A Novel Method That Allows SNP Discrimination with 160:1 Ratio for Biosensors Based on DNA-DNA Hybridization

Highly sensitive (high SBR) and highly specific (high SNP discrimination ratio) DNA hybridization is essential for a biosensor with clinical application. Herein, we propose a method that allows detecting multiple pathogens on a single platform with the SNP discrimination ratios over 160:1 in the dynamic range of 101 to 104 copies per test. The newly developed SWAT method allows achieving highly sensitive and highly specific DNA hybridizations. The detection and discrimination of the MTB and NTM strain in the clinical samples with the SBR and SNP discrimination ratios higher than 160:1 indicate the high clinical applicability of the SWAT.

Sphingomonas laterariae sp. nov., isolated from a hexachlorocyclohexane-contaminated dump site

A Gram-staining-negative, non-motile, cream-coloured and rod-shaped bacterium, designated strain LNB2T, was isolated from a hexachlorocyclohexane-contaminated dump site in the village of Ummari, in northern India. The taxonomic position of the novel strain was investigated by using a polyphasic approach. In a phylogenetic analysis based on 16S rRNA gene sequences, strain LNB2T appeared to be most closely related to Sphingomonas haloaromaticamans A175T (98.0 % sequence similarity) and Sphingomonas histidinilytica UM2T (97.3 %). In DNA–DNA hybridizations, the levels of DNA–DNA relatedness between the novel strain and S. haloaromaticamans A175T and S. histidinilytica UM2T were found to be low (8.6 % and 5.6 %, respectively). The genomic DNA G+C content of strain LNB2T was 61.0 mol%. The novel strain’s predominant fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C14 : 0 2-OH, C17 : 1ω6c and 11-methyl C18 : 1ω7c. The major ubiquinone was Q-10, the predominant polyamine was homospermidine, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, sphingoglycolipid, phosphatidylethanolamine and phosphatidyldimethylethanolamine. Based on the phylogenetic, biochemical and chemotaxonomic evidence and the results of the DNA–DNA hybridizations, strain LNB2T represents a novel species of the genus Sphingomonas , for which the name Sphingomonas laterariae sp. nov. is proposed. The type strain is LNB2T ( = MTCC 10873T = CCM 7880T = DSM 25432T).

Arcobacter trophiarum sp. nov., isolated from fattening pigs

In the course of a longitudinal study elucidating the dynamics of Arcobacter populations in pigs, 16 isolates of Gram-reaction-negative, rod-shaped, slightly curved, non-spore-forming bacteria were grouped by amplified fragment length polymorphism analysis into a distinct phenon within the genus Arcobacter. Fragments were generated for all isolates in a genus-specific PCR assay, but no amplicon was obtained in a species-specific multiplex-PCR test. Numerical analysis of the whole-cell protein profiles also showed that all isolates clustered in a single group that was distinct from related members of the genus Arcobacter. DNA–DNA hybridizations between two representative strains, designated 64T and 122, of the isolates obtained exhibited a mean DNA–DNA relatedness of 72 %. DNA–DNA hybridizations between strains 64T and 122 and reference strains of other animal-related bacteria of the genus Arcobacter revealed binding values of 47 % or less. The DNA G+C contents of the two representative strains were 28.5 and 28.4 mol%, respectively, and analysis of three marker genes identified Arcobacter cryaerophilus, A. thereius, A. cibarius and A. skirrowii as their closest phylogenetic neighbours. Strains 64T and 122 could be distinguished from other members of the genus Arcobacter by means of biochemical tests for catalase and urease activities, nitrate reduction, indoxyl acetate hydrolysis, lack of growth at 37 °C, growth in 2 % (w/v) NaCl, growth on 0.1 % sodium deoxycholate and non-supplemented Campylobacter charcoal-deoxycholate base medium and resistance to cephalothin (32 mg l−1) and cefoperazone (64 mg l−1). Additionally, a PCR assay was developed for the detection and identification of strains 64T and 122, which represent a novel species of the genus Arcobacter, for which the name Arcobacter trophiarum sp. nov. is proposed. The type strain is strain 64T (=LMG 25534T =CCUG 59229T).