rDNA Internal Spacer Analysis Research Articles

Potential use of molecular-typing methods for the identification and characterization of salmonella enterica serotypes isolated in the poultry production chain

Salmonella is widespread in nature and can be found in all links of the poultry production chain. Due to its high impact on meat processing, techniques for the rapid detection and reproducible characterization of Salmonella serotypes in foods are needed. The present study investigated the potential of molecular profiling to identify and differentiate 15 Salmonella serotypes isolated from the poultry production chain, based on 5 primers by random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC-PCR), amplification of rDNA internal spacer analysis (RISA), and amplified ribosomal DNA restriction analysis (ARDRA) of 16S-23S rRNA internal spacer region (ISR) cleaved with Alu I and Hha I restriction enzymes. Three isolates of each serotype were analyzed for the identification of similar and different profiles. Dendrograms were constructed from molecular profiles using the UPGMA method (unweighted pair-group method for the arithmetic averages) and the software program WinBoot. The present study indicates the usefulness of RISA and ARDRA of the 16S-23S rRNA intergenic spacer region (ISR) for systematic, epidemiological, and diagnostic purposes. Since these techniques can be used for the differentiation of serotypes, they are highly promising for the characterization of Salmonella serotypes and intra-serotypes. Data indicate that these techniques may be used to produce more consistent, reliable, and reproducible results in the identification and epidemiological study (traceability) of Salmonella in the poultry industry.

Effects of municipal solid waste compost amendments on soil enzyme activities and bacterial genetic diversity

Municipal solid waste (MSW) composts have been used to maintain the long-term productivity of agroecosystems and to protect the soil environment from overcropping, changes in climatic conditions and inadequate management; they also have the additional benefit of reducing waste disposal costs. Since MSW may contain heavy metals and other toxic compounds, amendments cannot only influence soil fertility, but may also affect the composition and activity of soil microorganisms. The effects of MSW compost and mineral N amendments in a 6-year field trial on some physical–chemical properties, enzyme activities and bacterial genetic diversity of cropped plots ( Beta vulgaris– Triticum turgidum rotation) and uncropped plots were investigated. The compost was added at the recommended and twice the recommended dosage (12, 24 t ha −1). Amendments of cropped plots with MSW compost increased the contents of organic C from 13.3 to 15.0 g kg −1 soil and total N from 1.55 to 1.65 g kg −1 soil. There were significant increases in dehydrogenase (9.6%), β-glucosidase (13.5%), urease (15.4%), nitrate reductase (21.4%) and phosphatase (9.7%) activities. A significant reduction in protease activity (from 3.6 to 2.8 U g −1 soil) was measured when a double dose of compost was added to the cropped plots. No dosage effect was detected for the other enzymes. Changes in the microbial community, as a consequence of MSW amendment, were minimal as determined using denaturing gradient gel electrophoresis, rDNA internal spacer analysis and amplified ribosomal DNA restriction analysis of bacteria, archaea, actinomycetes, and ammonia oxidizers. This indicates that there was no significant variation in the overall bacterial communities nor in selected taxonomic groups deemed to be essential for soil fertility.

Use of the 16S–23S ribosomal genes spacer region in studies of prokaryotic diversity

The description of microbial diversity by molecular culture-independent techniques most often involves the amplification of the 16S rRNA by PCR gene and either analysis of the diversity of amplified molecules (community fingerprinting) that allows the simultaneous study of many samples or the cloning and sequencing of a significant amount of amplification products. The fact that between the 16S and the 23S genes in the ribosomal operon there is a spacer extremely variable in both sequence and length provides an excellent tool to simplify both approaches. The spacer can be amplified almost as easily as the 16S rDNA taking advantage of conserved nucleotide stretches at the 5′ end of the 23S gene and the amplicon can contain different amounts of the 16S rDNA choosing primers at the different conserved areas within this gene. Identified by the acronym RISA (rDNA internal spacer analysis), the spacer addition provides a marker of highly variable size allowing standard separation of the amplification products and the sequence of this hypervariable region is useful in the fine discrimination of operational taxonomic units.