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

Abstract

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.

The Secretion of Toxins and Other Exoproteins of Cronobacter: Role in Virulence, Adaption, and Persistence

“…As is the case for many enteric pathogens, another common feature possessed among Cronobacter species is that they carry plasmids, which are known to be involved in contributing to genomic plasticity, bacterial virulence, and survival [40]. In general, such plasmids have been found among members of the Enterobacteriaceae that encode a diversity of virulence factors, such as harboring genes for antibiotic resistance, toxins, adherence factors, and secretion systems (types 3, 4, and 6), and it is thought that plasmid-borne virulence genes (or gene clusters) are acquired through horizontal gene transfer (HGT) [40][41][42][43][44][45][46]. In silico analysis of such plasmids harbored by Cronobacter species suggests that this common theme holds true here as well [47].…”

“…pESA3-like plasmids (synonymous with plasmids pCTU1, pCS2, pCSK29544_1, and pSP291-1) are thought to be examples of a prototypic virulence plasmid that was originally described by Franco et al [47]. Plasmid pEAS2 is similar to pCTU2, which are conjugative plasmids, and pCTU3 is a plasmid that contains gene clusters involved in heavy metal (Ag, Cu, and Arsenic) efflux (efflux pumps such as RND EPs); and maintenance of these plasmids seem to be under tight control by multiple toxin-antitoxin genes of both type I and type II classes of toxin-antitoxin (TA) genes [46,52,53,102,145]. TA genes are thought to help stabilize plasmids and mobile genetic elements or genetic cassettes, and they participate in the response to stressful growth conditions.…”

“…Activation of TAs in response to stress is thought to control the metabolic load of cells within a population by eliminating part of the population (through outer membrane lysis); this is followed by the surviving cells entering a physiological resting or dormant state [146]. Interestingly, a type II toxin-antitoxin system HipA family toxin gene (WP_041460783) is located just downstream of the iucABCD/iutA Cronobactin siderophore and the TonB-dependent siderophore receptor genes (WP_041460784.1) [46,47]. There are also chromosomally located TAs which have been shown to control several bacterial processes, like biofilm formation, survival during infection of eukaryotic cells, defense against invading bacteriophages and entrance and exit into persistence [146].…”

Profiling the Virulence and Antibiotic Resistance Genes of Cronobacter sakazakii Strains Isolated From Powdered and Dairy Formulas by Whole-Genome Sequencing

“…They are also involved in a persistence phenotype in some species, such as E. coli and Salmonella (Deter et al, 2017;Walling and Butler, 2019). Finkelstein et al (2019) found, in preliminary studies of C. sakazakii isolates, that two typical TA genes, fic and hipA, followed species-specific evolutionary lines. When expanding their focus to evaluate the presence of five TA in C. sakazakii, they found that some strains contained either a toxin or an antitoxin component but not both.…”

Genomic Characterization of Cronobacter spp. and Salmonella spp. Strains Isolated From Powdered Infant Formula in Chile

“…The TA genes play a fundamental role in the physiology of bacterial stress response, such as in stabilizing horizontally acquired mobile genetic elements and participating in a persistence phenotype in some species, including E. coli and Salmonella ( Deter et al, 2017 ; Walling and Butler, 2019 ). Finkelstein et al (2019) noted in preliminary studies with C. sakazakii isolates that 2 typical toxin genes, fic and hipA , followed the evolutionary lines of the species and that C. sakazakii ST1 strains were the only strains containing the 22 TA homologs.…”