Denaturing Gradient Gel Electrophoresis Technology Research Articles

Evaluating the qualities of probiotic products by a DGGE-based procedure

The quality of the probiotics is determined by the microbiological composition and can thus be verified by analyzing this characteristic. The use of the Denaturing Gradient Gel Electrophoresis (DGGE) allows the detection of each microorganism through the corresponding band on the gel, which can be considered as the "bar code" of that microorganism. This "bar code” principle demonstrates the potential for the application of DGGE technology to test microbial probiotics with or without a bacterium as registered to assess whether they are qualified. As such, in this study, we experiment the application of DGGE method to test 10 commercial probiotic products, with a focus on products consisting of only bacteria which dominate the market. First, we performed DNA genome extraction and amplification of 550 bp DNA of the 16S rDNA by PCR. Then, the amplicon was analysed on polyacrylamide gels containing a 30% to 60% linear denaturing gradient of urea and formamide. Microbial composition of 7 of the 10 products showed a strong correlation between DGGE results with product labeling as well as isolation of micoorganism. This study thus demonstrates the potential application of DGGE technology in evaluating the qualities of probiotic products.

Polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE): A promising tool to diagnose bacterial infections in diabetic foot ulcers

AimThe diagnosis of diabetic foot infections is difficult due to limitations of conventional culture-based techniques. The objective of this study was to evaluate the contribution of denaturing gradient gel electrophoresis (DGGE) in the microbiological diagnosis of diabetic foot ulcers in comparison to conventional techniques, and also to evaluate the need to perform a biopsy sample for this diagnosis. MethodsTwenty diabetic patients (types 1 and 2) with foot ulcers (grades 1–4) were included. After debridement of their wounds, samples were taken in duplicate by surface swabbing and deep-tissue biopsy. The samples were analyzed by conventional culture and by a new molecular biology tool, DGGE technology. ResultsPolymerase chain reaction (PCR)–DGGE led to the identification of more bacteria than did conventional cultures (mean: 2.35 vs 0.80, respectively). In 11 cases, the technology detected pathogenic species not isolated by classical cultures. PCR–DGGE also identified significantly more pathogenic species at deep levels compared with species detected at superficial levels (87% vs 58%, respectively; P=0.03). In 9/20 cases, pathogenic bacteria were detected only in deep samples, revealing the need to perform tissue biopsy sampling. ConclusionDGGE, achievable in 48h, could be a useful technique for the bacteriological diagnosis of diabetic foot infections. It may help to identify pathogenic bacteria in deeply infected ulcers, thereby contributing to a more appropriate use of antibiotics.

Distribution of Endophytic Bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. from Soils Contaminated by Polycyclic Aromatic Hydrocarbons

The distributions of endophytic bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. grown in soils contaminated with different levels of polycyclic aromatic hydrocarbons (PAHs) were investigated with polymerase chain reaction followed by denaturing gradient gel electrophoresis technology (PCR-DGGE) and cultivation methods. Twelve types of PAHs, at concentrations varying from 0.16 to 180 mg·kg−1, were observed in the roots and shoots of the two plants. The total PAH concentrations in Alopecurus aequalis Sobol obtained from three different PAH-contaminated stations were 184, 197, and 304 mg·kg−1, and the total PAH concentrations in Oxalis corniculata L. were 251, 346, and 600 mg·kg−1, respectively. The PCR-DGGE results showed that the endophytic bacterial communities in the roots and shoots of the two plants were quite different, although most bacteria belonged to Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. A total of 68 endophytic bacterial strains were isolated from different tissues of the two plants and classified into three phyla: Firmicutes, Proteobacteria and Bacteroidetes. In both plants, Bacillus spp. and Pseudomonas spp. were the dominant cultivable populations. With an increase in the PAH pollution level, the diversity and distribution of endophytic bacteria in the two plants changed correspondingly, and the number of cultivable endophytic bacterial strains decreased rapidly. Testing of the isolated endophytic bacteria for tolerance to each type of PAH showed that most isolates could grow well on Luria-Bertani media in the presence of different PAHs, and some isolates were able to grow rapidly on a mineral salt medium with a single PAH as the sole carbon and energy source, indicating that these strains may have the potential to degrade PAHs in plants. This research provides the first insight into the characteristics of endophytic bacterial populations under different PAH pollution levels and provides a species resource for the isolation of PAH-degrading endophytic bacteria.

A novel integrated step-feed biofilm process for the treatment of decentralized domestic wastewater in rural areas of China

For wastewater treatment in rural areas, a novel three-stage step-feed wastewater treatment system, combined with a drop-aeration biofilm process, was tested in the laboratory to investigate its performance in removing suspended solids (SS), chemical oxygen demand (COD), NH 4 +-N, total nitrogen (TN), and total phosphorus (TP). The removal rates of SS, COD and NH 4 +-N were 90%, 80%, and 90% in effluent concentrations less than 10 mg/L, 50 mg/Land8mg/L, respectively. The TP removal rate was less satisfactory. The C/N ratio in the raw wastewater was often less than 3.5, and the removal efficiency of TN was therefore limited. A carbon-release batch experiment was carried out to measure the feasibility of enhancing denitrification at low influent C/N ratios. The result showed that the C/N could be over 9.0 in the supernatant. Polymerase chain reaction denaturing gradient gel electrophoresis technology was used to reveal the changes in the bacterial community during different stages of the integrated step-feed biofilm process. The results showed that banding patterns and the distribution of dominant bands for the same experimental period in different aerobic zones were similar. Phylogenetic analysis indicated that lanes 10, 11 and 12, which presented three aerobic zones at the same operation period, had the closest phylogenetic relationship among the lanes.

Analysis of bacterial communities on aging flue-cured tobacco leaves by 16S rDNA PCR–DGGE technology

Many microorganisms, growing on aging flue-cured tobacco leaves, play a part in its fermentation process. These microflora were identified and described by culture-dependent methods earlier. In this study we report the identity of the microflora growing on the tobacco leaf surface by employing culture-independent methods. We have amplified microbial 16S rDNA sequences directly from the leaf surface and used denaturing gradient gel electrophoresis (DGGE) to identify bacterial community on the tobacco leaves. Our culture-independent methods for the study of microbial community on tobacco leaves showed that microbial community structures on leaves of variety Zhongyan 100, NC89 and Zhongyan 101 were similar between 0 and 6 months aging, and between 9 and 12 months aging, while the similarity is low between 0 and 6, and between 9 and 12 months aging, respectively. There were certain similarities of bacterial communities (similarity up to 63%) among the three tobacco varieties for 0 to 6 months aging. Five dominant 16S rDNA DGGE bands A, B, C, D and E were isolated, cloned, and sequenced. They were most similar to two cultured microbial species Bacteriovorax sp. EPC3, Bacillus megaterium, and three uncultured microbial species, respectively.

Evaluation of Freeze-Dried Kefir Coculture as Starter in Feta-Type Cheese Production

The use of freeze-dried kefir coculture as a starter in the production of feta-type cheese was investigated. Maturation of the produced cheese at 4 degrees C was monitored for up to 70 days, and the effects of the starter culture, the salting method, and the ripening process on quality characteristics were studied. The use of kefir coculture as a starter led to increased lactic acid concentrations and decreased pH values in the final product associated with significantly higher conversion rates compared to salted rennet cheese. Determination of bacterial diversity at the end of the ripening process in salted kefir and rennet cheeses by denaturing gradient gel electrophoresis technology, based on both DNA and RNA analyses, suggested a potential species-specific inhibition of members of the genera Staphylococcus and Psychrobacter by kefir coculture. The main active microbial associations in salted kefir cheese appeared to be members of the genera Pseudomonas and Lactococcus, while in salted rennet cheese, Oxalobacteraceae, Janthinobacterium, Psychrobacter, and Pseudomonas species were noted. The effect of the starter culture on the production of aroma-related compounds responsible for cheese flavor was also studied by the solid-phase microextraction-gas chromatography-mass spectrometry technique. Kefir coculture also appeared to extend the shelf life of unsalted cheese. Spoilage of kefir cheese was observed on the 9th and 20th days of preservation at 10 and 5 degrees C, respectively, while spoilage in the corresponding rennet cheese was detected on the 7th and 16th days. Microbial counts during preservation of both types of unsalted cheese increased steadily and reached similar levels, with the exception of staphylococci, which were significantly lower in unsalted kefir cheese. All types of cheese produced with kefir as a starter were approved and accepted by the panel during the preliminary sensory evaluation compared to commercial feta-type cheese.