PCR-based Denaturing Gradient Gel Electrophoresis Research Articles

Analysis of the Intestinal Microbiota in Autistic Patients by Denaturing Gradient Gel Electrophoresis

To analyze the differences in the composition of the intestinal microbiota between the autistic and healthy children, we selected 45 autistic and 20 control children aged 2 to 9 years to collect their fecal samples. The total microbial genome DNA of each fecal sample was extracted, and the V3 regions of microbial 16S rRNA genes were amplified. The intestinal microbial composition of both study groups was detected by PCR-based denaturing gradient gel electrophoresis. Quantity One and Biodap software were used to analyze the diversity and similarity of bacterial populations, and SPSS software was used for statistical analysis. The denaturant gradient gel electrophoresis profiles documented significant differences in the composition of intestinal microflora between the autism and control groups. Analysis of the excised bands demonstrated the abundance of bacteria species assigned to the genus Escherichia/Shigella in the gastrointestinal tract of the autism group but a low content in the control group. An opposite result was obtained for the Bacteroides genus. These data indicate that intestinal microbial composition may is correlated with the occurrence of the autism.

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.

Effects of feeding Original XPC™ to broilers with a live coccidiosis vaccine under industrial conditions: Part 2. Cecal microbiota analysis

Biological supplements in poultry feed are of continued interest due to the improvements in growth performance, protection from pathogen invasion, and benefits in overall host health. The fermentation metabolites of Diamond V Original XPC™ (XPC) have previously been shown to improve commercial performance and reduce Salmonella in poultry. The current study sought to characterize the cecal microbiota using culture-independent analysis based on 16S rRNA gene in Coccivac-D sprayed broilers supplemented with XPC and/or Salinomycin (SAL). Ross 708 male broilers (n = 640) were assigned to one of 4 treatments: Cocci-vaccine (T1), Cocci-vaccine + XPC (T2), Cocci-vaccine + SAL (in the grower diet only) (T3), and Cocci-vaccine + SAL (in the grower diet only) + XPC (T4). Analysis with a PCR-based denaturing gradient gel electrophoresis (DGGE) indicated a shift in the microbial populations present at the various sampling ages — 16, 28, and 42 days. Phylogenetic analysis indicated further consistency in microbial communities directly related to bird age. Identification of microbial communities present and the assessment of their respective quantities using an Illumina MiSeq indicated treatment with XPC had no significant impact on microbial diversity (Chao1 index, observed operational taxonomic unit (OTU) and phylogenetic diversity (PD) whole tree). Sampling age revealed significantly greater diversity at 16 and 28 d (P < 0.05) as compared to the 42 d for the Shannon diversity index, while showing significantly decreased richness and diversity in the 42 d sampling age (Chao1 and observed OTU; P < 0.05). The results of the current study indicate that the chicken intestinal microbiota are impacted more by temporal changes rather than by the feed additive studied.

Fingerprinting and Identification of Bacteria Present in UASB Granules Used to Treat Winery, Brewery, Distillery or Peach-lye Canning Wastewater

The effective operation of the anaerobic digestion process in an upflow anaerobic sludge blanket (UASB) bioreactor is dependent on the microbial composition of the UASB granules. The granules contain a consortium of bacteria, with a specific metabolic function for each group, contributing to the overall efficiency and stability of the bioreactor. The aim of this study was to fingerprint and identify the bacteria present in four different types of South African UASB granules that are used to treat winery, brewery, distillery and peach-lye canning wastewaters. This was done by combining conventional microbiological platings with PCR-based denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. Each granule type showed distinct PCR-based DGGE fingerprints with unique bands, while other bands were found to be present in all the granules, regardless of the wastewater being treated. Sixty-eight different bacteria (40 pure isolates and 28 clones) were partially sequenced and identified from the winery, brewery, distillery and peach-lye canning granules. Thirty-five percent of the identified bacteria represented the unculturable bacteria and 65% represented the culturable bacteria, which included members of the following genera: Bacillus, Pseudomonas, Bacteroides, Enterococcus, Alcaligenes, Clostridium, Shewanella, Microbacterium, Leuconostoc, Sulfurospirillum, Acidaminococcus, Vibrio, Aeromonas, Nitrospira, Synergistes, Rhodococcus, Rhodocyclus and Syntrophobacter. A DGGE marker was successfully constructed, representing members of the bacterial consortium in UASB granules.

PCR-based DGGE Identification of Bacteria Present in Pasteurised South African Fruit Juices

The contamination of pasteurised fruit juice products by thermophilic acidophilic bacteria (TAB) has become a concern for producers. The aim of this study was to identify the bacteria present in South African fruit juices before and after pasteurisation, using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE). Alicyclobacillus acidoterrestris was found to be present in apple, pear, white grape and aloe vera juice. White grape juice was found to contain Alicyclobacillus pomorum, while two uncultured bacteria in the orange, apple, mango and pear juices were presumptively identified as members of the genus Bacillus, and one uncultured bacteria was identified as being closely related to Alcaligenes faecalis. The results emphasise the need for rapid and accurate detection of TAB in food products.

Microbial Diversity of Ground Beef Products in South Korean Retail Market Analyzed by PCR-DGGE and 454 Pyrosequencing

ABSTRACTThe purpose of this study was to assess the microbial community in ground beef purchased from local stores in the north central area of South Korea. A total of 31 ground beef samples showed 3.77 ± 5.16 × 105 CFU/g on aerobic plate counts, and PCR-based denaturing gradient gel electrophoresis was performed to assess the microbial community, which revealed a diverse profile among the products. Four of these samples were selected for further analyses via 454 pyrosequencing. Pseudomonas was the most frequently identified genus in terms of relative abundance (4.54–73.8%), and other bacteria such as Lactobacillus, Acinetobacter, and Carnobacterium were also identified in the ground beef. The presence of bacteria can affect the spoilage of ground beef products depending on the microbial populations, and understanding these populations may help limit spoilage and/or foodborne illness. This study illustrates the microbial diversity occurring among Korean ground beef products, even within a local region, and provides a molecular approach for potential ground beef microbial quality assessment.

Assessment of cecal microbiota, integron occurrence, fermentation responses, and Salmonella frequency in conventionally raised broilers fed a commercial yeast-based prebiotic compound

Prebiotics are defined as nondigestible food ingredients that can stimulate the growth of one or more beneficial bacteria in the gastrointestinal tract. The Biolex® MB40 is a commercial prebiotic that contains mannanoligosaccharides. The aims of this study were to evaluate the effects of prebiotic Biolex® MB40 on cecal microbiota of conventionally raised chickens using PCR-based denaturing gradient gel electrophoresis (DGGE) and assessing Salmonella prevalence. Chickens were randomly selected and distributed into three groups; a negative control (NC) and two treatment groups (T1 and T2). The NC group was fed a non-medicated feed, while the treatment groups were fed either T1 or T2, 0.05% antibiotic (BMD50) or 0.2% Biolex® MB40 respectively. During the study, cecal contents and bird feed were plated on selective media for Salmonella, yeast and mold prevalence analysis. Ten chickens from each group were randomly selected at 1, 2, 4 and 6 wk and ceca were extracted for DNA isolation for PCR-based DGGE. Also, short-chain fatty acids (SCFAs) were analyzed from collected cecal material by gas chromatography. Only 4.2% of the samples were Salmonella positive. Presence of class 1 integron from cecal material were analyzed by PCR and 97.5% of the cecal samples were positive for integron presence, but no class I integrons were detected in the Salmonella isolates. According to the PCR-based DGGE analysis, the T2 group exhibited a cecal microbial population pattern that was similar to the T1 group prior to wk 4 and the T2 group appeared to be almost identical with the NC group after wk 4 but T2 exhibited less Bacteroides rodentium prior to wk 4. Overall results showed that the commercial prebiotic, MB40 did not lead to a detectable reduction of Salmonella but the general frequency of Salmonella was minimal in all treatments. However, feeding an MB40 supplement did result in similar DGGE band patterns as the T1 group indicating that cecal microbiotia were potentially similar in these 2 groups. Overall, it appears that MB40 (T2) exhibited similar DGGE-cecal population patterns as BMD50 (T1) which suggests that these treatments may have influenced the populations in a comparable fashion.

Bacterial diversity of gut content in sea cucumber (Apostichopus japonicus) and its habitat surface sediment

This study investigated the bacterial diversity of gut content of sea cucumber (Apostichopus japonicus) and its habitat surface sediment in a bottom enhancement area using PCR-based denaturing gradient gel electrophoresis (DGGE) technique. Bacterial diversity evaluation showed that the value of the Shannon-Wiener index of gut content in different intestinal segments of A. japonicus varied between 2.88 and 3.00, lower than that of the surrounding sediment (3.23). Phylogenetic analysis showed that bacterial phylotypes in gut content and the surrounding sediment of A. japonicus were closely related to Proteobacteria includingγ-, α-, δ- and ɛ-proteobacteria, Bacteroidetes, Firmicute, and Actinobacteria, of which γ-proteobacteria were predominant. These results suggested that the sea cucumber A. japonicus was capable of feeding selectively, and PCR-DGGE was applicable for characterizing the bacterial community composition in gut content and the surrounding sediment of sea cucumber. Further investigation targeting longer 16S rDNA gene fragments and/or functional genes was recommended for obtaining more information of the diversity and function of bacterial community in the gut content of sea cucumber.

Assessment of Gastrointestinal Microflora in Pasture Raised Chickens Fed Two Commercial Prebiotics

Prebiotics include nondigestible carbohydrate dietary additives and other biological components that stimulate the growth of one or more types of bacteria in the gastrointestinal tract that are beneficial to the host. These bacteria can inhibit colonization of pathogenic bacteria by producing antimicrobial substances and competing for niches within the gut. In this study, we evaluated the effects of Biolex® MB40 and Lieber® ExCel which are commercial prebiotics derived from brewer’s yeast cell walls. The two prebiotics were added to Genetically Modified Organism (GMO)- free chicken feeds with each group consisting of 1) control (no prebiotic), 2) Biolex® MB40 with 0.2% and 3) Leiber® ExCel with 0.2%. Feeds were supplemented with the prebiotics during the 8 weeks of the entire experimental period. At 8 weeks, a total of 15 birds from each group were randomly selected for necropsy. The PCR-based Denaturing Gradient Gel Electrophoresis (PCR-based DGGE) technique was utilized to compare microbial populations in control and treatment groups. Feeds supplemented with either Biolex® MB40 or Leiber® ExCel prebiotics exhibited more consistent microbial populations (i.e. relatedness) compared to the control group. For Biolex® MB40 supplemented group, all samples were clustered with over 74% of relatedness. The Leiber® ExCel supplemented group exhibited 77% relatedness among 4 samples with the exception of one outlier. According to sequencing results, Bacteriodes salanitronis were consistently detected in all groups, and Barnesiella ciscericola and Firmicutes were identified in both treatment groups. In addition, the class 1 integron gene prevalence was evaluated and frequencies of 93.3% for the control, 73.3% for the Biolex® MB40 and 73.3% for the Leiber® ExCel treatment group of chickens were observed. Last, Campylobacter concentrations were significantly lower in both treatment groups compared to the control group.

Comparing Spatial and Temporal Diversity of Bacteria in a Chlorinated Drinking Water Distribution System

Temporal and spatial drinking water bacterial diversity was evaluated through bulk water sampling from three locations, at varying distances from the treatment plant, in a chlorinated distribution system serving 250,000 people. Most bacteria were identified as alpha-, beta-, and gamma-Proteobacteria based on quantitative polymerase chain reaction assays targeting these bacterial classes as well as bacteria. Temporal variability is correlated with seasonal changes in the chlorine concentration as shown by the positive relationship between the hypochlorite dose and alpha-Proteobacteria. Analysis of variance of alpha-, beta-, and gamma-Proteobacteria and bacteria quantities for all sites and months suggests that temporal patterns are stronger than spatial patterns in this system population. Band patterns from PCR-based denaturing gradient gel electrophoresis using bacterial primers also reveal the importance of temporal variability as samples clustered by months more than by locations. These results demonstrate the consistent presence of alpha-, beta-, and gamma-Proteobacteria at different locations within a particular distribution system and suggest these bacterial classes may be useful indicators of system conditions.

PCR-based DGGE fingerprinting and identification of the microbial population in South African red grape must and wine

&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Aim&lt;/strong&gt;: The aim of this study was to evaluate the microbial population present in red grape must and wine using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE).&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Methods and results&lt;/strong&gt;: Red wine from the cultivars Pinotage and Merlot were produced and samples taken throughout alcoholic and malolactic fermentation (MLF). PCR fragments were resolved by DGGE and unique fingerprints were obtained for the bacteria and yeasts present in the wines. &lt;em&gt;Lactobacillus plantarum&lt;/em&gt;, &lt;em&gt;Enterobacter&lt;/em&gt; &lt;em&gt;sakazakii&lt;/em&gt; (&lt;em&gt;Cronobacter&lt;/em&gt; sp.) and &lt;em&gt;Pantoea agglomerans&lt;/em&gt; were present in the Pinotage during both alcoholic and MLF, and in both inoculated and spontaneous fermentations. &lt;em&gt;E. sakazakii&lt;/em&gt; (&lt;em&gt;Cronobacter&lt;/em&gt; sp.) and &lt;em&gt;P. agglomerans&lt;/em&gt; were also observed in the Merlot wines during alcoholic fermentation as well as MLF. &lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt; was the most dominant yeast observed in Pinotage, and was the only yeast observed in Merlot. This yeast was observed until the end of MLF.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Conclusions&lt;/strong&gt;: Results showed that the microbial flora that participates in the winemaking process is more diverse than commonly thought.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Significance and impact of the study&lt;/strong&gt;: This method may serve as an alternative to conventional microbiological methods for the identification of the microbial species in red grape must and wine.&lt;/p&gt;

Bacterial Diversity in Biological Filtration Plant for the Removal of Iron and Manganese from Groundwater

Bacterial diversity of the microbial consortia in a biological filtration plant for the elimination of iron (Fe) and manganese (Mn) from groundwater in Joyo City, Kyoto, Japan, was studied. PCR-based denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S ribosomal RNA (rRNA) genes represented at least 15 signals, and nucleotide sequences of the dominant fragments showed similarities to Gallionella and Nitrospira. Phylogenetic analysis using the obtained nucleotide sequences of the 16S rRNA gene clone library showed the presence of the bacteria related to Hyphomicrobium, Gallionella, and Sideroxydans, which are supposed to be involved in Fe and Mn removal. In contrast, no 16S rRNA gene clone affiliated with the genus Leptothrix, which have been regarded as a major Fe- and Mn-oxidizer in biological filtration system, was observed. Though a large number of 16S rRNA gene clones closely related to Nitrospira was obtained, no clone showed high sequence similarity to known ammonium oxidizing bacteria (AOB). However, PCR-amplification of the ammonia monooxigenase gene (amoA) of AOB and ammonia oxidizing archaea (AOA) indicated the presence of both AOB and AOA in this biological filtration plant.

圧力処理キムチの変性剤濃度勾配ゲル電気泳動による菌叢解析

高静水圧は発酵の制御に有望な技術だと考えられている．高静水圧の効果を把握するため，キムチ発酵における微生物叢の挙動をPCR増幅したDNAの変性密度勾配ゲル電気泳動とゲルから抽出したDNAの塩基配列シーケンシングにより調べた．キムチからLactobacillus，LeuconostocおよびWeissella属を含む11種の乳酸菌を分離した．60日間のキムチ発酵過程では，pHは3.9まで二段階で低下した．乳酸菌濃度は15日目に最大になり，その後3.2×108 cfu/mlに保たれた．乳酸菌の中ではL. sakeiが一定して認められ，L. plantarumが21日目以降に見られた．酵母の菌濃度は15日目に最大となり1.4×108 cfu/mlに達し，27日目以降は検出限界以下へと減少した．酵母ではKazachstania servazziiが圧倒的に優占化していた．発酵21日目に200 MPa，60分の高圧処理をキムチに加えたところ，以降のpH低下が見られなかった．乳酸菌と酵母の菌濃度は急激に低下し，それぞれ8.3×105 cfu/mlならびに検出限界以下となり，乳酸菌は緩やかに回復したが，酵母は速やかな回復を見せた．しかし，高圧処理の有無による微生物叢の明確な違いは観察されなかった．発酵開始時におけるL. sakeiとK. servazziiの添加も試みたが，明確な効果は見られなかった．

Comparative Mesophilic and Thermophilic Anaerobic Digestion of Palm Oil Mill Effluent Using Upflow Anaerobic Sludge Blanket

The effects of organic loading rate and operating temperature on the microbial diversity and performances of upflow anaerobic sludge blanket (UASB) reactors treating palm oil mill effluent (POME) were investigated. The following two UASB reactors were run in parallel for comparison: (1) under a mesophilic condition (37 degrees C) and (2) under a mesophilic condition in transition to a thermophilic condition (57 degrees C). A polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis showed that the microbial population profiles significantly changed with the organic loading rate (OLR) and the temperature transition from the mesophilic to the thermophilic condition. Significant biomass washout was observed for the mesophilic UASB when operating at a high organic loading rate (OLR) of 9.5 g chemical oxygen demand (COD)/L.d. In contrast, the thermophilic UASB can be operated at this OLR and at a temperature of 57 degrees C with satisfactory COD removal and biogas production. The PCR-based DGGE analysis suggested that the thermophilic temperature of 57 degrees C was suitable for a number of hydrolytic, acidogenic, and acetogenic bacteria.

Dynamics of Seed-Borne Rice Endophytes on Early Plant Growth Stages

Bacterial endophytes are ubiquitous to virtually all terrestrial plants. With the increasing appreciation of studies that unravel the mutualistic interactions between plant and microbes, we increasingly value the beneficial functions of endophytes that improve plant growth and development. However, still little is known on the source of established endophytes as well as on how plants select specific microbial communities to establish associations. Here, we used cultivation-dependent and -independent approaches to assess the endophytic bacterrial community of surface-sterilized rice seeds, encompassing two consecutive rice generations. We isolated members of nine bacterial genera. In particular, organisms affiliated with Stenotrophomonas maltophilia and Ochrobactrum spp. were isolated from both seed generations. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) of seed-extracted DNA revealed that approximately 45% of the bacterial community from the first seed generation was found in the second generation as well. In addition, we set up a greenhouse experiment to investigate abiotic and biotic factors influencing the endophytic bacterial community structure. PCR-DGGE profiles performed with DNA extracted from different plant parts showed that soil type is a major effector of the bacterial endophytes. Rice plants cultivated in neutral-pH soil favoured the growth of seed-borne Pseudomonas oryzihabitans and Rhizobium radiobacter, whereas Enterobacter-like and Dyella ginsengisoli were dominant in plants cultivated in low-pH soil. The seed-borne Stenotrophomonas maltophilia was the only conspicuous bacterial endophyte found in plants cultivated in both soils. Several members of the endophytic community originating from seeds were observed in the rhizosphere and surrounding soils. Their impact on the soil community is further discussed.

Rapid Detection of rpoB Mutations in Rifampin Resistant M. tuberculosis from Sputum Samples by Denaturing Gradient Gel Electrophoresis

Objective: To establish a rapid detection method for identifying rpoB mutations associated with rifampin (RIF) resistance in sputum specimens.Methods: We detected rpoB mutations directly in 90 sputum specimens collected from suspected tuberculosis patients using PCR-based denaturing gradient gel electrophoresis (DGGE) and compared these results with those obtained by rpoB sequencing and conventional drug susceptibility testing.Results: The positive detection rate of Mycobacterium tuberculosis (M. tuberculosis) was 52.2% by Acid-Fast Bacilli staining and 72.2% by conventional mycobacterial culture. In contrast, the positive rate was significantly higher (93.3%) by PCR-based detection of the rpoB gene in the same specimens. Furthermore, 75% of the tested specimens presented abnormal patterns compared with the wild-type pattern (standard H37Rv strain) analysed by DGGE. A total of 12 different patterns, representing 12 different rpoB mutations, were observed in the 63 abnormal patterns. The match rate of rpoB mutations detected by DGGE reached 96.9% when compared to DNA sequencing.Conclusion: Our findings indicate that PCR-based DGGE is a rapid and reliable bio-technique for direct detection of rpoB mutations associated with RIF resistance in the sputum of suspected tuberculosis patients.

Evidence of compositional differences between the extracellular and intracellular DNA of a granular sludge biofilm

Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules. Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR-based denaturing gradient-gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA. Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm. The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms.

In Vitro Communities Derived from Oral and Gut Microbial Floras Inhibit the Growth of Bacteria of Foreign Origins

The gastrointestinal (GI) tract is home to trillions of microbes. Within the same GI tract, substantial differences in the bacterial species that inhabit the oral cavity and intestinal tract have been noted. While the influence of host environments and nutritional availability in shaping different microbial communities is widely accepted, we hypothesize that the existing microbial flora also plays a role in selecting the bacterial species that are being integrated into the community. In this study, we used cultivable microbial communities isolated from different parts of the GI tract of mice (oral cavity and intestines) as a model system to examine this hypothesis. Microbes from these two areas were harvested and cultured using the same nutritional conditions, which led to two distinct microbial communities, each with about 20 different species as revealed by PCR-based denaturing gradient gel electrophoresis analysis. In vitro community competition assays showed that the two microbial floras exhibited antagonistic interactions toward each other. More interestingly, all the original isolates tested and their closely related species displayed striking community preferences: They persisted when introduced into the bacterial community of the same origin, while their viable count declined more than three orders of magnitude after 4 days of coincubation with the microbial flora of foreign origin. These results suggest that an existing microbial community might impose a selective pressure on incoming foreign bacterial species independent of host selection. The observed inter-flora interactions could contribute to the protective effect of established microbial communities against the integration of foreign bacteria to maintain the stability of the existing communities.Electronic supplementary materialThe online version of this article (doi:10.1007/s00248-010-9711-9) contains supplementary material, which is available to authorized users.

Phytoplankton diversity in the East China Sea and Yellow Sea measured by PCR-DGGE and its relationships with environmental factors

Relationships between phytoplankton community composition and environmental variables in the East China Sea (ECS) and Yellow Sea (YS) were investigated using geochemical and molecular microbiology methods. The diversity of phytoplankton was characterized using cultivation-independent PCR-based denaturing gradient gel electrophoresis (DGGE). Groups resulting from unweighted pair-group method with arithmetic averages clustering of the DGGE profiles showed good consistency with the eco-environmental characteristics of the sea area they belonged to. Additionally, the clustering results based on DGGE fingerprinting and those based on morphological compositions were practically identical. The relationship of phytoplankton diversity to environmental factors was statistically analyzed. Temperature, dissolved inorganic nitrogen (DIN), and silicate-Si were found significantly related to the phytoplankton community composition. Canonical correspondence analysis (CCA) was performed to reveal the relationship between community composition and these three environmental factors. Generally, values of the ECS are clearly separated from those of the YS in the CCA biplot, due to mainly the effect of temperature and DIN.

Identification of changes in the composition of ileal bacterial microbiota of broiler chickens infected with Clostridium perfringens

We previously reported that Clostridium perfringens in vivo proliferation and alpha-toxin gene expression were highly correlated, both progressing in a parabolic curve pattern during the development of necrotic enteritis (NE). The present study investigated the response of dominant ileal bacteria in abundance to C. perfringens infection using PCR-based denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (QPCR) techniques. Chickens were fed antibiotic-medicated (bacitracin, 55mg/kg) or non-medicated diets, and were challenged with C. perfringens through the diet at 18 days of age. Ileal digesta was collected daily before and after the challenge for 5 days. Bacterial profiles of PCR-DGGE from both bacitracin-treated and untreated chickens responding to clostridial infection were analyzed by the principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Treatment-related differences in PCR-DGGE bacterial profiles on day 2 post-infection (PI) were detected. Subsequent QPCR assays identified changes in the abundance of lactobacilli, L. aviarius in particular. Changes in both populations correlated negatively with the population of C. perfringens in bacitracin-untreated group of chickens that displayed a high incidence of NE lesions. The results indicated that L. aviarius was suppressed by C. perfringens infection. This observation warrants further studies on the mechanisms underlying the ecological change and to assist in further development of novel probiotics to control NE disease.