Polymerase Chain Reaction-based restriction fragment length polymorphism analysis of the 16S-23S ribosomal genes spacer region in sphingomonads. (c2008)

Abstract

The ability of sphingomonads present in drinking water to cause community and hospital acquired opportunistic infections has raised the need to establish rapid, reproducible, and feasible assays that could screen and identify sphingomonads to the genus, species and subspecies level. In this study, a total of 129 samples with yellow-orange- pigmented colonies were isolated from drinking water in Lebanon and were divided into 10 biotypes based on colony morphology. PCR-RFLP analysis of the 16S-23S ITS was done on 18 ATCC reference strains and 20 sphingomonas-specific 16S rRNA gene PCR positive isolates representing the ten biotypes to investigate the level of 16S-23S ITS (Intergenic Transcribed Spacer) region polymorphism and thus its ability to discriminate between the different sphingomonads. The first step was PCR amplification of the ITS, using two universal primers that target the constant regions flanking the ITS. This was followed by RFLP (restriction fragment length polymorphism) of the amplified ITS using 3 restriction endonucleases: Hinfl, AiuI, and Bsp1431., ITS size ranged between 400-11 00 bp and was not variable enough to differentiate between Sphingomonas, Sphingobium, Nousphirngobium, and Sphingopuxix. However, analysis of restriction products revealed differences in the number and size of bands obtained. Sixteen distinct banding patterns were recognized among the reference strains and twelve among the drinking water isolates. Each of the four genera had a unique and reproducible restriction pattern, but were also variable among species of the same genus, and strains of the same species. Several isolates had restriction patterns that were similar to those generated by the reference strains, and isolates having the same colony morphology generated the same restriction pattern. This study revealed that the ITS PCR-RFLP is a rapid technique that can be used to generate molecular fingerprints of sphingomonads. To the extent of our knowledge, this study is the first comprehensive record of the different 16S-23S ITS sizes that can be found in the four major sphingomonad genera (Sphingomonas, Sphingobium, Nousphirngobium, and Sphingopuxix ). Moreover, this is the first study that describes the use of 16S-23S ITS PCR-RFLP for subtyping the different sphingomonad species. However, employing a polyphasic approach, based on both biotyping and molecular fingerprinting, proved efficient in overcoming problems usually faced when attempting to identify organisms especially those of environmental origin.