Evaluation of Ciprofloxacin's Impact on Antimicrobial Resistance in Escherichia coli: A Randomized, Placebo-Controlled Pilot Study

The primary objective of this study is to quantify the individual shifts in the **ciprofloxacin** minimum inhibitory concentration (MIC) distribution of **Escherichia coli** in the human gastrointestinal tract and to compare these shifts between the intervention and placebo groups. This is clinically relevant as it aims to assess the potential for low-dose ciprofloxacin to induce antimicrobial resistance in **E. coli**, a significant concern in public health due to the increasing prevalence of resistant bacterial strains.

Secondary objectives include:

• Describing per individual the MIC distributions, both at baseline and after administration of the intervention/placebo.
• Describing per participant the percentage of inhibited **E. coli** at prespecified concentrations of ciprofloxacin, both at baseline and after intervention/placebo.
• Determining if low-dose ciprofloxacin ingestion in humans can induce reduced susceptibility to ciprofloxacin in **E. coli**, defined as a ciprofloxacin MIC ≥ 0.125 mg/L.
• Repeating all above-mentioned objectives and endpoints for "non-**E. coli** coliforms" (pink colonies).
• Describing the concentration of ciprofloxacin in feces at baseline and day 30.

The clinical trial focuses on **antimicrobial resistance in Escherichia coli** and aims to quantify shifts in ciprofloxacin MIC distribution in the human gastrointestinal tract. The study population includes both male and female participants, aged 18 years and older. Participants are required to be in general good health, specifically not taking immunosuppressive agents and not clinically immunocompromised at the time of randomization. Females of childbearing potential must practice effective contraception and have a negative pregnancy test at screening. The trial includes a vulnerable population, although specific details about the total number of participants or lifestyle considerations such as diet or physical activity are not provided by the sponsor.

The clinical trial is designed as a **single-blind**, placebo-controlled, single-center, randomized controlled pilot study. The primary objective is to assess whether low-dose **ciprofloxacin** can induce antimicrobial resistance in **Escherichia coli** within the human gastrointestinal tract. The trial will involve a comparison between the intervention group receiving ciprofloxacin and a placebo group, with the primary endpoint being the quantification of shifts in the ciprofloxacin minimum inhibitory concentration (MIC) distribution of E. coli. The trial is expected to run from October 1, 2023, to July 30, 2024, with a maximum treatment period of 28 days for each participant.

Participants will undergo a series of study visits, beginning with an inclusion (screening) visit to confirm eligibility based on criteria such as age, consent, and health status. Females of childbearing potential must practice effective contraception and have a negative pregnancy test at screening. Following randomization, participants will attend follow-up visits to monitor the effects of the intervention or placebo, with assessments including the percentage of inhibited E. coli at specified ciprofloxacin concentrations and the concentration of ciprofloxacin in feces. The end-of-study visit will conclude the participant's involvement, with data collection on the primary and secondary endpoints.

Participant involvement is expected to last up to 30 days, including the treatment period and follow-up assessments. Conditions that may lead to early termination from the study include withdrawal of consent, adverse events, or non-compliance with study procedures. The study aims to provide insights into the potential for low-dose ciprofloxacin to induce antimicrobial resistance, contributing to the understanding of safe maximum residue limits (MRLs) and the prevention of further antimicrobial resistance emergence.

The clinical trial involves the administration of **Ciprofloxacin Kabi 200 mg/100 ml solution for infusion**, which is a commercially available pharmaceutical product. This medication is formulated as a **solution for infusion** and contains the active substance **ciprofloxacin**, a chemical compound classified under the ATC code J01MA02. The solution is intended for **oral use** after being transformed into a syrup form. This transformation is achieved by adding **SyrSpend SF pH 4 Liquid** to the Ciprofloxacin Kabi solution, resulting in a syrup with a concentration of 360 µg/5 mL of ciprofloxacin. The maximum daily dose is 392 µg, with a total maximum dose of 10,976 µg over a treatment period of up to 28 days. The administration schedule and participant compliance are monitored throughout the trial to ensure adherence to the dosing regimen.

**SyrSpend SF** is utilized in the trial as a non-experimental treatment to facilitate the oral administration of ciprofloxacin. Although it does not contain an active pharmaceutical ingredient, it serves as a vehicle to convert the infusion solution into a syrup suitable for oral consumption. The use of SyrSpend SF is critical in maintaining the stability and palatability of the ciprofloxacin syrup, ensuring that the medication can be effectively administered to participants. Compliance with the administration of SyrSpend SF is monitored alongside the primary treatment to ensure the integrity of the trial's dosing protocol.

Efficacy in this clinical trial will be assessed by evaluating the shifts in the **ciprofloxacin** minimum inhibitory concentration (MIC) distribution of *Escherichia coli* in the human gastrointestinal tract. The primary endpoint involves calculating the individual difference in median ciprofloxacin MIC after intervention compared to baseline, or alternatively, determining the maximum absolute distance between the two individual empirical cumulative distributions using the Kolmogorov-Smirnov test statistic. Secondary endpoints include analyzing individual MIC distributions, the percentage of inhibited *E. coli* at prespecified ciprofloxacin concentrations per participant and time point, and the percentage of inhibited *E. coli* with ciprofloxacin MIC ≥ 0.125 mg/L. These assessments will be conducted at baseline and after the administration of the intervention or placebo. Additionally, the concentration of ciprofloxacin in feces will be measured on day 0 and day 30. The trial aims to quantify these shifts to compare the effects between the intervention and placebo groups, thereby providing insights into the potential induction of antimicrobial resistance.