Evaluation of in vitro and in vivo antibiotic efficacy against a novel bioluminescent Shigella flexneri

Abstract

Shigella spp., the bacteria responsible for shigellosis, are one of the leading causes of diarrheal morbidity and mortality amongst children. There is a pressing need for the development of novel therapeutics, as resistance of Shigella to many currently used antibiotics is rapidly emerging. This paper describes the development of robust in vitro and in vivo tools to study antibiotic efficacy against Shigella flexneri. A novel bioluminescent S. flexneri strain (S. flexneri lux1) was generated, which can be used in a mammalian epithelial cell co-culture assay to evaluate antibiotic intracellular and extracellular efficacy. In addition, the S. flexneri lux1 strain was used with an intraperitoneal (IP) murine model of shigellosis to test the efficacy of ciprofloxacin and ampicillin. Both antibiotics significantly reduced the observed radiance from the gastrointestinal tissue of infected mice compared to vehicle control. Furthermore, plated gastrointestinal tissue homogenate confirmed antibiotic treatment significantly reduced the S. flexneri infection. However, in contrast to the results generated with tissue homogenate, the radiance data was not able to distinguish between the efficacy of ampicillin and ciprofloxacin. Compared to traditional methods, these models can be utilized for efficient screening of novel antibiotics aiding in the discovery of new treatments against shigellosis.

Figure 1

Characterization of S. flexneri lux1 calibration curves. The log transformed S. flexneri lux1 relative luminescence units (RLU) were plotted as a function of log transformed CFUs. The limit of detection was 7,500 CFUs with the Perkin Elmer Envision system (a). The experiment was repeated and analyzed with a Perkin Elmer IVIS Spectrum (b). With the IVIS Spectrum, the limit of detection was 47,315 CFUs. Linear regression was used to identify an association between the luminescence and CFUs. Each point represents the average of three technical replicates.

Figure 2

Characterization of S. flexneri lux1 growth kinetics. An overnight culture of S. flexneri lux1 was diluted 1:100 and to a nominal OD600 of 0.05. The cultures (n = 3 for each dilution) were shaken at 37 °C and aliquots were removed at multiple time points. The OD600 (a) and luminescence (b) were measured at each time point. Values are means ± SD. A significant positive association (R² = 0.89, P < 0.0001) between the luminescence and OD600 was observed when the log transformed relative luminescence units (RLUs) were plotted against the corresponding log transformed OD600 values (c). Linear regression was used to identify an association between the luminescence and OD600.

Figure 3

Time course analysis of gentamicin’s effect on extracellular and intracellular S. flexneri. A mammalian cell co-culture time course assay with HCT-8 cells and S. flexneri lux1 was used to monitor S. flexneri intracellular (red square) and extracellular (blue circle) growth and invasion (a). The time course assay was repeated with S. flexneri lux1 in the presence of gentamicin for 0.5 (b), 2 (c), or 4 (d) hours. The RLU values of the gentamicin-treated wells were normalized to wells that contained vehicle control. Values are means ± SD (n = 8). The solid line and dashed lines represent the normalized mean ± SD of the vehicle control (extracellular samples) at each time point, respectively. The vehicle control relative luminescence unit SD for extracellular and intracellular samples were similar at each time point.

Figure 4

Mammalian cell co-culture assay with S. flexneri lux1 in the presence of ampicillin or ciprofloxacin. The luminescence of the intracellular (red square) and extracellular (blue circle) S. flexneri was measured after incubation with either ampicillin (a–c) or ciprofloxacin (d–f) for 0.5 hours (a,d), 2 hours (b,e), and 4 hours (c,f). The values of the antibiotic treated wells were normalized to wells that contained vehicle control. Values are reported as mean ± SD (n = 8). The solid line and dashed lines represent the normalized mean ± SD of the vehicle control (extracellular samples) at each time point, respectively. The vehicle control relative luminescence unit SD for extracellular and intracellular samples were similar at each time point. A black circle with a solid line represents a significantly larger reduction in luminescence generated by ciprofloxacin compared to ampicillin at the corresponding concentration. In contrast, a black circle with a dashed line represents a significantly larger reduction in luminescence generated by ampicillin compared to ciprofloxacin at the corresponding concentration.

Figure 5

Shigella infection in the SI and cecum/LI of adult mice 24 hours after IP infection with S. flexneri lux1. Mice were administered vehicle control or 5 × 10⁷ CFU S. flexneri lux1 (n = 3 mice/group), and their weights were monitored over a 24-hour period (a). To determine if gastrointestinal tissue from infected mice had detectable luminescence at 24 hours post infection, the small and large intestines were collected from mice administered vehicle control or S. flexneri lux1 (n = 3 mice/group) and were imaged with an IVIS Envision Spectrum (b). There was significantly higher radiance (P < 0.05) measured from the gastrointestinal tissue of mice administered S. flexneri lux1 compared to mice administered vehicle control. For the control and infected mice, the weights and tissue radiance were compared with a Student’s two tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Efficacy of ciprofloxacin and ampicillin in the mouse model of Shigella infection. Adult mice were infected IP with S. flexneri lux1 (5 × 10⁷ CFU/mouse) and treated orally with vehicle control, ciprofloxacin (40 mg/kg), or ampicillin (50 mg/kg). The gastrointestinal tract was removed from the mice 24 hours post infection, and the level of S. flexneri infection was determined by IVIS (a,b) and plating tissue homogenate (c). The average background radiance is represented with a solid line in panel b and the dashed lines are the SD. For the tissue homogenates, the dashed line is the lower limit of detection (LLOD). For both the IVIS data and the plated tissue homogenate data, an ANOVA analysis with post-hoc comparison identified a significant (P < 0.05) reduction in the infection of antibiotic treated mice compared to vehicle treated controls. However, a Student’s two tailed t-test only identified a significant difference between the antibiotic treated groups with data generated from the plated tissue homogenate (P < 0.0001). To determine if there was an association between the tissue radiance and the CFUs, the average radiance measurements of the SI (blue circle) and cecum/LI (red triangle) were plotted against their corresponding homogenate CFU (d). There was a significant, positive association between the log transformed observed radiance and the log transformed measured CFU (SI, R^2 =0.63, P < 0.0001, cecum/LI, R² = 0.48, P < 0.01). Experiments were performed in duplicate on separate days (total n = 10 mice/per group, with n = 5 mice/group for each duplicate). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7

Ciprofloxacin dose response in the mouse model of Shigella infection. Previous studies have established a target ciprofloxacin AUC/MIC ratio of 250 for the treatment of Gram-negative bacterial infections in humans. The efficacy of ciprofloxacin oral doses predicted to generate AUC/MIC values ranging from 22–900 were evaluated in the mouse model of Shigella infection. In agreement with the target AUC/MIC ratio of 250, infected mice with predicted ciprofloxacin AUC/MIC values of ≤225 had detectable S. flexneri infection in the large intestine and/or small intestine. In contrast, mice receiving ciprofloxacin doses of 20 and 40 mg/kg BID (AUC/MIC ratios of 450 and 900, respectively) had no detectable S. flexneri in their gastrointestinal tissue. Values are reported as mean ± SD (n = 3 mice/group).