PCR-denaturing Gradient Gel Electrophoresis Profiles Research Articles

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.

The effect of irrigation with oil-polluted water on microbial communities in estuarine reed rhizosphere soils

The effect of long-term drip irrigation with oil-polluted water on microorganisms in reed rhizosphere soils was assessed. During five months of irrigation with oil-polluted water, n-alkanes and polycyclic aromatic hydrocarbons (PAHs) occurred primarily in topsoils. A petroleum origin was indicated by the predominance of n-alkanes with 13–26 carbons. Naphthalene, fluorene, phenanthrene and pyrene were the predominant components of the detected PAHs. After five months of irrigation, the oil contaminants decreased the abundances of bacteria, fungi and actinomycetes in the reed rhizosphere soils but promoted the growth and reproduction of hydrocarbon degraders. The counts of bacteria, actinomycetes and fungi at a depth of 20cm in soils irrigated with oil-polluted water were only 1/4, 1/10 and 1/2, respectively, of those without oil-polluted water. A Pearson correlation analysis confirmed that ORP, PAHs and n-alkanes had substantial effects on the microbial populations. PCR-denaturing gradient gel electrophoresis profiles revealed that the structure of the bacterial community was generally less affected, but the dominant flora was changed. Several members of the phyla Proteobacteria and Bacteroidetes formed a stable, dominant community structure after five months of irrigation with oil-polluted water. The growth and reproduction of certain members of the phyla Firmicutes, Actinobacteria and Cyanobacteria were clearly decreased by oil pollution. Oil pollution tended to simplify the functions present in the microorganism communities.

Biological treatment of steroidal drug industrial effluent and electricity generation in the microbial fuel cells

The single chamber microbial fuel cells (MFCs) were used to treat steroidal drug production wastewater (SPW) and generate electricity simultaneously. The results indicated that the maximum COD removal efficiency reached 82%, total nitrogen and sulfate removal rate approached 62.47% and 26.46%, respectively. The maximum power density and the Coulombic efficiency reached to 22.3Wm−3 and 30%, respectively. The scanning electron microscope showed that the dominant microbial populations were remarkably different in morphology on the surface of SPW and acetate-fed anodes. PCR-denaturing gradient gel electrophoresis profiles revealed that the microbial community structure fed with different concentrations of SPW presented a gradual succession and unique bacterial sequences were detected on the SPW and acetate-fed anodes. This research demonstrates that MFCs fed with SPW achieved a high efficiency of power density and simultaneous nutrient removal, and the dominant microorganisms on the anode were related to the types and the concentrations of substrates.

Efficacy of Rifaximin Vaginal Tablets in Treatment of Bacterial Vaginosis: a Molecular Characterization of the Vaginal Microbiota

Bacterial vaginosis (BV) is a common vaginal disorder characterized by an alteration of the vaginal bacterial morphotypes, associated with sexually transmitted infections and adverse pregnancy outcomes. The purpose of the present study was to evaluate the impact of different doses of rifaximin vaginal tablets (100 mg/day for 5 days, 25 mg/day for 5 days, and 100 mg/day for 2 days) on the vaginal microbiota of 102 European patients with BV enrolled in a multicenter, double-blind, randomized, placebo-controlled study. An integrated molecular approach based on quantitative PCR (qPCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) was used to investigate the effects of vaginal tablets containing the antibiotic. An increase in members of the genus Lactobacillus and a decrease in the BV-related bacterial groups after the antibiotic treatment were demonstrated by qPCR. PCR-DGGE profiles confirmed the capability of rifaximin to modulate the composition of the vaginal microbial communities and to reduce their complexity. This molecular analysis supported the clinical observation that rifaximin at 25 mg/day for 5 days represents an effective treatment to be used in future pivotal studies for the treatment of BV.

Investigation of the early intestinal microflora in premature infants with/without necrotizing enterocolitis using two different methods

The pathophysiology of necrotizing enterocolitis (NEC) is multifactorial, and gastrointestinal bacteria are thought to play an important role. In this study, the role of microflora in the gastrointestinal tract of neonates with NEC was assessed by comparing cases with controls. Of the 163 neonates, 21 developed NEC. The risk of NEC decreased by 8% with each additional day of gestational age. Typically, very few bacterial species could be cultured from the fecal specimens obtained. Gram-positive (G(+)) bacteria dominated the samples in the NEC group, whereas in the control group mixed flora of G(+) and Gram-negative (G(-)) bacteria were isolated. Surprisingly, molecular analysis using PCR-DGGE profiles did not confirm these differences. Our data suggest that G(+) bacteria in the intestine may play a role in the development of NEC in premature infants. One hundred and sixty three neonates born at <30 weeks of gestation were enrolled. Fecal samples taken during the first month of life were subjected to culture and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). A total of 482 fecal samples were examined.

Distinct commensal bacteria associated with ingesta and mucosal epithelium in the gastrointestinal tracts of calves and chickens

The primary aim of this study was to determine whether distinct gastrointestinal tract (GIT) microbial communities are established within ingesta and on mucosal surfaces of dairy calves and chickens to evaluate whether the principle of microbial segregation is of broad biological significance. Multivariate analysis of the predominant bacterial PCR-denaturing gradient gel electrophoresis profiles and estimated bacterial populations were compared in rumen, jejunum, ileum, cecum, and colon ingesta and matching mucosal tissues. Samples collected from 3-week old (n = 8) and 6-month old (n = 8) calves revealed that the predominant mucosa-associated bacteria were distinct from those inhabiting ingesta, and bacterial diversity varied significantly among the GIT regions. The estimated bacterial populations displayed significant regional differences for bovine mucosal (P = 0.05) and for ingesta (P = 0.03) only at 6 months of age. This indicates an established segregation of the enteric bacterial population throughout the GIT in weaned calves. Analysis of ileal and cecal bacterial profiles in chickens confirmed that the segregation of commensal bacteria between ingesta and the mucosal tissue was a common biological phenomenon. Our study provides some fundamental understanding of the impact of sample type (mucosa vs. ingesta), region, and host age on commensal bacterial establishment and segregation throughout the GIT.

Relationship between rumen methanogens and methane production in dairy cows fed diets supplemented with a feed enzyme additive

To investigate the relationship between ruminal methanogen community and host enteric methane (CH(4) ) production in lactating dairy cows fed diets supplemented with an exogenous fibrolytic enzyme additive. Ecology of ruminal methanogens from dairy cows fed with or without exogenous fibrolytic enzymes was examined using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analyses and quantitative real-time PCR (qRT-PCR). The density of methanogens was not affected by the enzyme additive or sampling times, and no relationship was observed between the total methanogen population and CH(4) yield (as g per head per day or g kg(-1) DMI). The PCR-DGGE profiles consisted of 26 distinctive bands, with two bands similar to Methanogenic archaeon CH1270 negatively correlated, and one band similar to Methanobrevibacter gottschalkii strain HO positively correlated, with CH(4) yield. Three bands similar to Methanogenic archaeon CH1270 or Methanobrevibacter smithii ATCC 35061 appeared after enzyme was added. Supplementing a dairy cow diet with an exogenous fibrolytic enzyme additive increased CH(4) yield and altered the composition of the rumen methanogen community, but not the overall density of methanogens. This is the first study to identify the correlation between methanogen ecology and host CH(4) yield from lactating dairy cows.

Rumen protozoa and methanogenesis: not a simple cause–effect relationship

Understanding the interactions between hydrogen producers and consumers in the rumen ecosystem is important for ruminant production and methane mitigation. The present study explored the relationships between rumen protozoa, methanogens and fermentation characteristics. A total of six donor sheep harbouring (F, faunated) or not (D, defaunated) protozoa in their rumens (D animals were kept without protozoa for a period of a few months (D-) or for more than 2 years (D+)) were used in in vitro and in vivo experiments. In vitro the absence of protozoa decreased NH3 and butyrate production and had no effect on methane. In contrast, the liquid-associated bacterial and methanogens fraction of D+ inocula produced more methane than D- and F inoculum (P<0·05). In vivo fermentation parameters of donor animals showed the same trend on NH3 and butyrate and showed that D+ animals were high methane emitters, while D- were the lowest (-35%). The concentration of dissolved dihydrogen measured after feeding followed the opposite trend. Methane emissions did not correlate with the relative abundance of methanogens in the rumen measured by quantitative PCR, but there was a trend for higher methanogens concentration in the solid-associated population of D+ animals compared with D- animals. In contrast, PCR-denaturing gradient gel electrophoresis profiles of methanogens' methyl coenzyme-M reductase A gene showed a clear clustering in liquid-associated fractions for all three groups of donors but fewer differences in solid-associated fractions. These results show that the absence of protozoa may affect differently the methanogen community and methane emissions in wethers.

Influence of the diet on the microbial diversity of faecal and gastrointestinal contents in gilthead sea bream (Sparus aurata) and intestinal contents in goldfish (Carassius auratus)

Fish intestinal microbiota changes with the diet and this effect is of particular interest considering the increasing substitution of fish meal by plant protein sources. The objective of this work was to study the effects of partial substitution of fish meal with lupin and rapeseed meals on gut microbiota of the gilthead sea bream (Sparus aurata) and in goldfish (Carassius auratus). Faecal, gastrointestinal and intestinal contents were characterized using culture-based and molecular methods. Vibrionaceae was high in faeces and in the intestine of sea bream, while a more diverse microbiota was retrieved from the stomach, where Bacillales and Flavobacteriaceae appeared to be influenced by the diet. PCR-denaturing gradient gel electrophoresis profiles revealed a high diversity of the microbiota transiting in the sea bream digestive tract, with a shift between gastric and intestinal communities, especially in the group fed with lupin meal. The goldfish was different, with a predominance of Aeromonas spp., Shewanella putrefaciens and Staphylococcus spp. among the aerotolerant-cultivable bacteria. The culture-independent methods revealed the presence of anaerobes like Cetobacterium somerae, and that of Vibrio spp., likely in a viable, but noncultivable state. There was a trend towards decreasing diversity in goldfish microbiota with the partial substitution by lupin, which seemed to inhibit some taxa.

Evaluation of rumen methanogen diversity in cattle fed diets containing dry corn distillers grains and condensed tannins using PCR-DGGE and qRT-PCR analyses

Condensed tannins (CT) and corn distillers grains from ethanol production have the potential to reduce enteric CH4 production from ruminants because of their anti-microbial properties and high lipid contents, respectively. However, their effects on the community of rumen methanogens in cattle rumens have not been investigated. Our objective was to evaluate effects of dry corn distillers grain with solubles (DDG) and CT from Acacia mearnsii added to the diet of cattle on rumen methanogens using PCR denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real time (qRT)-PCR. Eight ruminally cannulated beef heifers were used in a replicated 4×4 Latin square design with four 35d periods. Diets were (g DDG dry matter [DM]/kg DM): control (Ctrl; 0 DDG), 200 DDG, 400 DDG and 400 DDG plus 25g CT (400 DDG+CT). Rumen digesta samples were collected before feeding on d 25 and 28 of each period from 4 sites in the rumen. Total DNA extracted from rumen digesta samples (i.e., 0.5ml liquid+0.5g solid pooled by heifer for each period) was used to generate amplicons of partial 16S rRNA genes. The PCR products obtained were subjected to PCR-DGGE. Detectable methanogenic PCR-DGGE profiles from periods 1 and 2 clustered while periods 3 and 4 formed a second cluster. A total of 25 distinct PCR-DGGE bands were identified, of which 16 bands were successfully cloned and sequenced. The sequences of 11 bands had 97–100% similarity with Methanobrevibacter sp., and this was supported by clustering of 10 of these bands with this species upon phylogenetic analysis of the sequences. The remaining bands clustered with Methanosphaera stadmanae. Although no effect of diet on total methanogenic PCR-DGGE profiles occurred, the proportion of bands 4, 5, 6, 7, 9 and 11 increased and bands 16 and 18 decreased with increased DDG in the diet. Bands 13 and 19 were in all diets except 400 DDG+CT. Quantitative RT-PCR analysis revealed that the copy numbers of total methanogenic 16S rRNA gene did not differ among diets. Findings from PCR-DGGE and qRT-PCR analysis suggest that inclusion of DDG or a mixture of DDG and CT altered the diversity of rumen methanogens without affecting total methanogen associated 16S rRNA copy numbers thereby indicating that copy numbers of particular species of methanogens may be required to explain the altered diversity of rumen methanogens.This article is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors; K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.

Microbial eco-physiological profiles to estimate the biological restoration of a trichloroethylene-contaminated soil

In the present study, the effectiveness of two plants, Medicago sativa L. and Dittrichia viscosa L., and a biostimulation method based on the use of an olive waste vermicompost, to restore the original quality of a trichloroethylene-contaminated soil was evaluated using eco-physiological profiles. These were designed in form of sun-ray plots by combining soil enzyme activities (dehydrogenase, alkaline phosphatase, β-glucosidase, urease and o-diphenol oxidase), bacterial population size via real-time PCR, Shannon diversity index values for PCR-denaturing gradient gel electrophoresis profiles, and genetic diversity θ (π) of the sequenced Proteobacteria of the different treatments. The eco-physiological profiles coupling biochemical and molecular parameters could be used as a valuable index for monitoring the success of a restoration scheme, estimating the quality of both contaminated and restored soils. Particularly remarkable was the interaction between vermicompost and D. viscosa, the only treatment that improved biochemical and microbiological restoration in such a way that an eco-physiological profile greater than that of the uncontaminated soil was noticed. The results showed the need to combine chemical analysis and microbiological measurements for evaluating the efficacy of soil remediation techniques.

Characterization of Variation in Rumen Methanogenic Communities under Different Dietary and Host Feed Efficiency Conditions, as Determined by PCR-Denaturing Gradient Gel Electrophoresis Analysis

Understanding ruminal methanogens is essential for greenhouse gas mitigation, as well as for improving animal performance in the livestock industry. It has been speculated that ruminal methanogenic diversity affects host feed efficiency and results in differences in methane production. This study examined methanogenic profiles in the rumen using culture-independent PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis for 56 beef cattle which differed in feed efficiency, as well as diet (the cattle were fed a low-energy diet or a high-energy diet). The methanogenic PCR-DGGE profiles detected were greatly affected by diet, and the major pattern changed from a community containing predominantly Methanobrevibacter ruminantium NT7 with the low-energy diet to a community containing predominantly Methanobrevibacter smithii, Methanobrevibacter sp. AbM4, and/or M. ruminantium NT7 with the high-energy diet. For each diet, the methanogenic PCR-DGGE pattern was strongly associated with the feed efficiency of the host. Diet-associated bands for Methanobrevibacter sp. AbM4 and M. smithii SM9 and a feed efficiency-related band for M. smithii PS were identified. The abundance of total methanogens was estimated by determining the numbers of copies of the 16S rRNA genes of methanogens. However, the size of the methanogen population did not correlate with differences in feed efficiency, diet, or metabolic measurements. Thus, the structure of the methanogenic community at the species or strain level may be more important for determining host feed efficiency under different dietary conditions.

Linkage of microbial ecology to phenotype: correlation of rumen microbial ecology to cattle's feed efficiency

Linkage of rumen microbial structure to host phenotypical traits may enhance the understanding of host-microbial interactions in livestock species. This study used culture-independent PCR-denaturing gradient gel electrophoresis (PCR-DGGE) to investigate the microbial profiles in the rumen of cattle differing in feed efficiency. The analysis of detectable bacterial PCR-DGGE profiles showed that the profiles generated from efficient steers clustered together and were clearly separated from those obtained from inefficient steers, indicating that specific bacterial groups may only inhabit in efficient steers. In addition, the bacterial profiles were more likely clustered within a certain breed, suggesting that host genetics may play an important role in rumen microbial structure. The correlations between the concentrations of volatile fatty acids and feed efficiency traits were also observed. Significantly higher concentrations of butyrate (P < 0.001) and valerate (P = 0.006) were detected in the efficient steers. Our results revealed potential associations between the detectable rumen microbiota and its fermentation parameters with the feed efficiency of cattle.

A preliminary study of the effect of probiotic Streptococcus salivarius K12 on oral malodour parameters

To determine whether dosing with bacteriocin-producing Streptococcus salivarius following an antimicrobial mouthwash effects a change in oral malodour parameters and in the composition of the oral microbiota of subjects with halitosis. Twenty-three subjects with halitosis undertook a 3-day regimen of chlorhexidine (CHX) mouth rinsing, followed at intervals by the use of lozenges containing either S. salivarius K12 or placebo. Assessment of the subjects' volatile sulphur compound (VSC) levels 1 week after treatment initiation showed that 85% of the K12-treated group and 30% of the placebo group had substantial (>100 ppb) reductions. The bacterial composition of the saliva was monitored by culture and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Changes in the PCR-DGGE profiles occurred in most subjects following K12 treatment. In vitro testing showed that S. salivarius K12 suppressed the growth of black-pigmented bacteria in saliva samples and also in various reference strains of bacteria implicated in halitosis. Administration of bacteriocin-producing S. salivarius after an oral antimicrobial mouthwash reduces oral VSC levels. The outcome of this preliminary study indicates that the replacement of bacteria implicated in halitosis by colonization with competitive bacteria such as S. salivarius K12 may provide an effective strategy to reduce the severity of halitosis.

Reduction of Colitis by Prebiotics in HLA-B27 Transgenic Rats Is Associated with Microflora Changes and Immunomodulation

HLA-B27 transgenic rats develop spontaneous colitis under specific pathogen-free conditions (SPF) but germ-free rats remain disease-free, emphasizing a role for intestinal bacteria in the pathogenesis of chronic intestinal inflammation. Prebiotics are dietary substances that affect the host by stimulating growth and/or activity of potentially health promoting bacteria. The aims of this study were to investigate whether prebiotics can prevent colitis in SPF HLA-B27 rats, and secondly, to explore mechanisms of protection. SPF HLA-B27 transgenic rats received orally the prebiotic combination long-chain inulin and oligofructose (Synergy 1), or not, prior to the development of clinically detectable colitis. After seven weeks, cecal and colonic tissues were collected for gross cecal scores (GCS), histologic inflammatory scores (scale 0-4), and mucosal cytokine measurement. Cecal and colonic contents were collected for analysis of the gut microbiota by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and fluorescent in-situ hybridization (FISH), and analysis of short-chain fatty acid composition. Prebiotic treatment significantly decreased GCS and inflammatory histologic scores in the cecum and colon. Prebiotic treatment also decreased cecal IL-1beta, but increased cecal TGF-beta concentrations. Inulin/oligofructose altered the cecal and colonic PCR-DGGE profiles, and FISH analysis showed significant increases in cecal Lactobacillus and Bifidobacterium populations after prebiotic treatment compared with water-treated rats. In conclusion, the prebiotic combination Synergy 1 reduced colitis in HLA-B27 transgenic rats, which effect was associated with alterations to the gut microbiota, decreased tissue proinflammatory cytokines and increased immunomodulatory molecules. These results show promise for prebiotics as primary or adjuvant maintenance therapy for chronic inflammatory bowel diseases.

Development of the intestinal bacterial composition in hospitalized preterm infants in comparison with breast-fed, full-term infants.

The establishment and succession of bacterial communities in hospitalized preterm infants has not been extensively studied. Because earlier studies depended on classical cultural techniques, their results were limited. This study monitored the establishment and succession of the neonatal microbiota in the first weeks of life by analyzing the 16S rDNA variety in fecal samples applying PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Fecal samples from 29 preterm infants hospitalized in a neonatal intensive care unit, including samples from antibiotic-treated infants and one with neonatal necrotizing enterocolitis, were subjected to PCR-DGGE analysis. Daily DGGE profiles from all preterm infants during the first 4 wk were obtained and analyzed. In addition, feces of 15 breast-fed, full-term infants and a variety of clinical bacterial isolates were examined and compared with the PCR-DGGE profiles of the preterm infants. During the first days of life, the DGGE profiles were rather simple but increased in their complexity over time. It became obvious that not only the intraindividual band-pattern similarity increased over time, but also the interindividual. During the observation period, similarity values (Cs) increased in each preterm infant from 0 to 80%, whereas interindividual Cs increased from 18.1 to 57.4%, revealing the acquisition of a highly similar bacterial community in these infants. In contrast, Cs-values obtained for breast-fed, full-term infants were rather low (11.2%). Escherichia coli, Enterococcus sp., and Klebsiella pneumoniae were the bacteria most commonly found in all preterm infants. The interindividual bacterial composition in hospitalized preterm infants is more similar in comparison with breast-fed, full-term infants and is not necessarily influenced by birth weight, diet, or antibiotic treatment.