P2-140 NGS Based Method for Enterobacteriaceae Discrimination and Reliability for Cronobacter spp. Identification

Tuesday, July 11, 2017
Exhibit Hall (Tampa Convention Center)
Sofia Nogueira , SGS Molecular , Lisboa , Portugal
Fan Mingzhen , Nestec Ltd, Nestlé Research Center , Lausanne , Switzerland
David Tomas Fornes , Nestlé , Lausanne , Switzerland
Sandra Chaves , SGS Molecular , Lisboa , Portugal
Introduction: : Enterobacteriaceae is a large bacterial family, including both harmless and pathogenic genera. Cronobacter spp. is a food-born pathogen associated with severe infections in premature infants. Over 90% of Cronobacter spp. infections in neonates are related to powdered infant formula (PIF). Presently, most sequencing methods used for bacteria identification are based on 16S rRNA gene which do not allow distinguishing among different genera within Enterobacteriaceae family. SGS Molecular developed a new method based on amplicon sequencing that allows Enterobacteriaceae differentiation in complex samples. 

Purpose: Apply the Enterobacteriaceae identification method for distinguishing Enterobacteriaceae strains, including Cronobacter spp. and close related non-Cronobacter genera in mixed samples.

Methods: A fusion-primer based method was developed, internally validated (validation was performed with 20 Enterobacteriaceae species and 19 mixtures) and adapted by SGS Molecular to become a beta version library preparation kit. The Cronobacter spp. study performed at Nestlé Research Center included 15 Cronobacter spp. isolated strains and 10 different mixtures of Cronobacter and non-Cronobacter spp. strains analised by triplicate. After preparation, libraries were sequenced using Ion torrent platform. Sequencing results were analyzed with a dedicated software developed by SGS Molecular.

Results: All the Enterobacteriaceae species tested during the method’s validation could be correctly identified. Considering Cronobacter spp. evaluation, all isolated strains were properly identified and in all mixtures Cronobacter spp. could be distinguished from non-Cronobacter spp. strains. With some few exceptions, the Enterobacteriaceae strains analyzed together with Cronobacter spp. were properly identified.

Significance: Although some improvements must be performed to this workflow, results obtained suggest that SGS Molecular’s kit for Enterobacteriaceae identification allows the distinction of species within this bacterial group, as well as the differentiation of Cronobacter spp. from non-Cronobacter spp. strains in mixed populations