Prevalence, Distribution, and Phylogeny of Type Two Toxin-Antitoxin Genes Possessed by Cronobacter Species where C. sakazakii Homologs Follow Sequence Type Lineages

Cronobacter species are a group of foodborne pathogenic bacteria that cause both intestinal and systemic human disease in individuals of all age groups. Little is known about the mechanisms that Cronobacter employ to survive and persist in foods and other environments. Toxin-antitoxin (TA) genes are thought to play a role in bacterial stress physiology, as well as in the stabilization of horizontally-acquired re-combinatorial elements such as plasmids, phage, and transposons. TA systems have been implicated in the formation of a persistence phenotype in some bacterial species including Escherichia coli and Salmonella. This project's goal was to understand the phylogenetic relatedness among TA genes present in Cronobacter. Preliminary studies showed that two typical toxin genes, fic and hipA followed species evolutionary lines. A local database of 22 TA homologs was created for Cronobacter sakazakii and a Python version 3 shell script was generated to extract TA FASTA sequences present in 234 C. sakazakii genomes previously sequenced as part of Center for Food Safety and Applied Nutrition's (CFSAN) GenomeTrakr project. BLAST analysis showed that not every C. sakazakii strain possessed all twenty-two TA loci. Interestingly, some strains contained either a toxin or an antitoxin component, but not both. Five common toxin genes: ESA_00258 (parDE toxin-antitoxin family), ESA_00804 (relBE family), ESA_01887 (relBE family), ESA_03838 (relBE family), and ESA_04273 (YhfG-Fic family) were selected for PCR analysis and the primers were designed to detect these genes. PCR analysis showed that 55 of 63 strains possessed three of these genes Sequence analysis identified homologs of the target genes and some of the strains were PCR-negative for one or more of the genes, pointing to potential nucleotide polymorphisms in those loci or that these toxin genes were absent. Phylogenetic studies using a Cronobacter pan genomic microarray showed that for the most part TAs follow species evolutionary lines except for a few toxin genes possessed by some C. malonaticus and C. universalis strains; this demonstrates that some TA orthologues share a common phylogeny. Within the C. sakazakii strains, the prevalence and distribution of these TA homologs by C. sakazakii strain BAA-894 (a powdered infant formula isolate) followed sequence-type evolutionary lineages. Understanding the phylogeny of TAs among the Cronobacter species is essential to design future studies to realize the physiological mechanisms and roles for TAs in stress adaptation and persistence of Cronobacter within food matrices and food processing environments.