Identification of Three related genera salmonella citrobacter and proteus using API 20E, 16S-23S rDNA intergenic transcribed spacer fingerprinting, and 16S rDNA sequencing.

Abstract

A total of 176 presumptive Salmonella strains isolated from different sources (food, environment, veterinary, and human) were analyzed by API 20E, serotyping, ITSF/ITSR-PCR and MINf/MINr-PCR. On the other hand, the 16S-23S rRNA intergenic transcribed spacer region was used to aid in their identification. Test results were confirmed by 16S rRNA sequencing. Of 176 suspected S. enterica isolates, API20E identified 105 (59.6%) as S. enterica, 42 (24%) as Citrobacter and 29 (16.4%) as Proteus. A total of 11 serotypes were found among 102 out of 105 Salmonella isolates. The remaining isolates were classified as untypeable by serotyping. When ITSF/ITSR-PCR and MINf/MINr-PCR were used, 101 out of 105 Salmonella isolates tested generated PCR products.Amplification of the spacer region between 16S-23S rRNA gene allowed discrimination of genera and facilitated species identification. Sequencing of 16S rRNA gene has identified 4 different species in proteus genus (P. mirabilis, P. penneri, P. vulgaris, and P. hausseri), 3 different species in Citrobacter genus (Citrobacter freundii, Citrobacter braakii, andCitrobacter youngae), and Salmonella enterica sub sp. enterica for all Salmonella isolates.The four suspect Salmonella isolates were definitively classified in Citrobacter genus. In conclusion, this study has demonstrated that API20E, serotyping associated toSalmonella specific PCR are accurate methods for S. enterica detection. In addition, PCR amplification of the 16S-23S spacer region was suitable tool for identification of bacteria at genera and species level. Key words: Salmonella, API 20E, serotyping, MINf/MINr-PCR, ITSF/ITSR-PCR, 16S-23S intergenic spacer regions (ITS)-PCR, 16S rRNA.