Abstract
Cronobacter strains harboring CRISPR-Cas systems are important foodborne pathogens that cause serious neonatal infections. CRISPR typing is a new molecular subtyping method to track the sources of pathogenic bacterial outbreaks and shows a promise in typing Cronobacter, however, this molecular typing procedure using routine PCR method has not been established. Therefore, the purpose of this study was to establish such methodology, 257 isolates of Cronobacter sakazakii, C. malonaticus, and C. dublinensis were used to verify the feasibility of the method. Results showed that 161 C. sakazakii strains could be divided into 129 CRISPR types (CTs), among which CT15 (n = 7) was the most prevalent CT followed by CT6 (n = 4). Further, 65 C. malonaticus strains were divided into 42 CTs and CT23 (n = 8) was the most prevalent followed by CT2, CT3, and CT13 (n = 4). Finally, 31 C. dublinensis strains belonged to 31 CTs. There was also a relationship among CT, sequence type (ST), food types, and serotype. Compared to multi-locus sequence typing (MLST), this new molecular method has greater power to distinguish similar strains and had better accordance with whole genome sequence typing (WGST). More importantly, some lineages were found to harbor conserved ancestral spacers ahead of their divergent specific spacer sequences; this can be exploited to infer the divergent evolution of Cronobacter and provide phylogenetic information reflecting common origins. Compared to WGST, CRISPR typing method is simpler and more affordable, it could be used to identify sources of Cronobacter food-borne outbreaks, from clinical cases to food sources and the production sites.
Highlights
The Cronobacter genus including C. sakazakii, C. malonaticus, C. dublinensis, C. turicensis, C. universalis, C. muytjensii, and C. condimenti comprises opportunistic foodborne pathogens that can cause rare but life-threatening diseases in neonates and immunecompromised infants, including meningitis, necrotizing enterocolitis, and septicemia (Iversen et al, 2008; Kucerova et al, 2011; Joseph et al, 2012a; Zeng et al, 2018a)
CRISPR1 and CRISPR2 loci were more conserved and active than others in all species; CRISPR2 had the largest average number of spacers. These results were in accordance with our previous study (Zeng et al, 2017, 2018b), and similar spacers were rarely detected among species, indicating intensive changes through adaptive acquisition and loss
1706 unique spacers were identified in C. sakazakii strains, and these were divided into 129 CRISPR types (CTs); CT15 (n = 7) was the most prevalent followed by CT6 (n = 4)
Summary
The Cronobacter (formerly Enterobacter sakazakii) genus including C. sakazakii, C. malonaticus, C. dublinensis, C. turicensis, C. universalis, C. muytjensii, and C. condimenti comprises opportunistic foodborne pathogens that can cause rare but life-threatening diseases in neonates and immunecompromised infants, including meningitis, necrotizing enterocolitis, and septicemia (Iversen et al, 2008; Kucerova et al, 2011; Joseph et al, 2012a; Zeng et al, 2018a). CRISPR Typing of Cronobacter gastroenteritis outbreak caused by C. sakazakii in a senior high school of China (Yong et al, 2018). This genus has been isolated from the environment, food, and clinical sources (Ueda, 2017; Yong et al, 2018; Zeng et al, 2018a; Li et al, 2019). A curated open access MLST database has been established for the genus with more than 2200 strains and associated metadata1 This database has enabled the recognition of certain Cronobacter clonal lineages within the genus as pathogenic variants, whereas others are primarily commensal organisms associated with the environment. WGST is a new method for subtyping bacteria, but its high costs still limit its application (Forsythe et al, 2014; Deng et al, 2015)
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