Raja 42, a novel gamma lactam compound, is effective against Clostridioides difficile

Abstract

Clostridioides difficile infection (CDI) is the primary cause of hospital-acquired diarrhea, and responsible for over 500,000 enteric infections a year in the United States alone. Although most patients with CDI are successfully treated with metronidazole or vancomycin, the high rate of recurrence is still a serious problem, in which case these antibiotics are usually not very effective. The primary objective of this research is to develop a potentially effective therapeutic agent against C. difficile that are resistant to metronidazole or vancomycin. The susceptibility to metronidazole and vancomycin was examined with 194 C. difficile clinical isolates. Sixty of these isolates chosen based on a variety of criteria were examined for their susceptibility against the 4-chloro-1-piperidin-1ylmethyl-1H-indole-2,3-dione compound (Raja 42), a novel isatin-benzothiazole analogue containing a gamma-lactam structure, as we previously found that this novel compound is effective against a variety of different bacteria. Most of the 60 isolates were resistant to ceftriaxone and ciprofloxacin, raising the possibility that they might have been exposed previously to these or structurally similar antibiotics (e.g., β-lactam and quinolone compounds). Among the isolates, 48 (80%) and 54 (90%) were susceptible to metronidazole and vancomycin, respectively. Raja 42 was found to be effective against most of the isolates, especially so against metronidazole-resistant C. difficile. Most importantly, five isolates that show resistance to metronidazole and vancomycin were sensitive to Raja 42. Thus, Raja 42, a gamma lactam antibiotic, has the potential to effectively control C. difficile strains that are resistant to metronidazole and vancomycin.

Fig 1. Distribution of metronidazole and vancomycin MIC against C. difficile clinical isolates.

(A) The chemical structure and name of Raja 42 are shown. (B-E) Metronidazole and vancomycin were determined for their MIC values on 194 C. difficile clinical isolates (S1 Table). The susceptibility of these isolates to metronidazole or vancomycin was determined according to the guideline recommended by EUCAST and CLSI. The MIC value of metronidazole against C. difficile is 2 μg/mL, according to which 12 of the isolates were resistant to metronidazole (B, C). Vancomycin MIC against C. difficile is 2 μg/mL (D, E).

Fig 2. Distribution of Raja 42 MIC values against C. difficile isolates.

(A) The average MIC measured using a 96-well format plotted for Raja 42 against C. difficile ATCC 9689 in triplicates. Note that due to the nature of the BHI medium used for culture, the broth was of a bright yellow colour and recorded as 0.5 turbidity when untreated. Similarly, the recording for treatments of 4.6 μg/mL-75 μg/mL displayed a similar turbidity of 0.5 allowing us to draw the conclusion that these were baseline levels in accordance to the untreated recording. The lowest concentration value (4.6 μg/mL) was stated as the MIC. (B) Determination of C. difficile ATCC 9689 MIC value using an agar plate dilution method. The MIC value of Raja 42 against ATCC 9689 C. difficile was determined to be 4.6 μg/mL (arrow). (C) The cut-off MIC value of Raja 42 against 60 C. difficile clinical isolates was 18.75 μg/mL. The isolates were treated with serial dilutions of Raja 42 ranging from 0.55 μg/mL to 150 μg/mL using 96-well plates. According to the EUCAST Epidemiological Cut-Off Finder (ECOFFinder), 97.5% subset ECOF value corresponds to the MIC value of 18.75 μg/mL. All assays were carried out in triplicate. The values presented are mean ± SEM (n = 3 independent experiments) p* and p** values are < 0.05 and < 0.001, respectively.