Defining Reference Sequences for Nocardia Species by Similarity and Clustering Analyses of 16S rRNA Gene Sequence Data

International audienceBACKGROUND: The intra- and inter-species genetic diversity of bacteria and the absence of 'reference', or the most representative, sequences of individual species present a significant challenge for sequence-based identification. The aims of this study were to determine the utility, and compare the performance of several clustering and classification algorithms to identify the species of 364 sequences of 16S rRNA gene with a defined species in GenBank, and 110 sequences of 16S rRNA gene with no defined species, all within the genus Nocardia. METHODS: A total of 364 16S rRNA gene sequences of Nocardia species were studied. In addition, 110 16S rRNA gene sequences assigned only to the Nocardia genus level at the time of submission to GenBank were used for machine learning classification experiments. Different clustering algorithms were compared with a novel algorithm or the linear mapping (LM) of the distance matrix. Principal Components Analysis was used for the dimensionality reduction and visualization. RESULTS: The LM algorithm achieved the highest performance and classified the set of 364 16S rRNA sequences into 80 clusters, the majority of which (83.52%) corresponded with the original species. The most representative 16S rRNA sequences for individual Nocardia species have been identified as 'centroids' in respective clusters from which the distances to all other sequences were minimized; 110 16S rRNA gene sequences with identifications recorded only at the genus level were classified using machine learning methods. Simple kNN machine learning demonstrated the highest performance and classified Nocardia species sequences with an accuracy of 92.7% and a mean frequency of 0.578. CONCLUSION: The identification of centroids of 16S rRNA gene sequence clusters using novel distance matrix clustering enables the identification of the most representative sequences for each individual species of Nocardia and allows the quantitation of inter- and intra-species variability

Diversity analysis of Streptomycetes and associated phosphotranspherase genes in soil

An attempt was made to verify the observation that Streptomyces griseus was prevalent in soil based on isolation work. A genus-specific PCR was developed for Streptomyces based on the housekeeping gene atpD and used to investigate species diversity within selected soils. The presence of S. griseus was investigated to determine coexistence of resistance-only streptomycin phosphotransferase (strA) in the same soil as streptomycin producers. Two additional PCR-based assays were developed; one specific for strA in association with production, the other for more diverse strA and other related phosphotranferases. Both the S. griseus atpD and strA genes were below the PCR detection limit in all soils examined. A number of more diverse phosphotransferase genes were amplified, a minority of which may be associated with streptomycin production. We conclude that neither streptomycin producers nor S. griseus are prevalent in the fresh or chitin and starch-amended soils examined (less than 0.1% of soil actinobacteria). One of the soil sites had received plantomycin (active ingredient: streptomycin) and diversity studies suggested that this altered the streptomycete populations present in the soil

Effects of lamb sucking on the bacterial flora of teat duct and mammary gland of ewes

We aimed (i) to determine differences in bacterial flora of teat duct and mammary gland of ewes before and after suckling, (ii) to evaluate factors potentially affecting those. We collected samples of teat duct material and mammary secretion from 11 ewes immediately before and after sucking by lambs, as well as 120 min later. We processed samples bacteriologically and compared changes in infection by the Sign Test. We isolated bacteria from 3.5% duct and 1.5% secretion samples before suckling. Respective figures post-suckling were 10.6% and 2.0%, and 120 min later 6.8% and 1.5%. We recorded differences in infection of duct samples before and after suckling in 40 cases; bacteria were isolated before suckling from six samples, whereas after it from 34 (p < 0.001). Also, we recorded differences in samples collected after suckling and 120 min later in 12 cases; bacteria were isolated immediately post-suckling from eight samples, whereas 120 min later from four (p = 0.375). No significant changes were seen for secretion. We found neither difference between ewes with single or twin lambs, nor among stages of lactation. Mostly, we isolated staphylococci: 70% of isolates before suckling, 80% of isolates after it, 91% of isolates 120 min later. After suckling we also isolated two Mannheimia haemolytica strains. Suckling predisposes to entrance of bacteria into the teat; however, increased teat infections did not result in mammary infections. Isolation of M. haemolytica post-suckling indicates that lambs act as source of infection for this pathogen