A Preliminary Study on Cross-Reactivity of Heat-Treated Quail and Hen's Egg White Proteins in Young Children.

We investigated the effects of different types of heat treatments on hen's egg white (HEw) and quail egg white (QEw) proteins and their cross-reactivity in young children. Crude extracts of raw and water-boiled HEw and QEw and commercially developed stone-baked HEw were prepared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was then performed. Patients diagnosed with HEw allergy were enrolled, and pooled sera were tested with each extract in an enzyme-linked immunosorbent assay (ELISA)-inhibition test. A skin prick test (SPT) and oral food challenge (OFC) were also performed. The median age of 12 patients was 2.5 years. SDS-PAGE results revealed strongly stained bands for the ovomucoid of boiled HEw and QEw, while stone-baked HEw displayed remarkable changes for all protein fractions. In the ELISA-inhibition test, pre-incubation of the sera led to a profound decrease, moderate decrease, and minimal decrease in the amount of IgE binding to boiled and raw HEw, boiled and raw QEw, and stone-baked HEw proteins, respectively. SPTs and OFC demonstrated cross-reactivity values of 41.7% (5/12) and 16.7% (2/12) for boiled QEw and stone-baked HEw, respectively. We observed moderate cross-reactivity between QEw and HEw. Boiling had a limited effect on altering egg allergenicity. Commercially developed, stone-baked HEw can be an alternative food for children with HE allergy.

Development of an S-layer gene-based PCR-DGGE assay for monitoring dominant Lactobacillus helveticus strains in natural whey starters of Grana Padano cheese.

Monitoring L. helveticus strain dynamics in natural whey starters is of great interest at the industrial level due to the key role that this bacterial population plays in Grana Padano cheese production. In this study, we aimed to develop a PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) assay based on the slpH locus, in parallel with performing culture-dependent analysis of whey samples using optimized media to maximize the number of isolated strains. We designed new primers targeting the slpH locus to amplify a gene region that would be suitable for PCR-DGGE analysis and discriminating strains. Our results confirmed that the developed PCR-DGGE method was rapid and reliable for monitoring the L. helveticus population in whey starter cultures. All sequences of bands detected in the PCR-DGGE profiles from whey samples showed high similarity to S-layer genes of L. helveticus, and perfectly matched with the slpH locus sequences of dominant strains. Overall, our findings indicated that the target region of the slpH locus was sufficiently heterologous to discriminate L. helveticus strains, and that our PCR-DGGE analysis provided a more accurate picture of the population composition of whey starters compared to culture-dependent techniques that often fail to isolate the most abundant strains.

Contribution of bacterial biodiversity on the operational performance of a styrene biotrickling filter.

Long-term operational stability of biotrickling filters (BTFs) degrading volatile organic compounds (VOCs) is dependent on both physicochemical as well as biological properties. Effects of increasingly stressful levels of air pollutants on the microbial structure of biofilms within BTFs are not well understood, especially for VOCs such as styrene. To investigate the relationship between biofilm biodiversity and operational stability, the temporal dynamics of a biofilm from a biotrickling filter subjected to stepwise increasing levels of air polluted with styrene was investigated using 16S rDNA pyrosequencing and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). As styrene contaminant loads were increased, microbial community composition was distinctly altered and diversity was initially reduced in early stages but gradually stabilized and increased diversity in later stages, suggesting a recovery and acclimatization period within the microbial community during incremental exposure of the pollutant. Although temporary reductions in known styrene-degrading bacterial genera (Pseudomonas and Rhodococcus) occurred under increased styrene loads, stable BTF performance was maintained due to functional redundancy. New candidate genera for styrene degradation (Azoarcus, Dokdonella) were identified in conditions of high styrene loads, and may have supported the observed stable BTF performance throughout the experiment. Styrene inlet load was found to be important modulator of community composition and may have been partly responsible for the observed temporary reductions of Pseudomonas. Notable differences between dominant genera detected via pyrosequencing compared to species detected by PCR-DGGE suggests that simultaneous implementation of both techniques is valuable for fully characterizing dynamic microbial communities.

Exploration of the key functional strains from an azo dye degradation microbial community by DGGE and high-throughput sequencing technology.

This study investigated a previously developed thermophilic microbial community with the ability to effectively degrade azo dyes. To identify the key microbes of this microbial community, a dilution-to-extinction approach was combined with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and Illumina high-throughput sequencing technology (HTST). Strains belonging to Tepidiphilus sp. almost disappeared from the degradation solution at dilution ratios above 10-7; furthermore, at this ratio, the diluted microbial community almost lost their decolorization ability, indicating this ratio as the critical point for effective azo dye decolorization. Strains belonging to Tepidiphilus sp. were indicated as possible key functional microbes of this microbial community for effective azo dye decolorization. Moreover, the synergistic action of other microbes, such as Anoxybacillus sp., Clostridium sp., and Bacillus sp., was suggested to further promote the decolorization process by secreting azoreductase and laccase. Caloramator spp. were found have the ability to degrade proteins and amino acids, which might promote the degradation process with further degradation microbes. The loss of these bacteria might diminish the synergistic relationships among different strains, which further results in the failure of efficient azo dye decolorization and degradation by this microbial community.

Evaluation of denaturing gradient gel electrophoresis (DGGE) and next generation sequencing (NGS) in combination with enrichment culture techniques to identify bacteria in commercial microbial-based products.

Identification of bacteria in new or existing commercial microbial-based products (MBPs) is important for compliance with government regulations and for human and environmental risk assessment. Research was performed to develop effective methods to identify bacteria present in a MBP using a combined approach of conventional enrichment culture technique and denaturing gradient gel electrophoresis (DGGE) followed by clonal sequencing or next generation sequencing (NGS). Genomic DNA obtained from un-enriched or enriched MBP in MacConkey broth, Azide Dextrose broth, Peptone Water mixed with Polymixine B and Gram Negative (GN) media under three different temperatures (22 °C, 28 °C and 37 °C) were sequenced in two methods for the V3 and V6 hypersensitive regions of 16S ribosomal DNA (rDNA) and compared. Enrichment followed by DGGE and clonal sequence analysis identified 20 bacterial genera in all enriched and un-enriched media. In contrast, NGS was able to identify 114 bacterial families and 134 genera both in V3 and V6 regions. In clonal sequence analysis, in comparison to the un-enriched MBP, the MacConkey broth enriched for Escherichia or Shigella and Morganella species, GN medium enriched for Proteus and Morganella species and Azide Dextrose broth enriched for Vagococcus and Enterococcus species at both 28 °C and 37 °C. Moreover, the enrichment facilitated NGS to record higher numbers of families and genera in all enrichment cultures, comparatively higher variations in V3 region than in V6. More prominently, NGS identified 14 genera and 9 species in the family Enterobacteriaceae compared to only 5 genera identified in the un-enriched control using V6 region variance in MacConkey broth at 28 °C. Increasing the temperature without enrichment identified specific families by V3 and V6 regions. This study indicates that the polyphasic approach with appropriate enrichment and incubation at different temperatures followed by NGS analysis is a promising method for the identification of viable, non-pathogenic or potential pathogenic bacteria in complex MBPs.

Nitinol as a suitable anode material for electricity generation in microbial fuel cells.

Nitinols (Nickel-titanium alloys) have a good electrical conductivity and biocompatibility with human tissue and bacteria and, therefore, can be effectively used as an anode material in bioelectrochemical systems. This paper aimed to use nitinols (at different Ni/Ti ratios) as an anode material for microbial fuel cells (MFCs) in order to achieve higher power density. The maximum power densities of the MFCs using NiTi-1, NiTi-2, and NiTi-3 electrodes were 555 mW/m2, 811 mW/m2, and 652 mW/m2, respectively. More bacterial adhesion was observed on the NiTi-2 electrode. Electrochemical impedance spectroscopy (EIS) results showed low charge transfer resistance at MFCs fabricated with NiTi. The biofilm observations indicate that bacterial attachment is better with NiTi-2 as compared with that on NiTi-1 and NiTi-3. The resulting mesopore and macropore rich structure significantly promote microbial colonization, enabling formation of compact electroactive biofilms with additional benefit from the excellent biocompatibility and chemical stability of NiTi-2. Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) results indicated that five groups of bacteria were the dominant phyla in the MFCs: environmental samples, b-proteobacteria, g-proteobacteria, d-proteobacteria, and CFB group bacteria. The high biocompatibility, electrical conductivity and stability of nitinols make them a more attractive anode material for MFCs.

Differentiation and quantification of the ochratoxin A producers Aspergillus ochraceus and Aspergillus westerdijkiae using PCR-DGGE.

Ochratoxin A (OTA) is a nephrotoxic, teratogenic, immunotoxic, and carcinogenic mycotoxin which is produced in tropical zones mainly by Aspergillus carbonarius, A. niger, A. ochraceus, and A. westerdijkiae. A. ochraceus and A. westerdijkiae species are phenotypically and genomically very close but A. westerdijkiae produce OTA at a very higher level than A. ochraceus. These species have been differentiated recently. The DNA primer pairs which were drawn so far are not specific and a genomic region of the same size is amplified for both species or they are too specific, and in this case, the DNA of a single species is amplified. To help preventing OTA contamination of foodstuffs, the PCR-DGGE (Denaturing Gradient Gel Electrophoresis) method was used to discriminate between A. ochraceus and A. westerdijkiae DNA fragments of the same size but with different sequences and thus faster access to a diagnosis of the toxigenic potential of the fungal microflora. The proposed methodology was able to differentiate A. westerdijkiae from A. ochraceus with only one primer pairs in a single run. A calibration based on initial DNA content was obtained from image analysis of the DGGE gels and a method of quantification of the two strains was proposed.

Berberine treatment reduces atherosclerosis by mediating gut microbiota in apoE-/- mice.

BACKGROUND: Berberine (BBR) has long been used for treating bacterial diarrhea due to its antimicrobial effect and is currently used to treat obesity, diabetes, hyperlipemia and atherosclerosis. Given the poor oral bioavailability of BBR, the mechanisms through which BBR mediates metabolic disorders are not well understood. The present study was designed to explore the role of BBR-induced gut microbiota modulation in the development of atherosclerosis. METHODS: Male apoE-/- mice were fed a high-fat diet (HFD) with or without the intragastric administration of BBR. Because mice are coprophagic and can transfer their gut microbiota to each other, we cohoused BBR-treated HFD-mice with non-BBR-treated HFD-fed mice. RESULTS: After 12 weeks of HFD feeding, compared with non-BBR-treated HFD-fed mice, BBR-treated HFD-fed mice exhibited a significant reduction in both atherosclerosis development and inflammatory cytokine expression. In addition, cohousing BBR-treated HFD-fed mice with non-BBR-treated HFD-fed mice decreased atherosclerosis development and inflammatory cytokine expression. The denaturing gradient gel electrophoresis and principal component analyses showed that the gut microbial profiles of BBR-treated HFD-fed mice were significantly different from those of HFD-fed mice but were similar to those of cohoused mice. The abundances ofFirmicutes and Verrucomicrobia in cohoused and BBR-treated mice were different from those in HFD-fed and normal chow-fed mice. Moreover, BBR reduced hepatic FMO3 expression and serum trimethylamine N-oxide levels. CONCLUSION: The antiatherosclerotic effect of BBR is related to alterations in gut microbiota compositions, indicating the potential therapeutic value of pharmacological approaches that may modulate the gut microbiota in treating atherosclerosis.

Identification of Lactic Acid Bacteria in Galchi- and Myeolchi-Jeotgal by 16S rRNA Gene Sequencing, MALDI-TOF Mass Spectrometry, and PCR-DGGE.

Jeotgal is a Korean traditional fermented seafood with a high concentration of salt. In this study, we isolated lactic acid bacteria (LAB) from galchi (Trichiurus lepturus, hairtail) and myeolchi (Engraulis japonicas, anchovy) jeotgal on MRS agar and MRS agar containing 5% NaCl (MRS agar+5% NaCl), and identified them by using 16S rRNA gene sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as culture-dependent methods. We also performed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) as a culture-independent method to identify bacterial communities. Five samples of galchi-jeotgal and seven samples of myeolchi-jeotgal were collected from different regions in Korea. A total of 327 and 395 colonies were isolated from the galchi- and myeolchi-jeotgal samples, respectively. 16S rRNA gene sequencing and MALDI-TOF MS revealed that the genus Pediococcus was predominant on MRS agar, and Tetragenococcus halophilus on MRS agar+5% NaCl. PCR-DGGE revealed that T. halophilus, Tetragenococcus muriaticus, and Lactobacillus sakei were predominant in both types of jeotgal. T. halophilus was detected in all samples. Even though the same species were identified by both culture-dependent and -independent methods, many species identified by the culture-dependent methods were not in the bacterial list identified by the culture-independent methods. The distribution of bacteria in galchi-jeotgal was more diverse than in myeolchi-jeotgal. The diverse LAB in galchi- and myeolchi-jeotgals can be further studied as candidates for starter cultures to produce fermented foods.

Characterization of the Microbiota in Air- or Vacuum-Packed Crisp Grass Carp ( Ctenopharyngodon idella C. et V.) Fillets by 16S rRNA PCR-Denaturing Gradient Gel Electrophoresis and High-Throughput Sequencing.

The microbial communities in air- and vacuum-packed crisp grass carp ( Ctenopharyngodon idella C. et V.) fillets have not been characterized during chilled storage. High-throughput sequencing of bacterial 16S rRNA has now revealed that the bacterial community in fresh fillets is diverse and distinct from that in spoiled samples. The predominant phylum was Proteobacteria, and 66 genera were identified. In fresh fillets, the most abundant genera were Acinetobacter (53.3%), Wautersiella (6.3%), unclassified Alcaligenaceae (4.4%), Stenotrophomonas (3.8%), unclassified Enterobacteriaceae (3.8%), and Enhydrobacter (3.6%). These genera diminished during chilled storage and sometimes disappeared. At the end of storage, Aeromonas and Pseudomonas were the most abundant. Similar results were obtained by PCR-denaturing gradient gel electrophoresis. These data provide detailed insight into the evolving bacterial communities in air- and vacuum-packed crisp grass carp fillets during storage, revealing Aeromonas and Pseudomonas as major spoilage organisms. These data may be useful for improvement of crisp grass carp quality and shelf life during chilled storage.

Advancement in LH-PCR methodology for multiple microbial species detections in fermented foods.

The length-heterogeneity PCR is a low throughput molecular biology methods explored to monitor bacteria populations in different environments. It could be more used in food microbiology analysis, not only for fingerprinting analysis, but it has been hampered until now by a limiting factor which relates to the high percentage of secondary peaks. With the aim to overcome this problem, different experiments were performed focusing on changing PCR parameters in order to obtain more specific amplicon patterns and also to reduce the complexity of community patterns. With this purpose, different annealing temperatures were tested on complex fermented food matrices taken from both animal and vegetable origin and also on the bacteria isolated from the same food source. In particular, the optimal annealing temperature identified for the fermented food samples is 59 °C and the optimal for bacterial strains varied between 63 °C and 65 °C. The approach allowed the modification of the LH-PCR protocol increasing the amplification efficiency and therefore the bacteria species discrimination. These temperatures also allowed the implementation of the previous LH-PCR published database. The modification in the level of accuracy of the LH-PCR technique could also allow an improvement in the relative species quantification by the peak area evaluation.

Use of PCR-denaturing gradient gel electrophoresis for the discrimination of Candida species isolated from natural habitats.

Candida species are opportunistic microbes that cause chronic infections for a human being. Therefore, the exact identification of Candida species is extremely important for improved therapeutic strategy against these species. Identification based on conventional methods cannot differentiate between some of yeasts species, hence PCR based molecular techniques and sequencing could be an alternative tool for the yeasts identification. A quick molecular method based on the polymerase chain reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) was applied for distinguishing strains belonging to the Candida species. Six different species designated as AH-20, AH-21, AH-22, AH-23, AH-24 and AH-25 were isolated from soil samples, and their exact identification was detected based on the D1/D2 domain of the 26S rRNA gene amplification and sequence determination. Alignment results and the comparison of 26S rRNA gene sequences of the isolates to 26S rRNA gene sequences available in the GenBank database, as well as the phylogenetic analysis, confirmed the accurate position of the isolates as Candida intermedia strain AH-20, Candida boidinii strain AH-21, Candida tropicalis strain AH-22, Candida mengyuniae strain AH-23, Candida maltosa strain AH-24 and Candida maltosa strain AH-25. Fragments of the D1/D2 domain of 26S rRNA gene were amplified using NL1-GC/LS2 primers and separated by the DGGE. Results showed that all Candida species reported in this study were well discriminated by a distinct band in the DGGE profile. Our results demonstrated that DGGE technique using NL1-GC/LS2 primers could use for the rapid discrimination of yeast strains belonging to the same genera.

Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques for analysing the influence of diet on ruminal bacterial diversity.

The objective of this study was to compare the automated ribosomal intergenic spacer analysis (ARISA) and the denaturing gradient gel electrophoresis (DGGE) techniques for analysing the effects of diet on diversity in bacterial pellets isolated from the liquid (liquid-associated bacteria (LAB)) and solid (solid-associated bacteria (SAB)) phase of the rumen. The four experimental diets contained forage to concentrate ratios of 70:30 or 30:70 and had either alfalfa hay or grass hay as forage. Four rumen-fistulated animals (two sheep and two goats) received the diets in a Latin square design. Bacterial pellets (LAB and SAB) were isolated at 2 h post-feeding for DNA extraction and analysed by ARISA and DGGE. The number of peaks in individual samples ranged from 48 to 99 for LAB and from 41 to 95 for SAB with ARISA, and values of DGGE-bands ranged from 27 to 50 for LAB and from 18 to 45 for SAB. The LAB samples from high concentrate-fed animals tended (p < 0.10) to show greater peak numbers and Shannon index values than those isolated from high forage-fed animals with ARISA, but no differences were identified with DGGE. The SAB samples from high concentrate-fed animals had lower (p < 0.05) peak numbers and Shannon index values than those from animals fed high-forage diets with ARISA, but only a trend was noticed for these parameters with DGGE (p < 0.10). The ARISA detected that animals fed alfalfa hay diets showed lower (p < 0.05) SAB diversity than those fed grass hay diets, but no differences were observed with DGGE. No effect of forage type on LAB diversity was detected by any technique. In this study, ARISA detected some changes in ruminal bacterial communities that were not detected by DGGE, and therefore ARISA was considered more appropriate for assessing bacterial diversity of ruminal bacterial pellets. The results highlight the impact of the fingerprinting technique used to draw conclusions on dietary factors affecting bacterial diversity in ruminal bacterial pellets.

Application of Denaturing Gradient Gel Electrophoresis to the Analysis of Bacterial Communities Associated With Asymptomatic and Symptomatic Pericoronitis.

PURPOSE: Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities associated with asymptomatic and symptomatic pericoronitis. The aim of the study was to compare the fingerprinting patterns of these 2 clinical conditions. MATERIALS AND METHODS: The microbiota of mandibular third molar pockets associated with asymptomatic or symptomatic pericoronitis cases were collected and profiled by the polymerase chain reaction DGGE method. Banding patterns were compared by cluster analysis techniques. RESULTS: Thirteen symptomatic pericoronitis and 7 asymptomatic pericoronitis samples were collected. Comparative analysis of the 2 clinical conditions showed bands that were common to the symptomatic and asymptomatic cases, but most DGGE bands appeared to be unique to the clinical condition. No single band occurred in all profiles. The mean number of bands detected in the 16S rDNA community profiles was 23.8 ± 4.2 (range, 19 to 34) for samples from symptomatic cases and 24.1 ± 2.4 (range, 21 to 29) for those from asymptomatic cases. Cluster analysis and multidimensional scaling analysis of the DGGE banding pattern showed a distinction in the similarity of banding patterns according to the presence or absence of symptoms. CONCLUSIONS: These results suggest that the diversity of pericoronal pocket microbiota in asymptomatic pericoronitis cases differs markedly from that of symptomatic cases.

Application of modified JDP-DGGE-based molecular genotyping method to predict Acanthamoeba genotype and to analyse community diversity in aquatic environments.

Acanthamoeba spp. are ubiquitous, opportunistic potential human pathogens, causing granulomatous amoebic encephalitis and keratitis. They are classified as protozoa, and they include at least 20 different genotypes (T1-T20) based on variation in the 18S rRNA gene. Acanthamoeba spp. are diverse in their production of toxins and in their ability to resist environmental factors. Therefore, it is necessary to develop a rapid genotyping method for Acanthamoeba spp. in aquatic environments. Although the denaturing gradient gel electrophoresis (DGGE) method for analysing microbial genotypes is potentially useful for rapid identification of aquatic environmental species, the technique has been compromised by artificial DGGE profiles in which many DNA fragments of identical sequences are segregated and displayed as different bands. The results indicate that PCR-DGGE genotyping with a GC clamp results in many segregated weaker bands of identical DNA sequences. In contrast, PCR-DGGE genotyping without a GC clamp displays genotype-dependent patterns in the major bands. Thus, DGGE without a GC clamp was performed to compare genotyping efficiency for Acanthamoeba in 21 water samples from rivers and reservoirs in Taiwan. Among them, four samples were found to demonstrate a banding pattern with more than one major band, and these band profiles of major bands were identical to those of positive controls. DNA cloning further confirmed that the sequences of the major bands were identical. In conclusion, more than two genotypes of Acanthamoeba in the four samples were identified by this method, suggesting that PCR-DGGE genotyping without a GC clamp is a useful approach for studying the diversity of Acanthamoeba communities. Graphical abstract.

Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods.

The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 102 to 105CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 102CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively.

Contribution of PCR Denaturing Gradient Gel Electrophoresis Combined with Mixed Chromatogram Software Separation for Complex Urinary Sample Analysis.

Complex samples are a challenge for sequencing-based broad-range diagnostics. We analysed 19 urinary catheter, ureteral Double-J catheter, and urine samples using 3 methodological approaches. Out of the total 84 operational taxonomic units, 37, 61, and 88% were identified by culture, PCR-DGGE-SS (PCR denaturing gradient gel electrophoresis followed by Sanger sequencing), and PCR-DGGE-RM (PCR- DGGE combined with software chromatogram separation by RipSeq Mixed tool), respectively. The latter approach was shown to be an efficient tool to complement culture in complex sample assessment.

Profiling 5-tolyltriazole biodegrading sludge communities using next-generation sequencing and denaturing gradient gel electrophoresis.

Efficient biodegradation of 5-tolyltriazole (5-TTri) in wastewater treatment would minimize its potential detrimental effects on aquatic systems. Therefore, in order to profile 5-TTri biodegrading activated sludge communities (ASC) by DGGE and NGS, acclimation experiments with (i) easily degradable substrates, and (ii) various complex substrates mimicking wastewater conditions were performed. DGGE revealed four genera: Aminobacter (family Phyllobacteriaceae), Flavobacterium (family Flavobacteriaceae), Pseudomonas (family Pseudomonaceae), and Hydrogenophaga (family Comamonadaceae). Metagenomics (DNA) revealed the dominant families Alcaligenaceae, Pseudomonadaceae and Comamonadaceae that also represented the most active families at the RNA level (metatranscriptomics), which might indicate their importance for 5-TTri biodegradation. ASC acclimation and the composition of the substrate significantly affected 5-TTri biodegradation and the development of biodegrading communities. Using acetate only, a moderate 5-TTri degrading community was detected with a very low biodiversity and Pseudomonas spp. as dominant organisms. In contrast, setups fed 'sludge supernatant' (a complex substrate) efficiently biodegraded 5-TTri and formed a more diverse microbial community but with Hydrogenophaga spp. as the dominant group. Finally, a hypothetical 5-TTri biodegradation pathway was constructed based exclusively on the detected, biodegradation-related, Hydrogenophaga spp. genes.

Rapid detection of mutations in erm(41) and rrl associated with clarithromycin resistance in Mycobacterium abscessus complex by denaturing gradient gel electrophoresis.

Clarithromycin resistance is increasing dramatically among Mycobacterium abscessus complex. The main resistance mechanisms are mutations in the erm(41) and rrl genes. Here we report PCR-based denaturing gradient gel electrophoresis (DGGE) as an alternative method for rapidly detection of mutations in erm(41) and rrl among M. abscessus isolates. Four primer sets targeting the full-length erm(41) gene and a 354bp fragment of the rrl gene were designed. A combination of 16 different DGGE patterns were observed for erm(41) gene, including 16 in M. abscessus subsp. abscessus and 1 in M. abscessus subsp. massiliense. Six DGGE patterns were obtained for rrl gene. Mutations in the erm(41) and rrl detected by DGGE were 100% identical to mutations detected by DNA sequencing. This is the first report to identify PCR-based DGGE as a practical, relatively inexpensive technique for rapidly detecting mutations in the erm(41) and rrl genes associated with clarithromycin resistance in M. abscessus complex.

Soil microbial community responses to acid exposure and neutralization treatment.

Changes in microbial community induced by acid shock were studied in the context of potential release of acids to the environment due to chemical accidents. The responses of microbial communities in three different soils to the exposure to sulfuric or hydrofluoric acid and to the subsequent neutralization treatment were investigated as functions of acid concentration and exposure time by using 16S-rRNA gene based pyrosequencing and DGGE (Denaturing Gradient Gel Electrophoresis). Measurements of soil pH and dissolved ion concentrations revealed that the added acids were neutralized to different degrees, depending on the mineral composition and soil texture. Hydrofluoric acid was more effectively neutralized by the soils, compared with sulfuric acid at the same normality. Gram-negative ß-Proteobacteria were shown to be the most acid-sensitive bacterial strains, while spore-forming Gram-positive Bacilli were the most acid-tolerant. The results of this study suggest that the Gram-positive to Gram-negative bacterial ratio may serve as an effective bio-indicator in assessing the impact of the acid shock on the microbial community. Neutralization treatments helped recover the ratio closer to their original values. The findings of this study show that microbial community changes as well as geochemical changes such as pH and dissolved ion concentrations need to be considered in estimating the impact of an acid spill, in selecting an optimal remediation strategy, and in deciding when to end remedial actions at the acid spill impacted site.