A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii (strain 1330^T, isolated from spiced meat purchased in Slovakia), two previously assigned to Cronobacter genomospecies 1 (strains NCTC 9529^T and 731, isolated from water and a leg infection, respectively) and two previously assigned to Cronobacter turicensis (strains 96 and 1435, isolated from onion powder and rye flour, respectively) was carried out. The analysis included phenotypic characterization, 16S rRNA gene sequencing and multilocus sequence analysis (MLSA) of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3036 bp). 16S rRNA gene sequence analysis and MLSA showed that strain 1330^T formed an independent phylogenetic lineage in the MLSA, with Cronobacter dublinensis LMG 23823^T as the closest neighbour. DNA–DNA reassociation and phenotypic analysis revealed that strain 1330^T represented a novel species, for which the name Cronobacter condimenti sp. nov. is proposed (type strain 1330^T = CECT 7863^T = LMG 26250^T). Strains NCTC 9529^T, 731, 96 and 1435 clustered together within an independent phylogenetic lineage, with C. turicensis LMG 23827^T as the closest neighbour in the MLSA. DNA–DNA reassociation and phenotypic analysis confirmed that these strains represent a novel species, for which the name Cronobacter universalis sp. nov. is proposed (type strain NCTC 9529^T = CECT 7864^T = LMG 26249^T).

The World Health Organization (WHO) has recognised all Cronobacter species as human pathogens. Among premature neonates and immunocompromised infants, these infections can be life-threatening, with clinical presentations of septicaemia, meningitis and necrotising enterocolitis. The neurological sequelae can be permanent and the mortality rate as high as 40–80 %. Despite the highlighted issues of neonatal infections, the majority of Cronobacter infections are in the elderly population suffering from serious underlying disease or malignancy and include wound and urinary tract infections, osteomyelitis, bacteraemia and septicaemia. However, no age profiling studies have speciated or genotyped the Cronobacter isolates. A clinical collection of 51 Cronobacter strains from two hospitals were speciated and genotyped using 7-loci multilocus sequence typing (MLST), rpoB gene sequence analysis, O-antigen typing and pulsed-field gel electrophoresis (PFGE). The isolates were predominated by C. sakazakii sequence type 4 (63 %, 32/51) and C. malonaticus sequence type 7 (33 %, 17/51). These had been isolated from throat and sputum samples of all age groups, as well as recal and faecal swabs. There was no apparent relatedness between the age of the patient and the Cronobacter species isolated. Despite the high clonality of Cronobacter, PFGE profiles differentiated strains across the sequence types into 15 pulsotypes. There was almost complete agreement between O-antigen typing and rpoB gene sequence analysis and MLST profiling. This study shows the value of applying MLST to bacterial population studies with strains from two patient cohorts, combined with PFGE for further discrimination of strains.

Aims: A kinetic 5′‐nuclease polymerase chain reaction (real‐time PCR) for the quantification of Escherichia coli was developed. Methods and Results: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non‐E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real‐time PCR, linear calibration lines were obtained in the range from 10² to 10⁸ CFU ml⁻¹ for three E. coli strains. Salmonella Enteritidis (10⁶ CFU ml⁻¹) or Citrobacter freundii (10⁶ CFU ml¹) had no effect on quantification of E. coli by the method. Conclusions: The developed real‐time PCR is suitable for rapid quantification of E. coli. Significance and Impact of the Study: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.