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

Center of Food Safety and Applied Nutrition, U. S. Food and Drug Administration, 8301 MuirKirk Rd, Laurel, MD 20708, USA
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Microorganisms 2019, 7(11), 554; https://doi.org/10.3390/microorganisms7110554
Received: 4 October 2019 / Revised: 4 November 2019 / Accepted: 9 November 2019 / Published: 12 November 2019
(This article belongs to the Special Issue Toxin-Antitoxin Systems I)
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. View Full-Text
Keywords: toxin–antitoxins; Cronobacter; microarray; phylogeny toxin–antitoxins; Cronobacter; microarray; phylogeny
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    Doi: 10.5281/zenodo.3473376
    Description: Supplemental Table 1. Cronobacter sakazakii strain names, sequence type (ST) assignment, source, country, and NCBI accession numbers used in the BLAST analysis experiment described in Supplemental Table 3. Supplemental table 2. One hundred and nine toxin-antitoxin Probe, probeset, probe species ID that the probe sequence came from, % similarity to BAA-894 allele, NCBI protein accession #, NCBI annotations, Loci and genome region (GR) which are located on the FDA Cronobacter microarray as described by Tall et al. (2015). Fifty Cronobacter strains were evaluated using these alleles and the results were used to develop the phylogenetic tree shown in Figure 1. Supplemental Table 3. Summary of local BLAST results using the 22 toxin-antitoxin loci against 234 C. sakazakii strains to identify which TA allele is present in each strain. Strains were identified as C. sakazakii by were identified using species-specific (rpoB and cgcA) PCR assays (Stoop et al., 2009; Lehner et al., 2012; Carter et al. 2013). Multi-locus sequence typing (MLST) analysis of the strains was carried out following the protocol described by Joseph et al. (2012b). Supplemental Figure 1.
MDPI and ACS Style

Finkelstein, S.; Negrete, F.; Jang, H.; Gangiredla, J.; Mammel, M.; Patel, I.R.; Chase, H.R.; Woo, J.; Lee, Y.; Wang, C.Z.; Weinstein, L.; Tall, B.D.; Gopinath, G.R. Prevalence, Distribution, and Phylogeny of Type Two Toxin-Antitoxin Genes Possessed by Cronobacter Species where C. sakazakii Homologs Follow Sequence Type Lineages. Microorganisms 2019, 7, 554. https://doi.org/10.3390/microorganisms7110554

AMA Style

Finkelstein S, Negrete F, Jang H, Gangiredla J, Mammel M, Patel IR, Chase HR, Woo J, Lee Y, Wang CZ, Weinstein L, Tall BD, Gopinath GR. Prevalence, Distribution, and Phylogeny of Type Two Toxin-Antitoxin Genes Possessed by Cronobacter Species where C. sakazakii Homologs Follow Sequence Type Lineages. Microorganisms. 2019; 7(11):554. https://doi.org/10.3390/microorganisms7110554

Chicago/Turabian Style

Finkelstein, Samantha, Flavia Negrete, Hyein Jang, Jayanthi Gangiredla, Mark Mammel, Isha R. Patel, Hannah R. Chase, JungHa Woo, YouYoung Lee, Caroline Z. Wang, Leah Weinstein, Ben D. Tall, and Gopal R. Gopinath. 2019. "Prevalence, Distribution, and Phylogeny of Type Two Toxin-Antitoxin Genes Possessed by Cronobacter Species where C. sakazakii Homologs Follow Sequence Type Lineages" Microorganisms 7, no. 11: 554. https://doi.org/10.3390/microorganisms7110554

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