Oral Presentation 14th Lorne Infection and Immunity 2024

Distribution of phenotypic antimicrobial resistance in the gastrointestinal microbiome. (#63)

Emily L Gulliver 1 2 , Jodee A Gould 1 2 , Emily L Rutten 1 3 , Linden J Gearing 1 2 , Wei Khoo 1 3 , Sean M Solari 1 2 , Remy B Young 1 4 , Jamia Hemphill 1 4 , Tamblyn Thomason 1 2 , Margarette Williamson 1 4 , Samuel C Forster 1 2
  1. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia
  2. Department of Molecular and Translational Science, Monash University, Clayton, Victoria, Australia
  3. Department of Molecular and Translational Science, Monash University, Clayton, Victoria, Australia
  4. Department of Microbiology, Monash University, Clayton, VIC, Australia

In 2019, 4.95 million deaths were associated with antimicrobial resistance (AMR) worldwide. The human gastrointestinal microbiome has been identified as a source of acquired resistance in pathogens. Previously, culture-independent metagenomics was the primary method for identifying antimicrobial resistance in gastrointestinal microbiota. However, this approach cannot identify novel AMR genes or determine their expression or functionality within the cell. To overcome these limitations, our study utilised novel culturing on YCFA media in anaerobic conditions coupled with microbial genomics to identify phenotypic antibiotic resistance in the microbiome. This method was used to culture 10 faecal samples from healthy individuals, with and without six common orally administered antimicrobials (Amoxicillin, Amoxicillin-Clavulanic acid, Cefalexin, Ciprofloxacin, Clindamycin and Doxycycline). The resultant 1058 colonies were picked, identified through 16S rRNA sequencing and resistance to each of the antimicrobials was confirmed in broth growth. Genes associated with resistance or sensitivity to the antimicrobials were identified through combining whole genome sequencing data and phenotypic (growth in broth) data of 98 isolates. There were 2050 genes associated with resistance to Amoxicillin-Clavulanic acid, 310 genes associated with resistance to Ciprofloxacin, 220 genes associated with resistance to Clindamycin, one gene associated with resistance to Doxycycline and no genes found to be associated with resistance to Amoxicillin or Cefalexin resistance. Of the genes associated with resistance, four are known antimicrobial resistance genes, including the Clindamycin resistance gene ermF. Using this as a positive control, the putative Clindamycin resistance genes NADPH-flavin oxidoreductase and an operon containing SAM-methyltransferase and tetR  were cloned into expression vectors in E. coli to determine difference in MIC. In this study advanced culturing coupled with microbial genomics has been used to identify putative, uncharacterised, antimicrobial resistance genes.