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    • ABT737 DNA microarray is one of the highly discriminatory

    2018-10-29

    DNA microarray is one of the highly discriminatory sequence-based molecular approaches that can quickly and accurately identify the relatedness of organisms by presence/absence of ABT737 in the pathogens. Low density microarray, targeting several major virulence genes has previously been proposed as a molecular tool to assess STEC [13]. Since then, microarray technology has been improved with the development of bioinformatics as well as whole genome sequencing (WGS) technology [11]. DNA microarray has been shown to provide advantage tools in the characterization of several major foodborne pathogens including Cronobacter sp. [14], E. coli[11,15–17], Listeria monocytogenes[18–20] or Clostridium botulinum[21]. We have shown here that after the isolates were obtained, we were able to obtain the genomic information of the isolates within 48h. The fast turnaround time is critically important not only for outbreak investigation, but is important for the regulatory perspective as well.
    Materials and methods
    Results and discussion We first screened all of the enrichments using BAX PCR for E. coli O157 and non O157 STEC. The results indicated that none of the samples has E. coli O157 strains. However, the raw chicken sample (002) was positive from the original E. coli non O157 STEC screening panel. Using the two recommended BAX PCR panels, we subsequently tested the sample to identify the Big Six group, finding that the sample was positive for E. coli serogroup O45. Following the FDA-BAM protocol, we were able to isolate two E. coli non-O157:H7 isolates from a raw chicken sample (002) (Table 1). Samples 002, from raw chicken which was positive for E. coli non O157 STEC screening, was further screened on Big-6 PCR panel. We found that the 002 broth as well as the isolates from which the two 002 isolates were obtained were positive for O45 serogroup (Fig. 1A). Interestingly, in addition to the positivity to the O45 group, sample 002 broth was also positive for O111 serogroup (Fig. 1B). This suggests that sample 002 may contaminated with both serogroups, O45 and O111. However, the Ct values for O111 is much higher than that of O45 (Table 3) suggesting that O45 may be predominant in the population and the culture based method may not be sensitive enough to enable us to isolate both strains. This indicates the advantage of molecular detection. Further biochemical testing (MUG, indole) and confirmatory testing with API20E and Prolex™ E. coli non-O157 latex kit strongly confirmed that both isolates were E. coli non-O157 serotype and of the O45 serogroup. Genomic DNA extracted from these two isolates were subjected to Microarray analysis. The analyzed results were obtained within approximately 48h after isolates had been received. In addition to the fast turnaround and less hands-on time relative to WGS, using microarray technology in the real-time outbreak investigation can be very important as it provides perspective on strains and the pathotype relationships [11]. DNA microarray analysis can be relatively quick to perform using R and Bioconductor software, available freely at https://www.bioconductor.org/. In general, there are two main methods, RMA and MAS5.0, for microarray analysis. In brief, RMA result reveals the intensity of labeled target DNA from the entire genome as they bind to the probes. MAS5.0 algorithm, on the other hand, statistically calculates the intensity of mismatched and matched probe/targets and reports the results as gene presence/absence [19]. We used both methods to characterize these isolates. The relatedness of these two isolates was determined by RMA and the results clearly indicated that the two isolates are almost identical (Fig. 2). Mas5.0 was used to obtain the gene content and serotype of the isolates. Traditionally, serotyping of E. coli was performed using pools of polyclonal antisera against these O- and H-antigens. However, the process is labor-intensive and time consuming. An alternative method is a molecular serotyping by detecting the presence of specific antigenic gene sequences. In this case, O-serotype is identified by the presence of a specific wzx and wzy genes whereas H-serotype is from fliC genes [26,30,31]. As the MAS5.0 analysis can be used to identify gene contents, we further examined the genes that encode for O- (wzx, wzy and wzm) and H- (fliC) antigens. The microarray results indicated that these isolates harbor wzx45 wzy45 and fliC8 with all other wzx, wzy, wzm and fliC genes absent. This result confirmed that the isolates are E. coli O45:H8. All of the stx and eae genes are absent suggesting that these two isolates are not pathogenic. In addition, some of other virulence genes encoding virulence proteins such as bundle-forming pilus (bfp) found in Enteropathogenic E.coli, or hemolysinA (hlyA) were also absent in both O45 isolates.