Azathioprine, Mercaptopurine, and 5-Aminosalicylic Acid Affect the Growth of IBD-Associated Campylobacter Species and Other Enteric Microbes

Abstract

Campylobacter concisus is a bacterium that is associated with inflammatory bowel disease (IBD). Immunosuppressive drugs including azathioprine (AZA) and mercaptopurine (MP), and anti-inflammatory drug such as 5-aminosalicylic acid (5-ASA) are commonly used to treat patients with IBD. This study aimed to examine the effects of AZA, MP, and 5-ASA on the growth of IBD-associated bacterial species and to identify bacterial enzymes involved in immunosuppressive drug metabolism. A total of 15 bacterial strains of five species including 11 C. concisus strains, Bacteroides fragilis, Bacteroides vulgatus, Enterococcus faecalis, and Escherichia coli were examined. The impact of AZA, MP, and 5-ASA on the growth of these bacterial species was examined quantitatively using a plate counting method. The presence of enzymes involved in AZA and MP metabolism in these bacterial species was identified using bioinformatics tools. AZA and MP significantly inhibited the growth of all 11 C. concisus strains. C. concisus strains were more sensitive to AZA than MP. 5-ASA showed inhibitory effects to some C. concisus strains, while it promoted the growth of other C. concisus strains. AZA and MP also significantly inhibited the growth of B. fragilis and B. vulgatus. The growth of E. coli was significantly inhibited by 200 μg/ml of AZA as well as 100 and 200 μg/ml of 5-ASA. Bacterial enzymes related to AZA and MP metabolism were found, which varied in different bacterial species. In conclusion, AZA and MP have inhibitory effects to IBD-associated C. concisus and other enteric microbes, suggesting an additional therapeutic mechanism of these drugs in the treatment of IBD. The strain dependent differential impact of 5-ASA on the growth of C. concisus may also have clinical implication given that in some cases 5-ASA medications were found to cause exacerbations of colitis.

Keywords: 5-aminosalicylic acid; Campylobacter concisus; anti-inflammatory drug; azathioprine; enteric bacteria; immunosuppressive drug; inflammatory bowel disease; mercaptopurine.

Figure 1

Effects of AZA, MP, and 5-ASA on the growth of C. concisus strains. The growth of C. concisus strains on horse blood agar plates containing different concentrations of AZA, MP or 5-ASA was expressed as the percentage of the colony forming unit (CFU) relative to the CFU of their respective negative control. The negative controls were the same C. concisus strains grown on HBA plates without drugs. Strains isolated from patients with CD, UC, gastroenteritis and healthy controls are colored in blue, red, brown and green respectively. ^*Indicates statistically significance (^*P < 0.05, ^**P < 0.01, ^***P < 0.001). ^#Indicates complete inhibition of growth. CD, Crohn's disease; UC, ulcerative colitis.

Figure 2

Effects of AZA, MP, and 5-ASA on the growth of B. fragilis, B. vulgatus, E. faecalis, and E. coli. The growth of B. fragilis, B. vulgatus, E. faecalis and E. coli was expressed as the percentage of the colony forming unit (CFU) relative to the CFU of their respective negative control. The negative controls were the same bacterial strains grown on horse blood agar plates without drugs. ^*Indicates statistically significance (^*P < 0.05, ^**P < 0.01, ^***P < 0.001). ^#Indicates complete inhibition of growth.

Figure 3

Comparison of the inhibitory effect of AZA and MP on C. concisus growth. C. concisus strains were more sensitive to AZA than MP. Six of the 11 C. concisus strains (P2CDO4, P20CDO-S2, 13826, P3UCO1, P14UCO-S1, and H12O-S1) exhibited significantly reduced growth in the presence of 10 μg/ml of AZA as compared to that of the same concentration of MP (P < 0.05). When AZA and MP were present at higher concentrations (50, 100, and 200 μg/ml), the growth of all 11 C. concisus strains were significantly lower in response to AZA than the same concentrations of MP (P < 0.01).

Figure 4

Identification of enzymes required for AZA and MP metabolism in C. concisus and other four enteric bacterial species. (A) The process of generating TGNs from AZA and MP. This part of the information was obtained from previous publications (Eklund et al., ; Meggitt et al., 2011). TGNs are purine analogs that inhibit DNA synthesis in immune cells which is the therapeutic mechanism of AZA and MP. GSH, glutathione; GS-imidazole, glutathionyl derivative of 1-methyl-4-nitroimidazole; GST, glutathione S-transferase; TPMT, thiopurine methyltransferase; 6-MMP, 6-methylmercaptopurine; HGPRT, hypoxanthine guanine phosphoribosyl transferase; XO, xanthine oxidase; TIMP, thioinosine monophosphate; MTIMP, methylthioinosine monophosphate; IMPD, inosine monophosphate dehydrogenase; TXMP, thioxanthine monophosphate; GMPS, guanosine monophosphate synthetase; TGNs, thioguanine nucleotides; TGMP, thioguanine monophosphate; TGDP, thioguanine diphosphate; TGTP, thioguanine triphosphate. (B) The presence of enzymes required for AZA and MP metabolism in C. concisus and other bacterial species. This part of the information was obtained in this study by bioinformatics analysis. N, not present. Protein IDs listed are NCBI locus (^#) or Uniprot protein ID (^*).