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. 2019 Oct 18;14(10):e0223975.
doi: 10.1371/journal.pone.0223975. eCollection 2019.

Population analysis of D6-like plasmid prophage variants associated with specific IncC plasmid types in the emerging Salmonella Typhimurium ST213 genotype

Claudia Silva  1 , Edmundo Calva  1 , Marcos Fernández-Mora  1 , José L Puente  1 , Pablo Vinuesa  2
Affiliations

Population analysis of D6-like plasmid prophage variants associated with specific IncC plasmid types in the emerging Salmonella Typhimurium ST213 genotype

Claudia Silva et al. PLoS One. .

Abstract

The Salmonella enterica serovar Typhimurium sequence type 213 (ST213) emerged as a predominant genotype in Mexico. It is characterized by harboring multidrug resistance (MDR) IncC plasmids (previously IncA/C) and the lack of the Salmonella virulence plasmid (pSTV). Here we show that the D6-like plasmid prophage is present in most of the ST213 strains. We used the reported nucleotide sequence of YU39 plasmid (pYU39_89) to design a PCR typing scheme for the D6-like plasmid prophages, and determined the complete nucleotide sequences for the D6-like prophages of three additional ST213 strains (YU07-18, SL26 and SO21). Two prophage variants were described: i) a complete prophage, containing homologous sequences for most of the genetic modules described in P1 and D6 phages, which most likely allow for the lytic and lysogenic lifestyles; and ii) an incomplete prophage, lacking a 15 kb region containing morphogenesis genes, suggesting that it is defective. The tail fiber gene inversion region was the most divergent one between D6 and pYU39_89 genomes, suggesting the production of a distinct set of tail fibers, which could be involved in host range preferences. A glutaminyl-tRNA synthetase gene (glnS), which could be involved in providing host cell increased fitness or plasmid maintenance functions, was found in all D6-like genomes. Population level analysis revealed a biogeographic pattern of distribution of these plasmid-phages and specific associations with variants of MDR IncC plasmids. Statistically significant associations were found between the two prophage variants (p75 or p89), the type of IncC plasmids (I or II) and geographic isolation regions (Sonora, San Luis Potosí, Michoacán and Yucatán). This work integrates results from molecular typing, genomics and epidemiology to provide a broad overview for the evolution of an emergent Salmonella genotype.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Linear representation of the pYU39_89 plasmid-phage, inferred functional modules and regions used for the PCR screening.
The pYU39_89 sequence (CP011430) (Calva, et al. 2015) was reannotated using the nomenclature for homologous genes in the P1 genome (Lobocka, et al. 2004) and the description of the D6 genome (Gilcrease and Casjens 2018). The names for the open reading frames are indicated inside, below or above the wide arrows. Inferred functional modules are indicated below and grouped by a solid line. The eight regions used for PCR detection are indicated in blue below as two thin arrows. The green dotted line indicates the region absent in the p75 plasmid-phage, and under the line are the three primer pairs used to distinguish the p89 and p75 variants. The predicted gene functions are listed in S1 Table.
Fig 2
Fig 2. Comparison of pYU39_89, D6 and P1 phage genomes.
A) Comparison between complete pYU39_89, D6 and P1 phage genomes. B) Comparison between the tail fiber gene inversion region of D6 and pYU39_89. The nucleotide identity of the homologous regions (percentage) is indicated by a gradient from dark to light grey; the scale is shown on the right side.
Fig 3
Fig 3. Comparison of the D6-like plasmid prophages from four Typhimurium ST213 strains.
A) The sequences of D6-like prophages were extracted from the complete genomes of strains YU39, YU07-18, SL26 and SO21. The accession numbers for pYU39_89, pYU07-18_89, pSL26_91 and pSO21_75 are CP011430, CP035549, CP032492 and CP032497, respectively. B) Detail of the variable region between complete and incomplete D6-like prophage variants. The primers for the three regions (base, for baseplate gene bplA; tail, for tail fibers gene gpU-gpU’; and p75, flanking the incomplete region) used to distinguish the variants are indicated in blue below two thin arrows. The scale shows the sequence (nucleotide) identity values.
Fig 4
Fig 4. Plasmid profiles and c1 Southern blot hybridization of representative Typhimurium ST213 strains.
A) Agarose gel showing the large plasmids for strains SO10, SL30, SO21, SO22, SO23 and YU07-18. The asterisks at the right side of the bands indicate the plasmid hybridizing with the c1 probe. B) Autoradiogram of the c1 Southern blot corresponding to the gel shown in panel A).
Fig 5
Fig 5. Schematic representation of the presence of IncC and D6-like plasmids in Typhimurium ST213 strains according to the isolation geographic location.
The isolation date and number of isolates (n) are shown in parenthesis. See Table 1 and text for details.
Fig 6
Fig 6. Graphic representation of an independence test among D6-like prophage variant, IncC plasmid type and geographic origin for 60 Typhimurium ST213 strains.
An association analysis was performed on the multi-way contingency table for four categorical variables: 1) D6-like prophage variant (complete or incomplete); 2) Geographic isolation region (MICH: Michoacán, SLP: San Luis Potosí, SON: Sonora or YUC: Yucatán); 3) IncC plasmid type (I or II); and 4) IncC plasmid carrying or lacking the blaCMY-2 region (CMY+ or CMY-). The height of the bars indicates the magnitude of the Pearson residuals, their width is proportional to the sample size and the color shading denotes the sign. Negative residuals are highlighted by dotted borders.
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Grants and funding

The research reported in this publication was supported by Consejo Nacional de Ciencia y Tecnología (FC-2015-2/879) to EC, including an investigator stipend for CS, and by Dirección General de Académico to JLP (PAPIIT IN215119) and PV (PAPIIT IN206318). The views expressed in this publication are solely those of the authors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.