Abstract
Cronobacter is a foodborne pathogen associated with severe infections and high mortality in neonates. The bacterium may also cause gastroenteritis, septicemia, and urinary tract and wound infectious in adults. A total of 15 Cronobacter isolates collected from 617 raw materials and environment samples from Powdered Infant Formula manufacturing factories during 2016 in Shaanxi, China, were analyzed for antimicrobial susceptibilities, species identification, biofilm formation, and whole-genome sequencing. The results showed that all 15 isolates were Cronobacter sakazakii, while the antimicrobial susceptibility test showed that all 15 C. sakazakii were pan susceptible. Most isolates were able to produce a weak biofilm, and two isolates from soil samples produced a strong biofilm formation. All isolates were classified into seven STs including ST4, ST40, ST64, ST93, ST148, ST256, and ST494, with ST64 (4/15, 26.7%) being dominant, and most were clinically related. The isolates harbored at least 11 virulence genes and two plasmids, with one isolate being positive for all virulence genes. Phylogenetic and ANI analysis showed strong clustering by sequence types and isolates from different sources or regions with a similar genomic background. The fact that isolates were obtained from raw materials and environment samples of PIF facilities shared a close phylogeny with one another suggests that cross-contamination events may have occurred between the processing room and external environments, which may give rise to a recurring risk of a continuous contamination during production.
Highlights
Cronobacter species are Gram-negative opportunistic pathogens that were originally referred to as yellow-pigmented Enterobacter cloacae according to its characteristics of yellow-pigment production and biochemical reactions (Urmenyi and White-Franklin, 1961)
Reference strains including C. sakazakii (ATCCTM 29544), C. malonaticus (DSM 18702), C. turicensis (DSM 18703), C. dublinensis subsp. dublinensis (DSM 18705), C. dublinensis subsp. lausannensis (DSM 18706), C. dublinensis subsp. lactaridi (DSM 18706), C. universalis (NCTC 9529), C. muytjensii (ATCCTM 51329), and a C. condimenti isolate from University College Dublin were included as positive controls for polymerase chain reaction (PCR) assays, while E. coli ATCC25922, Enterobacter cloacae CMCC45301, and Salmonella CMCC14028 were included as negative controls
A total of 15 Cronobacter strains were recovered from 617 samples including lactose powder, soil, and disk sedimentation and swabs taken from an air inlet, drain, and shoe soles of staff working in two powdered infant formula (PIF) production facilities in Shaanxi, China, 2016 (Table 1 and Figure 1)
Summary
Cronobacter species are Gram-negative opportunistic pathogens that were originally referred to as yellow-pigmented Enterobacter cloacae according to its characteristics of yellow-pigment production and biochemical reactions (Urmenyi and White-Franklin, 1961). When subsequently reclassified, they were renamed as Enterobacter sakazakii following data from DNA hybridization. WGS of Cronobacter in PIF studies, biochemical reactions, and antibiotic sensitivity tests Later again, they were further reclassified by Iversen in 2008 based on 16S rRNA gene sequencing, ribotyping, DNA– DNA hybridization, and fluorescence labeled-amplified fragment length polymorphism fingerprinting (Iversen et al, 2008). In 2002, the International Commission on Microbiological Specifications for Food classified Cronobacter as “severe risk for a restricted population, representing a threat of death or chronic sequelae” (ICMSF (International Commission on Microbiological Specifications for Foods), 2002)
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