Abstract:
Background: The prevalence of Bacteroides fragilis isolates resistant to first-line beta-lactam drugs
is increasing, resulting in reduced treatment efficacy. Investigating the bacterial transcriptome
and proteome can uncover links between bacterial genes and resistance mechanisms. In this
study, we experimentally assessed in vitro the transcriptional and proteomic profiles of B. fragilis
exposed to SICs of meropenem, an effective antimicrobial agent, collected from patients with
intra-abdominal diseases at Astana City Hospital, Kazakhstan.
Methods: B. fragilis was cultured in brain heart infusion broth and sub-cultured every 48 h for 8
days in media with and without meropenem. Total RNA was extracted from the liquid cultures
using a commercial RNeasy mini kit, and strand-specific RNA sequencing (RNA-seq) was performed on the DNBSEQ platform. Raw RNA-seq data were retrieved from BioProject No.
PRJNA531645 and uploaded to the NCBI Sequence Read Archive (accession no. SRX22081155).
Proteins of B. fragilis were extracted and separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis, followed by analysis of the eluted peptides using liquid
chromatography–tandem mass spectrometry. Cluster analysis utilised the Database for Annotation, Visualisation, and Integrated Discovery.
Results: The subinhibitory concentration (SIC) of meropenem was determined to be 0.5 μg/L
(minimum inhibitory concentration: 1). Mapping of reads to the reference genome identified
2477 expressed genes in all B. fragilis BFR KZ01 samples. Ten differentially expressed genes
(DEGs) were common across comparison groups during and post-antibiotic exposure (wMEM vs.
MEM2 and MEM2 vs. rMEM8); however, no substantially enriched Gene Ontology terms were
identified. The cluster analysis highlighted a significant enrichment cluster (W-0560 oxidoreductase) of DEGs following antibiotic withdrawal. In total, 859 B. fragilis proteins were identified,
with the expressions of three proteins, 3-oxoacyl-[acyl carrier protein] reductase, acetyl-CoA
carboxylase biotin carboxylase subunit, and beta-ketoacyl-ACP synthase III, being upregulated
in the enriched protein folding category. Notably, chaperone proteins such as FKBP-type peptidylprolyl cis-trans isomerases (involved in the cis-trans isomerisation of prolyl peptide bonds) and
GroES (a co-chaperone functioning with GroEL) were also identified.
