Development of a Custom-Designed, Pan Genomic DNA Microarray to Characterize Strain-Level Diversity among Cronobacter spp.

Abstract

Cronobacter species cause infections in all age groups; however neonates are at highest risk and remain the most susceptible age group for life-threatening invasive disease. The genus contains seven species:Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite an abundance of published genomes of these species, genomics-based epidemiology of the genus is not well established. The gene content of a diverse group of 126 unique Cronobacter and taxonomically related isolates was determined using a pan genomic-based DNA microarray as a genotyping tool and as a means to identify outbreak isolates for food safety, environmental, and clinical surveillance purposes. The microarray constitutes 19,287 independent genes representing 15 Cronobacter genomes and 18 plasmids and 2,371 virulence factor genes of phylogenetically related Gram-negative bacteria. The Cronobacter microarray was able to distinguish the seven Cronobacter species from one another and from non-Cronobacter species; and within each species, strains grouped into distinct clusters based on their genomic diversity. These results also support the phylogenic divergence of the genus and clearly highlight the genomic diversity among each member of the genus. The current study establishes a powerful platform for further genomics research of this diverse genus, an important prerequisite toward the development of future countermeasures against this foodborne pathogen in the food safety and clinical arenas.

Advancements in Microarray Utility for Detection and Tracking of Foodborne Microbes in the Genomic Era

“…It is noteworthy that such genetic differences revealed by microarray were confirmed by WGS [20]. Additionally, microarray can be used to detect virulence genes, antibiotic resistance genes, and other genetic traits [23,28]. For instances, multidrug resistance (MDR) phenotype was found among Salmonella Newport isolates; the microarray analysis identified the genes that coded for the MDR phenotype, indicating the versatility of microarray [19].…”

“…For instance, during the last decade, scientists at the Center of Food Safety and Applied Nutrition (CFSAN), FDA in conjunction with Affymetrix, Inc., have developed several microarray assays [25] for the investigation of genetic diversity and phylogenetic relatedness of Escherichia coli O157:H7 [23,26], STEC [22,27], Salmonella [19], Listeria monocytogenes [21], Cronobacter [28][29][30] and enteric viruses [15,17].…”

“…Tall et al [28] showed another example of how a pan genomic microarray approach can assist in foodborne investigations as a highly discriminatory characterization and identification tool for source attribution of Cronobacter. Surveillance studies have shown that Cronobacter contaminates a multitude of foods and environments, including water, infant foods (such as powdered infant formula (PIF) and follow-up formulas), dried milk protein products, cheese, licorice, candies, dried spices, teas, nuts, herbs, filth and stable flies, and PIF or milk powder production facilities and household environments [33].…”

“…The microarray was developed during 2009-2013 through the leveraging of WGS efforts of a five-member International Cronobacter consortium. The microarray was able to distinguish the seven Cronobacter species from one another and from non-Cronobacter species [28] as shown in Figure 2. n=126) and phylogenetically-related strains, which were generated from the gene-difference matrix shown in Table 1 as reported by Tall et al [28].…”

“…The microarray was able to distinguish the seven Cronobacter species from one another and from non-Cronobacter species [28] as shown in Figure 2. n=126) and phylogenetically-related strains, which were generated from the gene-difference matrix shown in Table 1 as reported by Tall et al [28]. The microarray experimental protocol as described by Jackson et al [26] was used for the interrogation of the strains and for the analysis.…”