Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains

Abstract

Background: Bacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome.

Results: We found a low genetic diversity for both housekeeping and accessory genes. Sequence type 19 (ST19) was supported as the founder genotype of STs 213, 302 and 429. We found a temporal pattern in which the derived ST213 is replacing the founder ST19 in the four geographic regions analyzed and a geographic trend in the number of resistance determinants. The distribution of the accessory genes was not random among chromosomal genotypes. We detected strong associations among the different accessory genes and the multilocus chromosomal genotypes (STs). First, the Salmonella virulence plasmid (pSTV) was found mostly in ST19 isolates. Second, the plasmid-borne betalactamase cmy-2 was found only in ST213 isolates. Third, the most abundant integron, IP-1 (dfrA12, orfF and aadA2), was found only in ST213 isolates. Fourth, the Salmonella genomic island (SGI1) was found mainly in a subgroup of ST19 isolates carrying pSTV. The mapping of accessory genes and multilocus genotypes on the dendrogram derived from macrorestiction fingerprints allowed the establishment of genetic subgroups within the population.

Conclusion: Despite the low levels of genetic diversity of core and accessory genes, the non-random distribution of the accessory genes across chromosomal backgrounds allowed us to discover genetic subgroups within the population. This study provides information about the importance of the accessory genome in generating genetic variability within a bacterial population.

Figure 1

Schematic representation of the molecular markers used to study core and plasmid accessory genes of Typhimurium from Mexico. A) The chromosomal variation was addressed by multilocus sequence typing using partial sequences of the seven housekeeping genes [53], denoted by boxes on the chromosome of strain LT2 [GenBank:AE006468] [46], and by macrorestriction analysis using the rarely cutting enzyme XbaI resolved by pulsed-field electrophoresis, represented by lines crossing the chromosome at several points. B) The presence of the Typhimurium virulence plasmid (pSTV) [GenBank:AE006471] was determined by PCR amplification of three genes involved in virulence spvC, rck and traT [19,28], and by Southern hybridisation on plasmid profiles using spvC as probe. C) The presence of the plasmid-borne cmy-2 gene, conferring resistance to extended spectrum cephalosporins [GenBank:NC_011079] [30,31], was determined by PCR and by Southern hybridisation on plasmid profiles. The chloramphenicol determinant floR was also assessed, since it has been reported that both resistances are often encoded by the same plasmid [48].

Figure 2

Schematic representation of the molecular markers used to study the integrons of Typhimurium from Mexico. A) Diagrammatic representation of the basic features of a class 1 integron [68]. The positions of the primers [see Additional file 3] used to amplify the different regions are shown by arrows. A class 1 integron consist of two conserved segments (5'-CS and 3'-CS) separated by a variable region that may contain an array of one or more gene cassettes. The 5'-CS includes the gene for the integrase (intI1), the promoters for the expression of the integrase (P_int) and the gene cassettes (P_c), and an adjacent attI recombination site, where the cassettes are integrated. Gene cassettes consist of a single promoter-less gene and a recombination site known as a 59-base element (59-be or attC), which is recognized by the site-specific recombinase (intI1). The 3'-CS includes qacEΔ1 and sul1 genes, determining resistance to quaternary ammonium compounds and to sulphonamide, respectively. The structure of the integron profiles found here, IP-1, IP-2, IP-3 and IP-4, are shown with their corresponding gene cassettes. B) Diagram of the regions of the Salmonella genome island 1 (SGI1) [43,44] that were studied. The positions of the primers [see Additional file 3] used to amplify the different regions are shown by arrows. The insertion of the island in the chromosome was detected by amplification of the right and left junctions; from the antibiotic resistance cluster the two integron-born gene cassettes (aadA2 and pse-1), floR and tetG were amplified.

Figure 3

Distribution of the percentage of Typhimurium STs according to the time period and geographic location.

Figure 4

Dendrogram depicting the relationships of Mexican Typhimurium strains based on XbaI restriction patterns resolved by PFGE. The fingerprints were clustered by the UPGMA algorithm using Dice coefficients with 1.5% band position tolerance. Detailed information about strains can be found in Additional file 2. The strain column depicts the nomenclature used in the MLST database for the MEXSALM collection. Abbreviations for the state column: YU, Yucatán; MI, Michoacán; SL, San Luis Potosí; SO, Sonora. Abbreviations for the source column: HE, human enteric; HS, human systemic; HA: human asymptomatic; PM, pork meat; SI, swine intestine; BM, beef meat; CM, chicken meat; BI, beef intestine. The strains positive for the presence of pCMY-2 or pSTV are indicated by a plus symbol (+), the two strains marked with a +' in the pSTV column are the strains for which rck could not be amplified. The nomenclature of integron profiles (IP1–IP4) is explained in the text. The five main clusters (I-V) are highlighted by dotted rectangles, and the four subgroups (a, b, c and d) in cluster I are indicated by oval boxes. Cophenetic values are shown for the clusters formed above 90% similarity.