with/without aac (3)‐IV (in the four gentamicin‐resistant isolates), sul 1 and/or sul 2 and/or sul 3 (in all trimethoprim/sulfamethoxazole resistant isolates). The majority of the resistant E. coli isolates (69.1%) belonged to phylogenetic group B2. Of the enterococci isolates E. faecium (n = 53), E. faecalis (n = 41), E. hirae (n = 4) and E. durans (n = 3) more than one‐fourth (28.7%) of the isolates were resistant to tetracycline; 21.8% were resistant to erythromycin and 8.9% were resistant to kanamycin. Resistance genes detected by PCR in enterococci included aph (3)′‐IIIa (in all kanamycin‐resistant isolates), aac (6′) (in all gentamicin‐resistant isolates), tet (M) and/or tet (L) (26 of 29 tetracycline‐resistant isolates), erm (B) (17 of 22 erythromycin‐resistant isolates). This survey showed that faecal bacteria such as E. coli and enterococci of healthy growing children's could be a reservoir of antimicrobial resistance genes. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Detection of OXA-type carbapenemases and integrons among carbapenem-resistant Acinetobactor baumannii in a teaching hospital in China

The increasing trend of carbapenem‐resistance (CR) and multi‐drug resistance (MDR) in A. baumannii worldwide has limited the therapeutic effectiveness of antibiotic therapy. The study was conducted to determine the prevalence of carbapenemases and integrons among the isolates of imipenem‐resistant A. baumannii (IRAB). A total of 71 non‐repetitive imipenem‐ resistant A. baumannii isolates were collected and tested for susceptibility to 17 antimicrobials. The modified Hodge test and EDTA‐disc synergy test were performed for the screening of carbapenemases and metallo‐β ‐lactamases (MBLs) production, respectively. Isolates were then subjected to multiplex‐PCR targeting genes encoding for OXA‐type carbapenemase, MBLs and integrases. Random amplified polymorphic DNA (RAPD) genotyping was performed to assess genetic relatedness. All isolates exhibited multi‐drug resistant phenotype. Colistin was the most active antimicrobial agent tested. Seventy‐one isolates (100%) demonstrated positive in the modified Hodge test. Thirty‐nine isolates showed positive in the EDTA‐disc synergy test, however, no MBL genes were detected. All strains possessed a bla_OXA‐51‐like gene. The co‐exis‐tence of bla_OXA‐51‐like/bla_OXA‐23‐like/intI1, bla_OXA‐51‐like/bla_OXA‐23‐like, bla_OXA‐51‐like/bla_OXA‐24‐like was detected in 91.6% (n = 65), 5.6% (n = 4), 2.8% (n = 2), respectively. Analysis of the genetic con‐text of bla_OXA‐23 showed the presence of ISAba1 upstream of bla_OXA‐23. No ISAba1 was detected upstream of bla_OXA‐51. Two different gene cassettes were found in these strains, and a high prevalence of aacA4, aadA1 and catB8 genes was observed. RAPD of 71 isolates showed 7 genotypes. The strains were mainly recovered from patients in intensive care unit, neurosurgery and department of respiratory disease. These ?ndings show that multi‐drug resistance in A. baumannii is a common problem. This study also shows a high distribution of bla_OXA‐23‐like and intI1 genes in imipenem‐resistant A. baumannii isolates. The clonal spread played an important role in the increase of OXA‐23 producing IRABs in the hospital environment. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Anti‐TB drug resistance levels and patterns among Mycobacterium tuberculosis isolated from newly diagnosed cases of pulmonary tuberculosis in Dar es Salaam,Tanzania

Anti‐tuberculosis drug resistance levels and patterns of Mycobacterium tuberculosis (Mtb) isolated from newly diagnosed tuberculosis (TB) patients in Temeke district in Dar es Salaam, Tanzania were investigated. A total of 226 Mtb isolates from 564 TB suspects with no previous history of anti‐TB treatment were tested for drug resistance against rifampicin, isoniazid, streptomycin and ethambutol on Lowenstein Jensen (LJ) medium using the proportion method. Of the 226 isolates, 22 (9.7%) were resistant to any one of the four anti‐TB drugs; nine (3.99%) isolates were isoniazid mono‐drug resistant and eight (3.54%) isolates were streptomycin mono‐drug resistant. Multi‐drug resistance, defined as resistance to both rifampicin and isoniazid, was observed in three (1.3%) isolates and two were also resistant to streptomycin and ethambutol. One (0.44%) isolate had poly resistance to isoniazid and streptomycin. The level of anti‐TB drug resistant Mtb in Temeke, an HIV endemic area, remained constant between 1995 and 2007. The level of resistance to any one of the four anti‐TB drugs was between 9.0% and 10%, resistance to individual drugs <4% and multi‐drug resistance <2%.     

Isolation and screening for alkaline thermostable xylanases

Screening of xylanase producing microorganisms from soil samples and hemicellulosic materials were carried out in three stages using wheat bran extract agar medium, xylan agar medium and liquid medium with xylan. One of the isolates, identified as Bacillus SSP-34 produced 100 times more xylanase activity than the other isolates. Maximum enzyme production by this isolate was observed after 102 hours of growth. The optimum pH for the crude xylanase preparation range from 6–8 and optimum temperature was at 50 °C. The enzyme was stable up to 20 minutes at 50 °C and at pH range 4.5–9.0 for more than 2 hours.     

Validation of 31 of the most commonly used immunohistochemical antibodies in cytology prepared using the cellient® automated cell block system

The Cellient^® cell block system has become available as an alternative, partially automated method to create cell blocks in cytology. We sought to show a validation method for immunohistochemical (IHC) staining on the Cellient cell block system (CCB) in comparison with the formalin fixed paraffin embedded traditional cell block (TCB). Immunohistochemical staining was performed using 31 antibodies on 38 patient samples for a total of 326 slides. Split samples were processed using both methods by following the Cellient^® manufacturer's recommendations for the Cellient cell block (CCB) and the Histogel method for preparing the traditional cell block (TCB). Interpretation was performed by three pathologists and two cytotechnologists. Immunohistochemical stains were scored as: 0/1+ (negative) and 2/3+ (positive). Inter‐rater agreement for each antibody was evaluated for CCB and TCB, as well as the intra‐rater agreement between TCB and CCB between observers. Interobserver staining concordance for the TCB was obtained with statistical significance (P < 0.05) in 24 of 31 antibodies. Interobserver staining concordance for the CCB was obtained with statistical significance in 27 of 31 antibodies. Intra‐observer staining concordance between TCB and CCB was obtained with statistical significance in 24 of 31 antibodies tested. In conclusions, immunohistochemical stains on cytologic specimens processed by the Cellient system are reliable and concordant with stains performed on the same split samples processed via a formalin fixed‐paraffin embedded (FFPE) block. The Cellient system is a welcome adjunct to cytology work‐flow by producing cell block material of sufficient quality to allow the use of routine IHC. Diagn. Cytopathol. 2014;42:1024–1033. © 2014 Wiley Periodicals, Inc.     

Some factors affecting bioconversion of whole bagasse into fungal biomass

Two isolated basidiomycetes mould cultures Polyporus BH₁ and Polyporus BW₁ and two standard white rot fungal cultures Pleurotus ostreatus and Polyporus versicolor were cultivated under submerged conditions on whole (untreated) bagasse for protein production. Some factors promoting better bioconversion were studied and selected. NORKRAN's medium at pH 5.0 with 1.0% bagasse of 80 mesh particle size using an appropriate quantity of inoculum, supplementing medium with an amino acid, without addition of any vitamin, etc. gave best results.     

Characterization of a new efficient low molecular weight Bacillus subtilis NRC 16 levansucrase and its levan

Screening of 18 bacterial honey isolates revealed that all the isolates were levansucrase producers. The most potent isolate that achieved the highest activity (45.66 U/ml) was identified as Bacillus subtilis NRC based on morphological examination and 16S rRNA. The results recorded the necessity of starch (5 g/L), baker's yeast (12.5 g/L), and AlCl₃ (5 mM) in improvement of the enzyme productivity. The Bacillus subtilis levansucrase was eluted as a single protein in one purification step. The enzyme molecular weight was (14 kDa). It showed its optimum activity at 45°C and could retain 60% of its activity after incubation at 50°C for 2 h. Its optimum activity was obtained at pH 8.2 and the enzyme showed great pH stability in both acidic and alkaline ranges. Unlike, most levansucrases all tested metals had an adverse effect in enzyme activity. The enzyme had antioxidant activities and were characterized as spherical micro‐ and nanoparticles by transmission electron microscopy. The effect of growth conditions and medium composition in levan structure and its fibrinolytic activity was evaluated.     

Isolation and characterization of biocides resistant bacteria from dental unit water line biofilms

Six biocides resistant isolates were isolated from dental unit water lines (DUWL) in Pakistan. All isolates could tolerate 150 μg ml^–1 of biocides (5.25% sodium hypocholrite, 35% H₂O₂, 4% tween 20, 1% povidine iodine, 0.2% chlorohexidine gluconate, 1% ethylene di‐amino tetra acetic acid and 1% phenol) on l‐agar and 100 μg ml^–1 in l‐broth. The growth rate of all isolates was determined by generating growth curves at 37 °C for 48 h. The isolates were found to differ in growth rates with lag phase varying from (4–6 h) in biocides supplemented media compared to 2–4 h in biocides free medium. They have wide temperatures (24–42 °C) and pH (5–9) ranges. Traditional ways of identification of bacteria by phenotypic characteristics were accomplished by phenotypic and biochemical characterization. Heavy metals and antimicrobial susceptibility tests indicated that all isolates examined were resistant to trimethoprim, chloramphenicol while sensitive to HgCl₂ and Pb (NO₃)₂. Almost all isolates were opportunistic pathogens. The 16S rRNA‐encoding genes from these six isolates were sequenced to confirm the identity of these isolates. 5 different genera (Bacillus, Achromobacter, Pseudomonas and Klebsiella) of bacteria were identified by 16S rDNA genes amplified from genomic DNA of biocides resistant DUWL biofilm isolates. Analysis of 16S rDNA genes revealed a much more clear identification of microrganisms than culture methods. However, different species of the same genera can have the same 16S rRNA gene sequence but are different due to phenotypic differences or different clinical manifestations. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)