Rapid and reasonable molecular identification of bacteria and fungi in microbiological diagnostics using rapid real-time PCR and Sanger sequencing

Analyses of short subunit gene sequences have been established for taxonomic classification and identification of bacteria and fungi. To produce partial bacterial ribosomal 16S rRNA and rpoB and fungal ribosomal ITS/LSU gene sequences for DNA sequencing, real-time PCR assays supplemented with the nucleic acid stain SYBR Green were created. Generation of PCR products was monitored based on amplification and melting curves. The PCR products were subsequently subjected to Sanger sequencing on demand for identification of bacteria and fungi in routine microbiological diagnostics within a period of two days. From a total of 78 bacterial isolates 40 (51%) or 67 (86%) could be identified at species level using only partial 16S rRNA or additionally rpoB gene sequences based on BLASTN (NCBI) database queries, respectively. Using partial 16S rRNA and rpoB gene sequencing unambiguous assignment was not possible for the closely related species of the Bacillus (B.) cereus group, Bordetella (B.) pertussis/B. parapertussis/B. bronchiseptica, Brucella spp., Enterobacter cloacae complex, Escherichia/Shigella spp., Staphylococcus (S.) hyicus/S. agnetis and Yersinia (Y.) pseudotuberculosis/Y. pestis. However, partial rpoB gene sequencing succeeded in identifying 27 bacterial isolates at species level in addition to 16S rRNA gene sequencing. Regarding ITS/LSU gene sequencing, best results could be achieved by ITS gene sequencing followed by LSU gene sequencing, resulting in 32 (63%) and 21 (43%) of a total of 51 fungal isolates that could be identified at species level, respectively. Insufficient identification at species level was observed for the genera Apiotrichum, Aspergillus, Cladosporium, Cryptococcus, Microsporum, Nannizziopsis, Penicillium, Trichosporon, and Tolypocladium included in this study. The concept of this procedure is suitable for rapid and reasonable molecular identification of bacteria and fungi within two days and is therefore applicable in routine microbiological diagnostic laboratories.