Prophage sequences defining hot spots of genome variation in Salmonella enterica serovar Typhimurium can be used to discriminate between field isolates

Abstract

Sixty-one Salmonella enterica serovar Typhimurium isolates of animal and human origin, matched by phage type, antimicrobial resistance pattern, and place of isolation, were analyzed by microbiological and molecular techniques, including pulsed-field gel electrophoresis (PFGE) and plasmid profiling. PFGE identified 10 profiles that clustered by phage type and antibiotic resistance pattern with human and animal isolates distributed among different PFGE profiles. Genomic DNA was purified from 23 representative strains and hybridized to the composite Salmonella DNA microarray, and specific genomic regions that exhibited significant variation between isolates were identified. Bioinformatic analysis showed that variable regions of DNA were associated with prophage-like elements. Subsequently, simple multiplex PCR assays were designed on the basis of these variable regions that could be used to discriminate between S. enterica serovar Typhimurium isolates from the same geographical region. These multiplex PCR assays, based on prophage-like elements and Salmonella genomic island 1, provide a simple method for identifying new variants of S. enterica serovar Typhimurium in the field.

FIG. 1.

Analysis of the microarray data by GACK. Test/reference ratios of the 24 S. enterica serovar Typhimurium isolates analyzed by microarray were assessed for the presence/absence of genes by using GACK software. The input data set was restricted to the 4,167 chromosomal features (SDT loci) expected to be present in one or more of the isolates. The heat map is plotted in the physical order of the SDT loci according to the order of the loci in the S. enterica serovar Typhimurium DT104 (NCTC 13348) genome. The presence of a gene in the test strain that is also present in the control strain (S. enterica serovar Typhimurium DT104 [NCTC 13348]) is shown in yellow, the absence or divergence of the gene from its presence in the control strain is shown in blue, and genes for which their presence was uncertain are in gray. The positions of prophage 1/ST104 and prophages 2 to 5 as well as SGI1 are indicated.

FIG. 2.

Schematic diagram of prophages 1 to 5 and SGI1, showing oligonucleotide target sites. The gray schematic diagrams represent the predicted open reading frames in the annotated genome of S. enterica serovar Typhimurium DT104 (NCTC 13348). The arrows show the location of the primer pairs P1 to P13, as listed in Table 3. The corresponding microarray results, processed by the gene-calling program GACK, are shown underneath each prophage or under SGI1. The columns represent the features harbored in each prophage or the genomic island. Yellow represents features that are present in the test strain as well as the control strain (S. enterica serovar Typhimurium DT104 [NCTC 13348]), blue represents genes that are in the control strain but are absent in the test strain, and gray indicates that the presence of the gene in the test strain is uncertain. The rows represent each S. enterica serovar Typhimurium isolate, in the following order: row 1, AWF002008; 2, AWX002821; 3, H20002800; 4, H20021651; 5, AWX000826; 6, AWX000841; 7, H20021958; 8, H20004019; 9, AWF009126; 10, AWF009147; 11, H20021856; 12, AWX003658; 13, AWX004816; 14, AWF007581; 15, H19963477; 16, H19992292; 17, AWX004814; 18, H20004467; 19, H20023824; 20 AWX006747; 21, AWX006748; 22, H20003919; 23, H20023530; 24, DT104 (NCTC 13348); 25, LT2 (ATCC 700220).