Phenotypic and Molecular Characterization of Carbapenem-Heteroresistant Bacteroides fragilis Strains

Abstract

Carbapenem-resistant Bacteroides fragilis strains usually emerge by an insertion sequence (IS) jump into the upstream region of the cfiA carbapenemase gene. However, intermediate or fully resistant cfiA-positive strains also exist. These do not have such IS element activations, but usually have heterogeneous resistance (HR) phenotypes, as detected by a disc diffusion or gradient tests. Heteroresistance is a serious antibiotic resistance problem, whose molecular mechanisms are not fully understood. We aim to characterize HR and investigate diagnostic issues in the set of cfiA-positive B. fragilis strains using phenotypic and molecular methods. Of the phenotypic methods used, the population analysis profile (PAP) and area under curve (AUC) measurements were the best prognostic markers for HR. PAP AUC, imipenem agar dilution and imipenemase production corresponded well with each other. We also identified a saturation curve parameter (quasi-PAP curves), which correlated well with these phenotypic traits, implying that HR is a stochastic process. The genes, on a previously defined ‘cfiA element’, act in a complex manner to produce the HR phenotype, including a lysine-acetylating toxin and a lysine-rich peptide. Furthermore, imipenem HR is triggered by imipenem. The two parameters that most correlate with the others are imipenemase production and ‘GNAT’ expression, which prompted us to suspect that carbapenem heteroresistance of the B. fragilis strains is stochastically regulated and is mediated by the altered imipenemase production.

An Archetypical Model for Engrafting Bacteroides fragilis into Conventional Mice Following Reproducible Antibiotic Conditioning of the Gut Microbiota

“…An assessment of the activity of antimicrobials against Bacteroides shows low resistance to metronidazole and carbapenems (imipenem) (<5–10%), while clindamycin was moderately active, with 10 to 60% resistance in strains of B. fragilis group [ 51 , 52 ]. Infections with clinical strains of the B. fragilis are routinely treated with metronidazole [ 53 ].…”

Detection of the antibiotic resistance genes content of intestinal Bacteroides, Parabacteroides and Phocaeicola isolates from healthy and carbapenem-treated patients from European countries

“…The cfiA-positive B. fragilis strains sometimes (1-5%) also have a particular phenotype, so-called heteroresistance, where more resistant subpopulations are in the cultures of the actual strains. An investigation of the molecular background of this latter resistance mechanism has just commenced [15]. The cfiA-positive subpopulation of the B. fragilis isolates also had a distinct phylogeny, and with typing methods, two genetic divisions were observed.…”

Detection of the antibiotic resistance gene content of normal microbiota Bacteroides, Parabacteroides and Phocaeicola isolates from European countries

“…The c A positive B. fragilis strains sometimes (1-5%) also have a particular phenotype, the so-called heteroresistance, where more resistant subpopulations are in the cultures of the actual strains. An investigation of the molecular background of this latter resistance mechanism has just commenced [15]. The c A-positive subpopulation of the B. fragilis isolates also have a distinct phylogeny; and with a typing method two genetic divisions were observed.…”