In vitro effects of sub‐inhibitory concentrations of amoxicillin on physiological responses and virulence determinants in a commensal strain of Escherichia coli

Abstract

Aims:The goal was to study the effects of sub-minimum inhibitory concentrations (sub-MICs) of amoxicillin (AMX) on various physiological responses and virulence determinants in a commensal strain of Escherichia coli. Materials and Results: The commensal strain was passaged under various sub-MICs of AMX and its effect on bacterial growth, motility, biofilm formation, expression of outer membrane proteins (OMPs) and cell adhesion was analysed. Bacterial growth was diminished at 1/2 and 1/4 MICs of AMX with significant reduction in growth rate. Using crystal violet (CV) assays and quantification of surface polysaccharides we observed strong biofilm formation, together with reduced swimming motility in E. coli at 1/2 MIC of AMX. Differential OMP expression upon AMX sub-MIC exposure coincided with enhanced cell adhesion to HT-29 cells in vitro. The results demonstrated that sub-MICs of AMX can stimulate unpredictable changes in commensal bacterial strains which can be a potent source for the propagation of antibiotic resistance. Conclusions:The study reports that AMX at 1/2 MIC significantly compromised bacterial growth and swimming motility, alongside inducing biofilm formation. This was also accompanied by upregulation of a single OMP which subsequently increased cell adhesion capabilities in E. coli at 1/2 MIC, thereby enhancing its colonization and survival abilities within the gut microsphere. Significance and Impact of the Study: For the first time, the effects of AMX sub-MICs on a commensal E. coli strain were described. The results corroborate on how antibiotics can act as stimulatory molecules and determine the pathogenicity of commensal bacteria in vivo that can disseminate resistance to other intestinal pathogens or microbes.

Subinhibitory concentrations of nalidixic acid alter bacterial physiology and induce anthropogenic resistance in a commensal strain of Escherichia coli in vitro

“…Antibiotics at sublethal concentrations have been demonstrated to alter the growth kinetics of several bacterial species (Reeks et al 2005;Chadha 2021). However, very little is known in this regard about commensal microorganisms.…”

“…Subsequent sub-MICs (1/4 and 1/8) did not show any significant changes in the growth kinetics of the commensal strain. This effect can be justified as the antibiotic gets diluted to empirically lower concentrations that are unable to hinder or retard bacterial growth under experimental conditions (Chadha 2021). In the presence of NA at 1/2 MIC, the doubling time increased significantly to 72Á53 AE 1Á187 min in comparison to antibiotic-free culture (AFC) (60Á17 AE 1Á899 min).…”

“…(Schulz 2002). Studies have implicated the role of differential OMP expression with increased cell adhesion upon amoxicillin exposure (Chadha 2021). Nevertheless, their association with nutrient uptake (as porins or siderophores) has also been established previously (Rossmanith et al 1991).…”

“…Biofilm formation under static conditions was examined using CV assay in a 96 well flat-bottom microtiter plate (Tarsons, Kolkata, India) (Chadha 2021). Sub-MICtreated cultures corresponding to A 600 value of 0Á5 McFarland standards (containing 1 9 10 8 cells per ml) were dispensed into LB-containing wells supplemented with NA at mentioned sub-MIC levels.…”

“…In this regard, antibiotic administration has been shown to disrupt intestinal homeostasis, eliminating both pathogenic and commensal bacteria, and inducing dysbiosis of the gut microflora (Konstantinidis et al 2020). However, the surviving commensals experience subminimum inhibitory concentrations (sub-MICs) upon antibiotic clearance from the body that exerts significant and unknown outcomes with time, including horizontal gene transfer, biofilm formation, enhanced cell adhesion and spontaneous mutations (Mlynek et al 2016;G€ um€ us ß et al 2018;Walsh et al 2019;Chadha 2021). This may also cause the passage of resistance from commensal strains to other microbial populations in the gut horizontally.…”