Additional file 1: Figure S1. of Multiple adaptive routes of Salmonella enterica Typhimurium to biocide and antibiotic exposure

MIC distributions to triclosan, chlorhexidine and benzalkonium chloride for 62 natural Salmonella isolates. The number of Salmonella isolates with reduced susceptibility to biocides analysed for gene expression are indicated above the arrows and MIC susceptibility values. Colors are according biocide distributions. (*) an isolate showed simultaneously reduced susceptibility to CHX and BKC. Other 6 isolates more susceptible for biocides were analysed for control (TRIS/CHXS/BKCS: 0.06-0.12/2-8/32-64 mg/L). Figure S2. XbaI digested-chromosomal DNA PFGE of several Salmonella mutants and its parental strain (5-35 s for 21 h). Figure S3. Growth curves of Salmonella mutants and the parental strain in plain LB at 37 °C with shaking. (DOCX 446 kb

Multiple adaptive routes of Salmonella enterica Typhimurium to biocide and antibiotic exposure

BACKGROUND: Biocides and antibiotics are used to eradicate or prevent the growth of microbial species on surfaces (occasionally on catheters), or infected sites, either in combination or sequentially, raising concerns about the development of co-resistance to both antimicrobial types. The effect of such compounds on Salmonella enterica, a major food-borne and zoonotic pathogen, has been analysed in different studies, but only few works evaluated its biological cost, and the overall effects at the genomic and transcriptomic levels associated with diverse phenotypes resulting from biocide exposure, which was the aim of this work. RESULTS: Exposure to triclosan, clorhexidine, benzalkonium, (but not to hypochlorite) resulted in mutants with different phenotypes to a wide range of antimicrobials even unrelated to the selective agent. Most biocide-resistant mutants showed increased susceptibility to compounds acting on the cell wall (β-lactams) or the cell membranes (poly-L-lysine, polymyxin B, colistin or toxic anions). Mutations (SNPs) were found in three intergenic regions and nine genes, which have a role in energy production, amino acids, carbohydrates or lipids metabolism, some of them involved in membrane transport and pathogenicity. Comparative transcriptomics of biocide-resistant mutants showed over-expression of genes encoding efflux pumps (sugE), ribosomal and transcription-related proteins, cold-shock response (cpeE) and enzymes of microaerobic metabolism including those of the phosphotransferase system. Mainly ribosomal, metabolic and pathogenicity-related genes had affected expression in both in vitro-selected biocide mutants and field Salmonella isolates with reduced biocide susceptibility. CONCLUSIONS: Multiple pathways can be involved in the adaptation of Salmonella to biocides, mainly related with global stress, or involving metabolic and membrane alterations, and eventually causing "collateral sensitivity" to other antimicrobials. These changes might impact the bacterial-environment interaction, imposing significant bacterial fitness costs which may reduce the chances of fixation and spread of biocide resistant mutants