Background

Terminal-Restriction Fragment Length Polymorphism (T-RFLP) is a technique used to analyze complex microbial communities. It allows for the quantification of unique or numerically dominant phylotypes in amplicon pools and it has been used primarily for comparisons between different communities. T-RFPred, Terminal-Restriction Fragment Prediction, was developed to identify and assign taxonomic information to chromatogram peaks of a T-RFLP fingerprint for a more comprehensive description of microbial communities. The program estimates the expected fragment size of representative 16S rRNA gene sequences (either from a complementary clone library or from public databases) for a given primer and restriction enzyme(s) and provides candidate taxonomic assignments.

Results

To show the accuracy of the program, T-RFLP profiles of a marine bacterial community were described using artificial bacterioplankton clone libraries of sequences obtained from public databases. For all valid chromatogram peaks, a phylogenetic group could be assigned.

Conclusions

T-RFPred offers enhanced functionality of T-RFLP profile analysis over current available programs. In particular, it circumvents the need for full-length 16S rRNA gene sequences during taxonomic assignments of T-RF peaks. Thus, large 16S rRNA gene datasets from environmental studies, including metagenomes, or public databases can be used as the reference set. Furthermore, T-RFPred is useful in experimental design for the selection of primers as well as the type and number of restriction enzymes that will yield informative chromatograms from natural microbial communities.

Background

Group A rotaviruses are the most common cause of severe diarrhea in infants and children worldwide and continue to have a major global impact on childhood morbidity and mortality. In recent years, considerable research efforts have been devoted to the development of two new live, orally administered vaccines. Although both vaccines have proven to confer a good protection against severe rotavirus gastroenteritis, these vaccines will have to be screened and may have to be updated regularly to reflect temporal and spatial genotype fluctuations. In this matter, the genetic characterization of circulating and new emerging rotavirus strains will need to be compulsory and accurate. An extended classification system for rotaviruses in which all the 11 genomic RNA segments are used, has been proposed recently. The use of this classification system will help to elucidate the role of gene reassortments in the generation of genetic diversity, host range restriction, co-segregation of certain gene segments, and in adaptation to a new host species.

Results

Here we present a web-based tool that can be used for fast rotavirus genotype differentiation of all 11 group A rotavirus gene segments according to the new guidelines proposed by the Rotavirus Classification Working Group (RCWG).

Conclusion

With the increasing sequencing efforts that are being conducted around the world to unravel complete rotavirus genomes of human and animal origin, this tool will be of great help to analyze and correctly classify the large amount of new data. The web-based tool is freely available at http://rotac.regatools.be.

Background

The increasing number of genomic sequences of bacteria makes it possible to select unique SNPs of a particular strain/species at the whole genome level and thus design specific primers based on the SNPs. The high similarity of genomic sequences among phylogenetically-related bacteria requires the identification of the few loci in the genome that can serve as unique markers for strain differentiation. PrimerSNP attempts to identify reliable strain-specific markers, on which specific primers are designed for pathogen detection purpose.

Results

PrimerSNP is an online tool to design primers based on strain specific SNPs for multiple strains/species of microorganisms at the whole genome level. The allele-specific primers could distinguish query sequences of one strain from other homologous sequences by standard PCR reaction. Additionally, PrimerSNP provides a feature for designing common primers that can amplify all the homologous sequences of multiple strains/species of microorganisms. PrimerSNP is freely available at .

Conclusion

PrimerSNP is a high-throughput specific primer generation tool for the differentiation of phylogenetically-related strains/species. Experimental validation showed that this software had a successful prediction rate of 80.4 – 100% for strain specific primer design.

Background

Simple computerized methods that analyse variability along alignments of nucleotide or amino acid sequences can be very useful in a clinical microbiology laboratory for two main purposes. First, to optimize primer selection, which is critical for the identification of infectious pathogens based on gene sequencing: primers must target conserved nucleotide regions bordering highly variable areas to ensure discrimination of species. Second, it can be of interest to reveal mutations associated with drug resistance of pathogen agents. Our aim was therefore to test easy and cost-free tools (SVARAP and aSVARAP) that require short hands-on work, little expertise, and which allow visual interpretation and statistical analysis of results.

Results

We first tested SVARAP to improve a strategy of identification of streptococci species of the Viridans Group targeting the groESL gene. Two regions with <500 nucleotides were identified, one being significantly more discriminant than one of a similar length used in a previous study (mean number of nucleotide differences between species, 113 (range: 12–193) vs. 77 (range: 14–109); p < 10-3). Secondly, aSVARAP was tested on reverse transcriptase (RT) sequences from 129 HIV-1 clinical strains to identify natural polymorphisms and drug-selected mutations emerging under nucleoside RT inhibitor (NRTI)-selective pressure. It revealed eleven of the 18 RT mutations considered in a reference HIV-1 genotypic NRTI-resistance interpretation algorithm.

Conclusion

SVARAP and aSVARAP are simple, versatile and helpful tools for analysis of sequence variability, and are currently being used in real practice in our clinical microbiology laboratory.

Background

Pseudomonas aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). With chronicity of infection, the organism resides as a biofilm, shows multi-drug resistance, diversifies its colony morphology and becomes auxotrophic. The patients have been found to be colonized with multiple genotypes. The present work was carried out to characterize P. aeruginosa isolated from children with cystic fibrosis using phenotypic and genotypic methods.

Results

We studied 56 patients with CF attending the Pediatric Chest clinic at All India Institute of Medical Sciences, New Delhi, India during August 1998-August 2001. These patients were regularly followed up at the clinic. Out of 56 patients, 27 were culture positive for P. aeruginosa where 8 were chronically infected (Group1) and 19 were intermittently colonized with the organism (Group2). Patients under Group1 had significantly higher rates of hospitalization, death and colonization with different colony morphotypes (p < 0.05). The isolates from Group1 patients were the positive producers of extended spectrum beta lactamase. A total of 5 auxotrophs were recovered from 2 patients where one was chronically infected with P. aeruginosa and the other was a recently enrolled patient. The auxotrophs had the specific requirement for methionine and arginine. Molecular typing revealed 33 ERIC-PCR (E1-E33) and 5 PCR-ribotyping (P1-P5) patterns. By ERIC-PCR, 4 patients were colonized with 2–4 genotypes and the remaining 23 patients were colonized with the single genotype.

Conclusion

With chronicity of infection, P. aeruginosa becomes multidrug resistant, diversifies its colony morphology, acquires mucoidity and shows auxotrophy for amino acids. The chronically infected patients can be colonized with multiple genotypes. Thus in a particular clinical set up, high index of suspicion should be there for diagnosis of CF patients so as to prevent the delay in diagnosis and management of CF patients.