Salmonella enterica sv. Typhimurium ATCC14028 wild-type and ΔrecA strains (Medina-Ruiz et al., 2010) and their ΔcheR mutant derivatives carrying plasmid pUA1127, harboring an eYFP::cheR fusion (Mayola et al., 2014), were used in this work.

Except when indicated, the cells were grown at 37°C on either Luria–Bertani (LB) plates containing 1.7% agar or on swarming plates (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.5% D-(+)-glucose, and 0.5% agar). These conditions are referred to in the following as non-swarming and swarming conditions, respectively.

When necessary, the plate media were supplemented with a sub-inhibitory concentration of amikacin (4 mg/L), cefotaxime (1.6 mg/L), chloramphenicol (2 mg/L), colistin (2.5 mg/L), tetracycline (4 mg/L), kanamycin (5 mg/L), ciprofloxacin (0.0065 mg/L), and/or trimethoprim (1 mg/L). In all cases, antibiotics were filtered and the corresponding antimicrobial was added to the media after the sterilization process when cooled down.

The minimal inhibitory concentrations (MICs) of the antibiotics used in this work for S. enterica ATCC14028 and the ΔcheR derivative with pUA1127 plasmid containing the eYFP::cheR fusion were determined by the standard microdilution method using tryptone broth (TB), as described previously (Wiegand et al., 2008). The obtained values for both strains were similar. Strain growth ability was tested in microtiter plate wells containing twofold serial dilutions of the antibiotic in TB (Supplementary Table S1). The sub-inhibitory concentration was defined as the antimicrobial concentration inhibiting growth by 20–30% compared to non-treated cells. Bacterial growth reduction was determined by measuring the optical density of bacterial cultures at 600 nm (Supplementary Figure S1) as described previously (Kohanski et al., 2010). Further, in all cases, it was confirmed that the established sub-inhibitory concentration reduced S. enterica viability by ∼30% after 2 h of treatment (Supplementary Table S2).

The swarming phenotype of wild-type S. enterica or its ΔcheR derivative was tested in the presence of the above-listed antimicrobial agents. In all cases, the observed swarming phenotype of the two strains was exactly the same. When needed, the S. enterica ΔrecA strain, which is unable to swarm (Medina-Ruiz et al., 2010), was also used as a non-motile control.

Swarming assays were carried out as described previously (Gómez-Gómez et al., 2007; Mayola et al., 2014; Irazoki et al., 2016b). Briefly, a single colony picked using a sterile toothpick from bacterial strains grown on LB plates was inoculated in the center of a freshly prepared swarming plate containing medium supplemented with the antimicrobial compound(s) of interest. The plates were incubated overnight at 37°C for 14 h and then imaged using a ChemiDoc^TM XRS+ system (Bio-Rad).

Chemoreceptor clustering assays were performed using S. enterica ΔcheR carrying plasmid pUA1127 containing the eYFP::cheR fusion under the control of an IPTG-inducible promoter (Ptac) (Mayola et al., 2014). The fusion protein served as a reporter of polar cluster localization (Kentner et al., 2006; Cardozo et al., 2010; Santos et al., 2014). The clustering experiments were carried out as described previously (Mayola et al., 2014; Irazoki et al., 2016b). Briefly, overnight cultures were grown under constant agitation at 30°C in TB supplemented with ampicillin and 25 μM IPTG. The day after, the cultures were diluted 1:100 in TB without antibiotics but with the addition of 25 μM IPTG to maintain the induction of the eYFP::cheR fusion construct. The cultures were incubated at 30°C until an OD₆₀₀ of 0.08–0.1 was reached, when the appropriate antimicrobial was added to each culture. Samples were collected at the indicated times and the cells were harvested by low-speed centrifugation for 15 min. The harvested cells were washed once using ice-cold tethering buffer (10 mM potassium-phosphate pH 7, 67 mM NaCl, 10 mM Na-lactate, 0.1 mM EDTA, and 0.001 mM L-methionine), resuspended in 20–100 μL of tethering buffer, and applied onto thin 1% agarose pads.

Fluorescence microscopy was performed using a Zeiss Axio Imager M2 microscope (Carl Zeiss Microscopy) equipped with a Zeiss AxioCam MRm monochrome camera (Carl Zeiss Microscopy) and a filter set for enhanced yellow fluorescent protein (eYFP; excitation BP500/25; beam splitter FT 515; emission BP535/30). Cell fields were photographed and the number of clusters then quantified using ImageJ software (National Institutes of Health). At least 500 cells were visually inspected. Each experiment was performed at least in triplicate using independent cultures, resulting in the examination of a minimum of 1500 cells from each growth condition.

Samples for RecA quantification were obtained by recovering the cells directly from the colony edge of the corresponding swarming plates, following the same procedure and conditions as described above. In this case, the cells were resuspended in sonication buffer (PBS 1×, cOmplete mini EDTA-free tablets, pH 7.3) and whole-cell lysates were obtained by sonication (two 30-s pulses of 20% amplitude, Digital sonifier^® 450, Branson). The supernatants were recovered, centrifuged (12000 g for 10 min), and the total protein concentration of each sample was then quantified using the Bradford method (protein reagent DyeR, BioRad). A standard curve was generated using bovine serum albumin (range: 1.5–200 μg/mL).

Pre-treated 96-well microtiter plates (Nunc-Immunoplate F96 Maxisorp, Nunc) were coated with serial dilutions of the whole-cell lysates. The RecA concentration in the polar cluster assays was determined by ELISA, as previously described (Irazoki et al., 2016b). A standard quantification curve was obtained using purified RecA protein. A rabbit anti-RecA antibody (ab63797, Abcam) served as the primary antibody, and a goat anti-rabbit-IgG horseradish-peroxidase-conjugated antibody (IgG, IgM, IgA, polyclonal antibody, YO Proteins) as the secondary antibody. The BD OptEIA TMB substrate reagent kit (BD Biosciences) was used as the developing solution. The plate reactions were read at 650 nm using a multi-plate reader (Sunrise, Tecan).

To evaluate the antibiotic susceptibilities of the cells, LB non-swarming or swarming plates supplemented when needed with the corresponding antibiotic were surface-inoculated using a sterile swab with either S. enterica ΔcheR pUA1127 or S. enterica ΔcheR ΔrecA pUA1127 freshly grown on LB plates. Antimicrobial susceptibility test disks (amikacin, 30 μg; trimethoprim, 25 μg; and tetracycline, 30 μg; Pronadisa) were placed in the middle of the inoculated plates. After a 14 h incubation at 37°C, the size of the bacterial growth inhibition zone was determined based on photographic images of the plates (ChemiDoc XRS + system, Bio-Rad). Each of these experiments was performed at least in triplicate. The images shown in the figures are representative of the entire image set.

The flagella were labeled as described previously (Turner et al., 2010) but with modifications. Cells from the corresponding swarming plates were collected in 0.5 mL of tethering buffer (described above), washed three times by centrifugation (1500 ×g for 10 min) at 15°C, and resuspended first in 1 mL and then in 0.5 mL of PBS. To label the flagella, 100 μL of PBS, 25 μL of 1 M NaHCO₃, and 0.5 μL of Cy3b dye (0.1 mg/μL) were added to the cell suspension. After a 1-h incubation at room temperature in the dark with gyrorotation, the labeled cells were washed three times with 1 mL of PBS before they were resuspended in 0.2–0.5 mL of tethering buffer.

To visualize the S. enterica ATCC14028 cells, they were immobilized and fixed on the same focal plane using thin 1% agarose pads in tethering buffer. Fluorescence microscopy was performed using a Zeiss Axio Imager M2 microscope (Carl Zeiss Microscopy) equipped with a Zeiss AxioCam MRm monochrome camera (Carl Zeiss Microscopy) and a filter set for Cy3b protein (excitation BP 546/12; beam splitter FT 560; emission BP 575-640). All fluorescence images were obtained at a 100× magnification under identical conditions. Each experiment was performed in triplicate using independent cultures. The images presented are representative of the entire image set. ImageJ software (National Institutes of Health) was used to prepare images for publication. In all cases, at least 100 cells were visually inspected. Each experiment was performed at least in triplicate using independent cultures, resulting in the examination of a minimum of 300 cells from each growth condition.

Data from the polar clustering assays and measurements of the RecA concentration were analyzed using a one-way analysis of the variance (ANOVA) with Prism (GraphPad). In all cases, the analyses were followed by Bonferroni multiple comparison post hoc tests. A p-value < 0.01 was considered to indicate statistical significance. The error bars in each of the figures indicate the standard deviation.

The effect on swarming of sub-inhibitory concentrations of the following antimicrobial agents differing in their mechanisms of action was analyzed: (i) inhibitors of translation (chloramphenicol, tetracycline, and the aminoglycosides kanamycin and amikacin); (ii) an inhibitor of cell-wall synthesis (the cephalosporin cefotaxime); (iii) an inhibitor of DNA replication (the fluoroquinolone ciprofloxacin); (iv) a disruptor of the outer cell membrane (colistin); and (v) an inhibitor of thymidine synthesis (trimethoprim). Swarming plates containing sub-inhibitory concentrations of the corresponding antimicrobial agent were inoculated with S. enterica wild-type strain in the middle of the plate. In all cases, it was determined that the antimicrobial concentration used produces about 30% reduction in cell viability when grown in liquid media (see Materials and Methods). In addition, the swarming ability of the wild-type strain and a ΔrecA mutant derivative, which is not able to swarm (Medina-Ruiz et al., 2010), were tested in the absence of any antimicrobial agent, as swarming and non-swarming controls, respectively (Figure 1A).

Our results indicated that the presence of kanamycin, amikacin, colistin, and tetracycline did not affect the swarming ability of S. enterica (Figure 1B); rather, the phenotype of the respective bacterial colonies was the same as that of wild-type non-treated cells (Figure 1A). By contrast, the addition of sub-inhibitory concentrations of cefotaxime, ciprofloxacin, trimethoprim, and chloramphenicol completely abolished swarming motility (Figure 1B). In these cases, the sub-inhibitory concentration of antimicrobial treatment gave rise to the same non-swarming phenotype as observed for non-treated ΔrecA cells (Figure 1A).

To identify the mechanism responsible for impaired swarming, the dynamics of chemosensory array assembly and the flagellation of swarming cells during antibiotic treatment were evaluated, since both are essential for the surface motility of S. enterica (Cardozo et al., 2010; Partridge and Harshey, 2013; Santos et al., 2014). The polar clustering assays were performed using cells grown in liquid media, since it has been previously described that the number of cells presenting polar clusters is similar regardless of whether they are grown in liquid or on swarming plates (Irazoki et al., 2016b).

Chemosensory arrays were observed using a S. enterica strain expressing the eYFP::cheR fusion under the control of an IPTG-inducible promoter (Supplementary Figure S2; Mayola et al., 2014; Irazoki et al., 2016b). This strain exhibits the same swarming phenotype as the wild-type strain. In concordance with their motility behavior, cells treated with the antimicrobials that allowed swarming (kanamycin, amikacin, tetracycline, and colistin) did not exhibit altered polar chemosensory array assembly (Figure 2B) compared to non-treated cells (Figure 2A). Consistent with this finding, there was no alteration in the RecA concentration in cells treated with the compounds that did not impair surface motility (Figure 2B).

However, among the agents that blocked swarming, treatment with cefotaxime, ciprofloxacin, and trimethoprim but not chloramphenicol induced a significant decrease in polar cluster assembly (Figure 2B and Supplementary Figure S2). Furthermore, the increase in the intracellular concentration of RecA mediated by cefotaxime, ciprofloxacin, and trimethoprim indicates the induction of the SOS response by these antibiotics (Figure 2B). The percentage of cells with polar clusters at the end of the corresponding treatment was similar to that observed in the ΔrecA strain (Figure 2A). Moreover, and as expected, when swarming cells were treated with antibiotic concentrations of cefotaxime, ciprofloxacin, and trimethoprim that did not induce the SOS response no swarming impairment was observed (Supplementary Figure S3). Previous reports showed that either the increase in the RecA concentration following SOS response induction by mitomycin-C treatment or the absence of this protein within ΔrecA mutant cells prevents polar chemosensory array formation due to CheW-RecA titration (Mayola et al., 2014; Irazoki et al., 2016a).

To visualize the flagella, the cells were directly labeled with Cy3b fluorescent dye, as previously described (Turner et al., 2010). As expected, treatment with the antimicrobial compounds that allowed swarming had no effect on cell flagellation, which was the same as in non-treated cells (data not shown). However, despite the inhibitory effect of cefotaxime, ciprofloxacin, and trimethoprim on swarming, cells treated with these compounds had the same flagellar phenotype as non-treated bacteria (data not shown). Only in cells treated with a sub-inhibitory concentration of chloramphenicol was there a clear reduction in the number of flagella (Figure 3), which explained the abolishment of swarming by this antibiotic.

S. enterica swarming cells exhibit elevated resistance to a variety of antibiotics, including those that target the cell envelope, protein translation, DNA replication, and transcription (Kim and Surette, 2003; Kim et al., 2003; Butler et al., 2010). To determine whether the antibiotics that impaired surface motility also inhibited the drug increased resistant phenotype, the antibiotic susceptibility of swarming cells to trimethoprim, amikacin, and tetracycline was tested in the presence or absence of sub-inhibitory concentrations of either non-swarming affecting or swarming impairing compounds (Figure 4A). These three antimicrobial agents (trimethoprim, amikacin and tetracycline) were selected based on the greater level of resistance to them exhibited by cells grown on swarming plates (Figure 4B) as well as their different modes of action (Brogden et al., 1982; Davis, 1987; Chopra et al., 1992).

Disk-diffusion sensitivity tests revealed that the increased-resistance phenotype was preserved in the presence of a sub-inhibitory concentration of kanamycin, which did not affect swarming behavior (Figure 4A). In this case, kanamycin-treated cells had an increased resistance to trimethoprim, amikacin, and tetracycline when growing on swarming plates, as occurred in the absence of treatment (Figure 4B). The same was observed when, instead of kanamycin, sub-inhibitory concentrations of either amikacin, colistin, or tetracycline were used (data not shown). These results confirmed that compounds unable to inhibit swarming also did not affect the increased swarming-associated drug resistance phenotype.

By contrast, this increased antibiotic resistance was dramatically abolished in S. enterica wild-type strain treated with a sub-inhibitory concentration of cefotaxime when growing on swarming plates (Figure 4A). Likewise, the recA defective mutant, which is unable to swarm (Medina-Ruiz et al., 2010), also did not increase its resistance sensitivity to trimethoprim, amikacin, and tetracycline when growing under swarming conditions (Figure 4B). Furthermore, the increased drug-resistance phenotype was also inhibited by other antimicrobials that hindered swarming as ciprofloxacin, trimethoprim, and chloramphenicol (data not shown).

Together, these data indicated that the restoration of antimicrobial sensitivity is directly associated with the absence of swarming and not specifically with the inhibition of chemosensory array assembly (induced by cefotaxime, ciprofloxacin, or trimethoprim) or the reduction in the number of flagella (chloramphenicol).

Combination antibiotic therapy takes advantage of possible synergistic effects between antibiotics (Tamma et al., 2012; Tängdén, 2014). For this reason, and considering the above results, we asked whether compounds that impaired swarming and hindered the increased drug-resistance phenotype maintained their effects when provided together with antimicrobials that did not alter motility. Thus, the cells were treated with sub-inhibitory concentrations of cefotaxime in combination with sub-inhibitory treatments of either kanamycin or colistin. Swarming ability, chemosensory cluster assembly, and the increased resistance phenotype were then examined.

As shown in Figures 5 and 6, combined treatment with sub-inhibitory concentration of the antimicrobial agents yielded the same results as obtained with cefotaxime alone (Figures 1, 2, 4). Thus, in all cases, cells treated with cefotaxime and colistin or cefotaxime and kanamycin were unable to swarm (Figure 5A). In both cases, the number of cells with assembled polar chemosensory arrays was reduced (Figure 5B), and the swarming-associated increased resistance phenotype abolished (Figure 6). The same results were obtained when, instead of kanamycin or colistin, other non-swarming-impairing compounds, such as tetracycline or amikacin, were used in the combined treatment or when cefotaxime was replaced by other swarming-impairing compounds, such as trimethoprim or ciprofloxacin (data not shown).

Taken together these observations showed that the presence of an antibiotic with no effect on swarming (kanamycin, amikacin, colistin, or tetracycline) did not alter the effects prompted by a swarming-impairing agent (cefotaxime, ciprofloxacin, or trimethoprim).