Diversity of the Cronobacter Genus as Revealed by Multilocus Sequence Typing

Abstract

Cronobacter (previously known as Enterobacter sakazakii ) is a diverse bacterial genus consisting of seven species: C. sakazakii , C. malonaticus , C. turicensis , C. universalis , C. muytjensii , C. dublinensis , and C. condimenti . In this study, we have used a multilocus sequence typing (MLST) approach employing the alleles of 7 genes ( atpD , fusA , glnS , gltB , gyrB , infB , and ppsA ; total length, 3,036 bp) to investigate the phylogenetic relationship of 325 Cronobacter species isolates. Strains were chosen on the basis of their species, geographic and temporal distribution, source, and clinical outcome. The earliest strain was isolated from milk powder in 1950, and the earliest clinical strain was isolated in 1953. The existence of seven species was supported by MLST. Intraspecific variation ranged from low diversity in C. sakazakii to extensive diversity within some species, such as C. muytjensii and C. dublinensis , including evidence of gene conversion between species. The predominant species from clinical sources was found to be C. sakazakii. C. sakazakii sequence type 4 (ST4) was the predominant sequence type of cerebral spinal fluid isolates from cases of meningitis.

Lack of continuity between Cronobacter biotypes and species as determined using multilocus sequence typing

“…However, with more sophisticated and accurate DNA based methods available for typing, the dependency on the biotyping has declined and therefore removes the subjectivity involved in the reporting of the results of these phenotypic tests. [8] …”

“…However, some malonate utilizing strains (biotypes 8b & 8c) remained in the C. sakazakii species, hence causing some confusion in the use of phenotyping for speciation. This short communication reviews the limitations of the continued use of the biotyping scheme in the light of an improved understanding of the phylogeny of the Cronobacter genus as revealed by multilocus sequence typing (MLST), and supported by recent whole genome sequence analysis across the genus [8,9].…”

“…The PCR primers and conditions used for the MLST were as previously described [8]. Background information on the strains and their MLST details are available with open access at http://www.pubmlst.org/cronobacter.…”

“…The MLST scheme was recently used in a polyphasic study, but without reference to biotypes, to describe two novel species C. universalis and C. condimenti [1]. The MLST scheme has also revealed a high level of clonality within the genus, the most notable being the strong association of C. sakazakii clonal complex 4 (composed of seven sequence types) with cases of neonatal meningitis [3,8,11]. Phylogenetic analysis based on 14 whole genome sequences across the seven Cronobacter species supported the phylogeny generated by MLST [8,9].…”

“…To overcome the subjectivity of phenotyping and microheterogeneties in 16S rDNA sequence analysis, we established a 7-loci multilocus sequence typing (MLST) scheme for the Cronobacter genus [8,10]. The MLST scheme is hosted by the University of Oxford (UK) as an open access, curated database at http://www.pubmlst.org/cronobacter and currently consists of 136 defined sequence types (ST) [2].…”