The Use of a Combined Bioinformatics Approach to Locate Antibiotic Resistance Genes on Plasmids From Whole Genome Sequences of Salmonella enterica Serovars From Humans in Ghana

Abstract

In the current study, we identified plasmids carrying antimicrobial resistance genes in draft whole genome sequences of 16 selected Salmonella enterica isolates representing six different serovars from humans in Ghana. The plasmids and the location of resistance genes in the genomes were predicted using a combination of PlasmidFinder, ResFinder, plasmidSPAdes and BLAST genomic analysis tools. Subsequently, S1-PFGE was employed for analysis of plasmid profiles. Whole genome sequencing confirmed the presence of antimicrobial resistance genes in Salmonella isolates showing multidrug resistance phenotypically. ESBL, either bla_TEM52-B or bla_CTX-M15 were present in two cephalosporin resistant isolates of S. Virchow and S. Poona, respectively. The systematic genome analysis revealed the presence of different plasmids in different serovars, with or without insertion of antimicrobial resistance genes. In S. Enteritidis, resistance genes were carried predominantly on plasmids of IncN type, in S. Typhimurium on plasmids of IncFII(S)/IncFIB(S)/IncQ1 type. In S. Virchow and in S. Poona, resistance genes were detected on plasmids of IncX1 and TrfA/IncHI2/IncHI2A type, respectively. The latter two plasmids were described for the first time in these serovars. The combination of genomic analytical tools allowed nearly full mapping of the resistance plasmids in all Salmonella strains analyzed. The results suggest that the improved analytical approach used in the current study may be used to identify plasmids that are specifically associated with resistance phenotypes in whole genome sequences. Such knowledge would allow the development of rapid multidrug resistance tracking tools in Salmonella populations using WGS.

Keywords: Ghana; Salmonella; multidrug resistance; plasmids; whole genome sequencing.

Figure 1

Alignment of IncFII(S)/IncFIB(S) plasmid homologs of Salmonella Enteritidis to a reference plasmid CP007529. The alignment was created with MAUVE. Top row shows the reference plasmid, and the rows below the reconstructed plasmids in Salmonella Enteritidis from the study. Each of the colored blocks outlines a region of the genome sequence that aligned to part of another genome, and is presumably homologous. Areas with no blocks were not aligned and contain sequence elements specific to a particular genome. The red box indicates the spv virulence- associated region in the reference plasmid. Black boxes surrounding a block in SE_113 and in SE_73 indicate antibiotic resistance gene blaTEM-1 and predicted IncN plasmid incorporation, respectively.

Figure 2

BLAST ring of IncFII(S)/IncFIB(S)/IncQ1 plasmid homologs of Salmonella Typhimurium and reference plasmid FN432031. Blast ring was created with GView. The middle ring is a reference plasmid, around which BLAST lanes are shown. Every lane corresponds to plasmid in each genome. The location of spv virulence-associated region, and antibiotic resistance genes are indicated outside the ring.

Figure 3

Alignment of IncN plasmid homologs of Salmonella Enteritidis to a reference plasmid JN102343. The alignment was created with MAUVE. The top row shows the reference plasmid, and the rows below the reconstructed plasmids in Salmonella Enteritidis from the study. Each of the colored blocks outlines a region of the genome sequence that aligned to part of another genome, and is presumably homologous. Areas with no blocks were not aligned and contain sequence elements specific to a particular genome. The black box in the reference plasmid surrounds a block found to contain antibiotic resistance genes strA, strB, tet(A), blaTEM-1B found in all isolates. The box in SE_102 indicate an insertion of sul1, dfrA15, which was found to be absent reference plasmid and in SE_15 isolate.

Figure 4

Alignment of TrfA/IncHI2/IncHI2A plasmid homologs of Salmonella Virchow and Salmonella Poona to a reference plasmid LN794248. The alignment was created with MAUVE. The top row shows the reference plasmid, and the rows below the reconstructed plasmids in Salmonella Virchow and Salmonella Poona from the study. Each of the colored blocks outlines a region of the genome sequence that aligned to part of another genome, and is presumably homologous. Areas with no blocks were not aligned and contain sequence elements specific to a particular genome. Black boxes in the reference plasmid surround blocks found to contain antibiotic resistance genes strA, strB, blaTEM-1B, sul2, tet(A), blaCTX-M-15, aac(3)-Iia, catB3, aac(6')Ib-cr, blaOXA-1, and dfrA14, which were present in S. Poona isolate from the study. The black box in the reconstructed plasmid in S. Poona indicates an insertion of QnrB1, which was absent in the reference plasmid.

Figure 5

BLAST ring of IncX1 plasmid homolog of Salmonella Virchow and a reference plasmid JQ269336. Blast ring was created with GView. The middle ring is the reference plasmid, around which a BLAST lane of the plasmid in S. Virchow is shown. The location of antibiotic resistance gene is indicated outside the ring.

Figure 6

PFGE gel of plasmid profiles in the examined Salmonella serovars. Lanes 1, 8, 15, 22, and 27 represent fragments generated by XbaI digestion of Salmonella Braenderup strain H9812. Fragment sizes are indicated on the left side of the gel. Lanes 2, 3, 4, 24, 25, and 26 represent plasmids of known sizes in Salmonella isolates from our own collection. Lanes 5, 6, 7, 9, 10, and 11 represent plasmids profiles of S. Enteritidis isolates 113, 15, 102, 14, 73, and 62, respectively. Lanes 12, 13, 14, 16, and 17 represent plasmid profiles of S. Typhimurium isolates 4233, 2256, 4829, B48, and 44, respectively. Lanes 18, 19 represent plasmid profiles of S. Virchow 590 and 645, respectively. Lanes 20, 21 and 23 represent plasmid profiles of S. Colindale 4770, S. Oakland 114, and S. Poona 323, respectively.