Bacterial strains

The prototype S. enterica serovar Typhimurium SL1344 [25] strain was exposed to biocides (TRI, CHX, BKC and SHC), and antibiotics (the β-lactam ampicillin, AMP; and the fluoroquinolone ciprofloxacin, CIP). The quantitative phenotype of this strain against diverse antimicrobials is summarized in Table 1.

Table 1 Susceptibility profiles of Salmonella mutants respect to SL1344 parental strain Full size table

Sixteen Salmonella spp. isolates from food-borne animals with reduced susceptibility to TRI (3 TRIR; MIC 1-2 mg/L), BKC (7 BKCR; MIC = 128 mg/L), CHX (1 being CHXR/BKCR, MIC = 16 mg/L (Additional file 1: Figure S1) used in a previous work [26], were investigated for their transcriptomic profiles. Such isolates, collected in a veterinary surveillance project in Europe, showed 13 different PFGE-types and belonged to Salmonella enterica subspecies enterica [serovars Anatum (n = 8), Hadar (n = 5), Dublin (n = 2)] and subspecies Typhimurium (n = 1).

Most of these strains were susceptible to antibiotics. A few number of isolates harbored plasmids that contained acquired genes coding for resistance to β-lactams (bla TEM-1 ), aminoglycosides (strA, strB), tetracycline (tetA, tetR) and quinolone (qepA). Plasmids from 6 isolates also carried genes encoding resistance to metals (As, Co).

Selection of mutants

A colony of S. enterica serovar Typhimurium SL1344 grown overnight in Luria Bertani (LB) plates was inoculated into LB-broth and LB supplemented with sub-inhibitory concentrations (1/2 × MIC) of biocides (TRI, CHX, BKC and SHC; Sigma-Aldrich, Inc., St. Louis, MO) or antibiotics (AMP and CIP) and further incubated overnight at 37 °C with shaking at 150 rpm. Subsequently, aliquots of 100 μl were plated onto LB plates containing a single biocide or a single antibiotic compound at concentrations ranging 2.5-33 × MIC and incubated at 30 °C. These primary selective plates were examined for growth during 7 days. A variable number of viable mutants (one per colony morphotype per plate) were tested for growth on secondary selective plates containing other biocides or antibiotics. The stability of mutants was evaluated after serial passages in non-selective LB broth (up to 50 generations). Mutants were named by the acronym name of the antimicrobial compound added to the broth cultures before plating, followed by the name and concentration of the compound added to the selective agar plates from where the mutant was retrieved. The mutants obtained from broth cultures not supplemented with any antimicrobial were designated as non-exposed (NE). Phenotypes of decreased and increased susceptibility to biocides and antibiotics appear represented by the super indexes “R” or “HS”, respectively. The colonial morphology in LB and blood agar plates was compared between mutants and the parental strain. The variability of XbaI-digested genomic DNA profiles of mutants and parental strain was assessed by pulsed field gel electrophoresis (PFGE) using standard protocols for DNA preparation, digestion and PFGE running conditions for Enterobacteriaceae [27].

Antimicrobial susceptibility testing

The minimal inhibitory concentrations (MICs) of biocides (TRI, CHX, BKC) and antibiotics (AMP; CAZ; CIP; erythromycin, ERY; gentamicin, GEN; chloramphenicol, CLO; tetracycline, TET) (BioMérieux, Marcy l’Etoile, France) were determined by both broth micro-dilution using E- test strips following CLSI guidelines. Escherichia coli ATCC10536 and Staphylococcus aureus ATCC6538 were used as control strains (3). Minimum bactericidal concentrations (MBCs) were determined by subculturing 10 μl from each well without visible bacterial growth when MIC was determined on Mueller-Hinton broth (Difco, Becton Dickinson, Maryland, USA). The minimal concentration yielding no-growth after overnight incubation at 37 °C was scored as the MBC. The susceptibility of wild type strains to AMP, streptomycin, sulphonamides, trimethoprim, nalidixic acid, CIP, CLO, TET, GEN and kanamycin was determined by disk diffusion.

Further, the susceptibility of mutants and the parental strain to 240 cell growth-inhibiting chemical compounds was screened using the Phenotype MicroArray PM11-PM20 in two independent experiments (Biolog, Hayward, CA, USA) as previously described [28]. The strains were grown overnight at 30 ° C on BUG agar (Biolog Universal Agar, Biolog Hayward California) and then, colonies were picked up with a sterile cotton swab and suspended in 15 ml of 1X inoculation fluid (IF-0a GN/GP Base, Biolog 74268). Cell density was adjusted to 85 % transmittance (T) on a Biolog turbidimeter. Inoculation fluid for PM11-20 was prepared mixing 100 ml of IF-10a GN Base (1.2X) (Biolog 74264), 1.2 ml of Biolog Redox Dye A (100X) (Biolog 74221), 0.6 ml of cell suspension at 85 % T, bringing to a final volume of 120 ml with sterile water. The mixture was inoculated in the PM plates (100 μl per well) and monitored automatically for color development every 15 min for 72 h at 30 °C in an Omnilog reader (Biolog). To identify phenotypes, the kinetic curves of both parental strain and mutants were compared using Omnilog-PM software (release OM_PM_109M). Such comparison was based on the half maximal inhibitory concentration (IC50) values for 4 concentrations of each antimicrobial, which is defined as the well at which a particular per-well parameter is the half of its maximal value over the concentration series; the reference parameter being the area under the curve. Raw data were filtered using differences of average area of mutant compared to control taking a difference of 1:3 (33 %) as significant.

Growth kinetics

The growth kinetics of both the parental strain SL1344 and biocide-tolerant mutants exhibiting various phenotypes was determined by measuring the optical density at 600 nm every 5–10 min for 24 h at 37 °C in Bioscreen C (ThermoLabsystems, Helsinki, Finland), adapting the method described by Foucault et al. [29]. Inocula in a concentration of 104 to 105 CFU/ml were obtained from a 1/1000 dilution of an overnight culture in fresh LB broth and aliquots of 400 μl were seeded in triplicate in a microtitre plate. Growth rates were determined in the interval estimated to be exponential using the GrowthRates 2.1 program [30]. The fitness cost (FC) reflects the relative growth rates, which were based on the individual growth rates of mutants relative to the parental strain. For each strain, data from growth rates were averaged and standard deviations calculated.

Whole genome sequencing (WGS)

Six mutants with different phenotypes were selected for whole genome sequencing. Genomic DNA was extracted from 1 ml of overnight cultures in plain LB broth using a Promega Wizard Genomic DNA Purification kit according manufacturer instructions. Genome sequencing was performed on Illumina MiSeq platform to obtain 100–200 bp paired-end reads. Reads were revised and corrected using Lighter software and further mapped against the genome of the SL1344 strain (GenBank acc. Number FQ312003) using Breseq v0.26.1 pipeline (http://barricklab.org/twiki/bin/view/Lab/ToolsBacterialGenomeResequencing). Single nucleotide polymorphisms (SNPs) detected in all mutants were not deemed confident and were excluded, because we cannot dismiss differences between the laboratory SL1344 strain used as wild-type in this work, and that corresponding to the canonical sequence in the GenBank database. Reads not found in all mutants were treated as deletions.

Transcriptome analysis

Array Design and Production

An array was designed to cover the complete genome of Salmonella enterica subsp. enterica serovar Typhimurium, as well as plasmids isolated from various Gram-negative microorganisms. Probe design was performed by the CustomArray Design Service (CustomArray Inc., Bothell, WA, USA) and included 12,005 capture probes (35–40 bp length), 326 quality control probes and 65 non-specific probes derived from plants, phages and unrelated bacterial sequences, and also 148 empty spots with no oligonucleotides. Arrays were synthesized on a CustomArray Synthesizer (CombiMatrix, Mukilteo, WA) and quality tested using the standard protocols provided by the manufacturer.

RNA extraction

Strains were grown overnight in 10 ml LB broth at 37 °C, 150 rpm. The cultures were diluted 1:100 in pre-warmed LB and grown to logarithmic phase (OD 570 = 0.5). 2 ml of the culture (5 x 108 – 1 x 109 colony forming units (UFC)/ml) were harvested in 4 ml of RNA protect reagent® (Qiagen GmbH, Hilden, Germany), incubated for 5 min at room temperature and centrifuged for 10 min at 5000 x g. Bacterial pellets were suspended in 200 μl of TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8) containing 1 mg/ml lysozyme (Sigma) and incubated for 5 min at room temperature, 600 rpm. Total RNA was then extracted using RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. Contaminating DNA was removed using DNA-free™ Kit (Applied Biosystems). The RNA isolation procedure was validated for RNA quality by testing RNA samples on an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA concentration and purity were determined by Nanodrop® ND-1000 spectrophotometer (Thermo Scientific). For each strain, at least 4 RNA samples were prepared from independent cultures.

RNA labelling and fragmentation

Isolated, unamplified RNA was labelled with Cy5, using ULS™ Labeling Kit for CombiMatrix arrays (Kreatech Biotechnology), according to the manufacturer’s instructions. RNA was fragmented with the RNA Fragmentation Reagents (Ambion®).

Array hybridization

12 K Custom arrays were hybridized with 2 μg of labelled, fragmented RNA, according to information provided by the manufacturer (Customarray/Combimatrix Incorporated). In brief, after pre-hybridization of the arrays, hybridization was performed at 45 °C for 16 hours in a hybridization buffer containing 25 % formamide. After washing steps, microarrays were scanned using Packard ScanArray4000 array scanner and software (ScanArray, version 3.1, Packard BioChip Technologies) with incremental laser power from 15 to 100 %. Data were extracted with Microarray Imager software (version 5.8.0, Combi Matrix) and spot intensity expressed as median intensity. After scanning, microarrays were striped using 12 K CustomArray™ Stripping kit, according to the manufacturer’s instructions. Quality of the stripping was verified by scanning the microarray at maximal laser intensity and repeated when necessary. Microarrays were used up to four times.

Data analysis

To adjust for difference in the amount and labelling efficiency of hybridized RNAs, the median fluorescence intensity values of all spots was determined for all laser intensities used during scanning. Scanning data with similar median fluorescence intensity were chosen for further analysis. Fluorescence values of spots with maximal intensity (signal saturation) at a given laser intensity were extrapolated by linear regression, using values gathered with lower laser intensity. For each set of arrays for a given strain, non-specific binding was determined from fluorescence values of the non-specific probes. The cut-off for specific binding was set as the upper 95 % confidence interval of the mean signal intensity of the non-specific probes. Probes were excluded when the mean values for the strains compared were under the cut-off value.

The fluorescence values were log 2 transformed and stage-wise quantitative normalization was performed for each set of comparison, using a script written in the statistical computing environment of R (R Development Core Team, 2011, version 3.3). To identify genes differentially regulated, we analyzed the transformed and normalized intensities determined by two methods, the Significance Analysis of Microarrays method (SAM, version 5.0, running under Shiny, a web-based interactive application framework for R environment, https://github.com/MikeJSeo/SAM) and “R”, comprising base package statistics and the attached LIMMA package (version 3.26.5). The presence of genes identified by both methods in the mutants analyzed was searched in the wild type strains included in the study and transcriptomic profiles were compared. The expression profiles of these genes were visualized in a heatmap built with the ‘pheatmap’ package in “R”.

Statistical analysis

In the SAM method, the delta value was set to obtain an average. False Discovery Rate (FDR) of 5 % and the fold change cut-off value was established as 1.5. In LIMMA analysis, genes with a fold change >1.5 and p < 0.05 were considered as differentially expressed. Only the genes identified as differentially expressed by both SAM and LIMMA were considered.

Availability of supporting data

The data sets supporting the results of this article are available in the ArrayExpress repository, (http://www.ebi.ac.uk/arrayexpress/) under accession numbers A-MEXP-2366 (S. Typhimurium combimatrix 12 K customarray design) and E-MTAB-2554 (microarray raw results).