16S rRNA gene based analysis of Enterobacter sakazakii strains from different sources and development of a PCR assay for identification

<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p><jats:italic>E. sakazakii</jats:italic> is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for <jats:italic>E. sakazakii</jats:italic> include the observation of yellow pigmentation of colonies and a positive "Equation missing"glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for <jats:italic>E. sakazakii</jats:italic> identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen <jats:italic>E. sakazakii</jats:italic> strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for <jats:italic>E. sakazakii</jats:italic> was developed and evaluated.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>By phylogenetic analysis of the new full-length 16S rRNA gene sequence data obtained we could show the presence of a second phylogenetic distinct lineage within the <jats:italic>E. sakazakii</jats:italic> species. The newly developed 16S rRNA gene targeting PCR system allows identification of <jats:italic>E. sakazakii</jats:italic> strains from both lineages. The assay's ability to correctly identify different <jats:italic>E. sakazakii</jats:italic> isolates as well as to differentiate <jats:italic>E. sakazakii</jats:italic> from other closely related <jats:italic>Enterobacteriaceae</jats:italic> species and other microorganisms was shown on 75 target and non-target strains.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>By this study we are presenting a specific and reliable PCR identification system, which is able to correctly identify <jats:italic>E. sakazakii</jats:italic> isolates from both phylogenetic distinct lines within the <jats:italic>E. sakazakii</jats:italic> species. The impact of this second newly described phylogenetic line within the <jats:italic>E. sakazakii</jats:italic> species in view of clinical and food safety aspects need further investigation.</jats:p> </jats:sec>