New pharmacokinetic in vitro model for studies of antibiotic activity against intracellular microorganisms

Abstract

The capacity for intracellular growth is an important survival strategy for a large group of common pathogens. Helicobacter pylori, the etiological agent for gastritis and duodenal ulcer, has been shown by both in vivo and in vitro studies to have the capacity to invade epithelial cells. In vitro models are used to study the effect of antibiotics on microoganisms. Most investigations are performed in broth culture or on agar plates, but kinetic models for bacteria in broth have been described. We present a new, kinetic model adapted for intracellular pathogens. A glass chamber, with a metal rack fitting Falcon cell culture inserts, was connected to a pump by rubber tubes. Different tube diameters and pump speeds were evaluated, and the assay was designed to mimic the half-lives of the antibiotics in vivo, i.e., 11.5 h for azithromycin, 5 h for clarithromycin, and 1 h for amoxicillin. Monolayers of HEp-2 cells were grown in the inserts for 2 days, after which H. pylori (clinical strain 88-23), was added to the system. Internalization was allowed for 12 h, and extracellular H. pylori cells were eradicated with gentamicin. The inserts were moved to the glass chamber, containing medium with 12.5 mg of either amoxicillin or azithromycin per liter or 2.4 mg of clarithromycin per liter. This represents 12.5, 50, and 80 times the extracellular minimum bactericidal concentration value, respectively. Samples were taken at 0, 2, 4, 6, 8, and 24 h. The HEp-2 cells were lysed, and intracellular bacteria were counted by plating. Inserts with infected cells grown in drug-free medium were included as controls for each time interval. A 3-log10 reduction of H. pylori was achieved in the experiments with azithromycin, and a 4-log10 reduction was achieved in the clarithromycin experiments, while no intracellular effect was seen when amoxicillin was used. The antibiotic concentrations at the sampling intervals were 12.5, 3.1, 0.8, 0.2, 0.05, and 0 mg/liter for amoxicillin; 12.5, 11.5, 10, 9, 8, and 3 mg/liter for azithromycin; and 2.4, 1.8, 1.4, 1, 0.8, and 0 mg/liter for clarithromycin. This new model for pharmacokinetic studies provides a useful tool, with applications for a broad range of microorganisms.

Clarithromycin Resistance of Helicobacter pylori Strains Isolated from Children’ Gastric Antrum and Fundus as Assessed by Fluorescent In‐situ Hybridization and Culture on Four‐Sector Agar Plates

“…Culture plates were divided into four sectors: the first contained only the culture medium (blank), the second 8 mg/L metronidazole dissolved in the medium, the third 2 mg/L of clarithromycin, and the fourth sector 4 mg/L of amoxicillin. These antibiotic concentrations were chosen as their break point minimum inhibitory concentration values for resistance [23–26]. Inoculated plates were placed in a case where microaerophilic environment was generated (with 5% O 2 , 10% CO 2 , and 85% nitrogen) and incubated at 37 °C until colonies were seen or up to a maximum of 7 days.…”

Evaluation of an intracellular pharmacokinetic in vitro infection model as a tool to assess tuberculosis therapy

“…These models are valuable tools frequently utilised in evaluating the activity of antimicrobial agents used in the treatment of aerobic and anaerobic bacteria [10][11][12][13]. A three-compartment pharmacokinetic infection model was first used by Hultén et al [8] to evaluate the activity of antimicrobial agents against the intracellular organism Helicobacter pylori.…”

“…A schematic depiction of the intracellular model, which was adapted from Hultén et al [8], is shown in Fig. 1.…”

In Vitro and Animal PK/PD Models

“…The activity of antibacterials against intracellular pathogens was also developed in the mid-1990s (Hulten et al 1996). A series of glass cell culture inserts containing 2-day grown monolayers of Hep-2 cells were connected to a pump with various tube diameters to simulate half-life of different drugs.…”