Inhibitory Concentrations of Ciprofloxacin Induce an Adaptive Response Promoting the Intracellular Survival of Salmonella enterica Serovar Typhimurium

Abstract

Antimicrobial resistance (AMR) is a pressing global health crisis, which has been fueled by the sustained use of certain classes of antimicrobials, including fluoroquinolones. While the genetic mutations responsible for decreased fluoroquinolone (ciprofloxacin) susceptibility are known, the implications of ciprofloxacin exposure on bacterial growth, survival, and interactions with host cells are not well described. Aiming to understand the influence of inhibitory concentrations of ciprofloxacin in vitro, we subjected three clinical isolates of Salmonella enterica serovar Typhimurium to differing concentrations of ciprofloxacin, dependent on their MICs, and assessed the impact on bacterial growth, morphology, and transcription. We further investigated the differential morphology and transcription that occurred following ciprofloxacin exposure and measured the ability of ciprofloxacin-treated bacteria to invade and replicate in host cells. We found that ciprofloxacin-exposed S. Typhimurium is able to recover from inhibitory concentrations of ciprofloxacin and that the drug induces specific morphological and transcriptional signatures associated with the bacterial SOS response, DNA repair, and intracellular survival. In addition, ciprofloxacin-treated S. Typhimurium has increased capacity for intracellular replication in comparison to that of untreated organisms. These data suggest that S. Typhimurium undergoes an adaptive response under ciprofloxacin perturbation that promotes cellular survival, a consequence that may justify more measured use of ciprofloxacin for Salmonella infections. The combination of multiple experimental approaches provides new insights into the collateral effects that ciprofloxacin and other antimicrobials have on invasive bacterial pathogens. IMPORTANCE Antimicrobial resistance is a critical concern in global health. In particular, there is rising resistance to fluoroquinolones, such as ciprofloxacin, a first-line antimicrobial for many Gram-negative pathogens. We investigated the adaptive response of clinical isolates of Salmonella enterica serovar Typhimurium to ciprofloxacin, finding that the bacteria adapt in short timespans to high concentrations of ciprofloxacin in a way that promotes intracellular survival during early infection. Importantly, by studying three clinically relevant isolates, we were able to show that individual isolates respond differently to ciprofloxacin and that for each isolate, there was a heterogeneous response under ciprofloxacin treatment. The heterogeneity that arises from ciprofloxacin exposure may drive survival and proliferation of Salmonella during treatment and lead to drug resistance.

Keywords: AMR; Salmonella; antimicrobial agents; cellular morphology; ciprofloxacin; confocal microscopy; transcriptomics.

FIG 1

Time-kill curves of S. Typhimurium isolates at different ciprofloxacin concentrations. S. Typhimurium isolates D23580 (A), SL1344 (B), and VNS20081 (C) were grown for 24 h in four concentrations of ciprofloxacin (0×, 1×, 2×, or 4× MIC) and subjected to CFU enumeration at 6 time points post-inoculation. Three biological replicates were performed, and each replicate was plotted independently. The average CFU per milliliter was calculated for each isolate and condition for the 24-h time point and plotted as means ± SDs. (D) D23580; (E) SL1344; (F) VNS20081. An ANOVA was performed to compare means at 24 h, and Dunnett’s test was performed to compare 24-h means of 1×, 2×, and 4× ciprofloxacin MIC to 0× (control).

FIG 2

Imaging of S. Typhimurium following 2 h of ciprofloxacin exposure. (A) D23580 (top), SL1344 (middle), and VNS20081 (bottom) were subjected to 4 concentrations of ciprofloxacin (0×, 1×, 2×, or 4× MIC) and stained and imaged using an Opera Phenix high-content microscope. Bacterial membranes were stained using CSA (red), nucleic acids were stained using DAPI (blue), and permeabilized dead cells were stained using SYTOX green (green). Imaging experiments were carried out in triplicates, with two technical replicates; images from one replicate shown. (B to D) The length of single bacteria (μm) was measured quantitatively based on image analysis, and these were plotted for each isolate and condition independently (D23580, panel B; SL1344, panel C; VNS20081, panel D). Bacterial lengths were plotted as median and interquartile ranges, and the mean ± SD was calculated for each condition compared to 0× MIC treatment. One-way ANOVAs were performed. ***, P < 0.0005. 0×-treated bacteria are in red; 1×-treated bacteria are in green; 2×-treated bacteria are in blue; and 4×-treated bacteria are in purple.

FIG 3

Bulk transcriptomics of S. Typhimurium following 2 h of ciprofloxacin treatment. S. Typhimurium isolates D23580 (A), SL1344 (B), and VNS20081 (C) were grown in medium containing either 0× or 2× MIC ciprofloxacin for 2 h, and RNA sequencing was performed. Differential gene expression was analyzed using DESeq2. The relative expression (log2 fold change) of each gene for 2× MIC ciprofloxacin versus 0× MIC ciprofloxacin was calculated for each isolate, and genes with an adjusted P value of <0.05 were plotted along the chromosome. Genes with a log₂ fold change of ≥2 are colored blue, and genes with a log₂ fold change of ≤−2 are colored red to highlight highly differentially expressed genes.

FIG 4

Bulk transcriptomics of S. Typhimurium D23580 under 4 different perturbations. S. Typhimurium D23580 was grown for 2 h in medium containing 0.5× ciprofloxacin MIC (A), 2× ciprofloxacin MIC (B), 1 μg/ml mitomycin C (C), or 1× azithromycin MIC (D) and subjected to RNA sequencing. Differential gene expression was analyzed using DESeq2. The relative expression (log₂ fold change) of each gene for treatment versus no treatment was calculated for each condition, and genes with an adjusted P value of <0.05 were plotted along the chromosome. Genes with a log₂ fold change of ≥2 are colored blue, and genes with a log₂ fold change of ≤−2 are colored red to highlight highly differentially expressed genes.

FIG 5

Transcriptomics of density gradient-separated S. Typhimurium D23580. S. Typhimurium D23580 was grown for 2 h in either 0× (NT) or 2× MIC ciprofloxacin and layered on sucrose gradients containing 25%, 50%, 60%, and 70% sucrose layers. Following density centrifugation, gradient-separated bacteria were subjected to RNA sequencing, and differential gene expression was analyzed using DESeq2. (A) Three comparisons were performed, and the log₂ fold change of relative gene expression was plotted as a heat map with upregulated genes in blue and downregulated genes in red. The comparisons were ciprofloxacin-treated 50% sucrose gradient versus NT (a), ciprofloxacin-treated 60% sucrose gradient versus NT (b), and ciprofloxacin-treated 60% sucrose gradient versus ciprofloxacin-treated 50% sucrose gradient (c). (B) For the comparison of ciprofloxacin-treated 50% sucrose gradient versus NT, significantly differentially expressed (P < 0.05) genes were plotted along the chromosome, and genes found within SPI-1 and SPI-2 are colored purple and blue, respectively. (C) The comparison of ciprofloxacin-treated 60% sucrose gradient versus NT was mapped along the chromosome as for panel B. (D) The comparison of ciprofloxacin-treated 60% sucrose gradient versus ciprofloxacin-treated 50% sucrose gradient as in panel B.

FIG 6

Cellular infections with S. Typhimurium D23580 following 2 h of ciprofloxacin exposure. S. Typhimurium D23580 was either not treated or treated with 2× MIC ciprofloxacin for 2 h prior to infection of macrophages (A) or HeLa cells (B). (Left) Bacterial internalization 1.5 h postinfection. (Right) Bacterial intracellular replication 6 h postinfection. Boxplots represent the means and interquartile ranges from four (macrophages) or three (HeLa cells) biological replicates of three technical replicates each. The means and SDs were calculated, and a Student’s paired t test was performed to calculate significance. *, P < 0.05; **, P < 0.005. (C) Transmission electron microscopy was performed using negatively stained D23580 either not treated (top panel) or treated with 2× MIC ciprofloxacin (bottom) for 2 h. Box inset shows extracellular matter in ciprofloxacin-treated culture. (D) Confocal images were taken of D23580 either not treated or treated with 2× ciprofloxacin MIC immediately following the initial 30-min infection of HeLa cells (top) or after the subsequent 1-h gentamicin treatment (bottom). HeLa cell membranes were stained with phalloidin (red), nucleic acids were stained with DAPI (blue), and bacteria were stained with CSA (green). Images of infected cells are compared to an uninfected control image for reference (left, same image used as comparator for 30 min and 1.5 h).