Fast DNA serotyping and antimicrobial resistance gene determination of salmonella enterica with an oligonucleotide microarray-based assay

Abstract

Salmonellosis caused by Salmonella (S.) belongs to the most prevalent food-borne zoonotic diseases throughout the world. Therefore, serotype identification for all culture-confirmed cases of Salmonella infection is important for epidemiological purposes. As a standard, the traditional culture method (ISO 6579:2002) is used to identify Salmonella. Classical serotyping takes 4-5 days to be completed, it is labor-intensive, expensive and more than 250 non-standardized sera are necessary to characterize more than 2,500 Salmonella serovars currently known. These technical difficulties could be overcome with modern molecular methods. We developed a microarray based serogenotyping assay for the most prevalent Salmonella serovars in Europe and North America. The current assay version could theoretically discriminate 28 O-antigens and 86 H-antigens. Additionally, we included 77 targets analyzing antimicrobial resistance genes. The Salmonella assay was evaluated with a set of 168 reference strains representing 132 serovars previously serotyped by conventional agglutination through various reference centers. 117 of 132 (81%) tested serovars showed an unique microarray pattern. 15 of 132 serovars generated a pattern which was shared by multiple serovars (e.g., S. ser. Enteritidis and S. ser. Nitra). These shared patterns mainly resulted from the high similarity of the genotypes of serogroup A and D1. Using patterns of the known reference strains, a database was build which represents the basis of a new PatternMatch software that can serotype unknown Salmonella isolates automatically. After assay verification, the Salmonella serogenotyping assay was used to identify a field panel of 105 Salmonella isolates. All were identified as Salmonella and 93 of 105 isolates (88.6%) were typed in full concordance with conventional serotyping. This microarray based assay is a powerful tool for serogenotyping.

Figure 1. Multiplex linear DNA amplification, labeling and hybridization of the ArrayStrips.

(a) Linear Multiplex Amplification starting from clonal RNA free genomic DNA, extracted DNA is internally labeled with biotin (Label [L]) and amplified in a linear multiplex PCR reaction; (b) Hybridization: the biotin labeled, single-stranded DNA product hybridizes specifically under stringent conditions to the corresponding probes. The resulting duplex is detected using a horse-radish peroxidase (Enzyme [E]) – streptavidin conjugate, which converts the substrate (Seramun green [S]) into a colored local precipitate. (c) Detection: the ArrayMate™ Reader (or ArrayTube™ Reader ATR 03) enables the visualization and subsequent automated analysis of the array image. The presence of a dark precipitated spot indicates successful hybridization; (d) Analysis: the assay specific software analysis script, supplied with the ArrayMate™ Reader (or ArrayTube™ Reader ATR 03), measures the signal intensity of each probe and determines with an assay specific algorithm which genes/alleles are present in the sample. (e) Genotype analysis: the PatternMatching software supplied with the ArrayMate™ Reader (or ArrayTube™ Reader ATR 03) is comparing the resulting pattern with a local database including 132 reference serovars previously sero- and genotyped, finally a report is given to which serovar the sample strain belongs with regard to the Kauffman-White Scheme.

Figure 2. Comparison for four different Salmonella serovars between theoretical predictions (white) and real hybridizations (black).

Arrows indicate the probes where differences were observed. For strain S. ser. Typhimurium LT2, the antigenic formula, the complete genome sequence (NC_003197.1), and a prediction for its hybridization pattern based on this sequence were available. The threshold matching score (MS) of 6.5 between two real hybridization experiments do not apply for this comparison, as theoretical predictions do not represent real results.

Figure 3. Result sheets of the PatternMatch software as generated by the ArrayMateTM Reader.

General information: sample ID, negative/positive staining control and marker detecting genus Salmonella (invA, galf, manC). Serotyping assignment section: two best hits according to the processed sample were given as matching scores (MS) and as percentage. Antibiotic resistance and virulence genotyping section: detectable resistance and virulence genes. The pattern looks slightly different because of positive probes for resistance genes (aadA1, aadA2, cmlA1, dfrA12, sul3, tem1, tetB) that usually are located on mobile genetic elements.