The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.

ObjectivesWidespread antimicrobial resistance often limits the availability of therapeutic options to only a few last-resort drugs that are themselves challenged by emerging resistance and adverse side effects. Apramycin, an aminoglycoside antibiotic, has a unique chemical structure that evades almost all resistance mechanisms including the RNA methyltransferases frequently encountered in carbapenemase-producing clinical isolates. This study evaluates the in vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii, and provides a rationale for its superior antibacterial activity in the presence of aminoglycoside resistance determinants.MethodsA thorough antibacterial assessment of apramycin with 1232 clinical isolates from Europe, Asia, Africa and South America was performed by standard CLSI broth microdilution testing. WGS and susceptibility testing with an engineered panel of aminoglycoside resistance-conferring determinants were used to provide a mechanistic rationale for the breadth of apramycin activity.ResultsMIC distributions and MIC90 values demonstrated broad antibacterial activity of apramycin against Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Morganella morganii, Citrobacter freundii, Providencia spp., Proteus mirabilis, Serratia marcescens and A. baumannii. Genotypic analysis revealed the variety of aminoglycoside-modifying enzymes and rRNA methyltransferases that rendered a remarkable proportion of clinical isolates resistant to standard-of-care aminoglycosides, but not to apramycin. Screening a panel of engineered strains each with a single well-defined resistance mechanism further demonstrated a lack of cross-resistance to gentamicin, amikacin, tobramycin and plazomicin.ConclusionsIts superior breadth of activity renders apramycin a promising drug candidate for the treatment of systemic Gram-negative infections that are resistant to treatment with other aminoglycoside antibiotics.

This study aimed to determine whether allelic variants of the FimH adhesin from Salmonella enterica confer differential bacterial binding to different types of mammalian cells [murine bone marrow-derived dendritic cells (DCs) and HEp-2 cells] and chicken leukocytes. Although the type 1 fimbriated S. enterica serovar Typhimurium strains AJB3 (SR-11 derivative) and SL1344 both aggregated yeast cells, only the former bound efficiently to DCs and HEp-2 cells. Type 1 fimbriaemediated binding to DCs having previously been shown to require the FimH adhesin and to be inhibited by mannose, FimH sequences from strains SL1344 and AJB3 were compared and found to differ by only one residue, asparagine 158 in SL1344 being replaced by a tyrosine in AJB3. The importance of residue 158 for FimH-mediated binding was further confirmed in recombinant Escherichia coli expressing S. enterica type 1 fimbriae with a variety of substitutions engineered at this position. Additional studies with the 'non-adhesive' FimH of a type 2 fimbriated S. enterica serovar Gallinarum showed that this FimH did not mediate bacterial binding to murine DCs or HEp-2 cells. However, the type 2 FimH significantly improved bacterial adhesion to chicken leukocytes, in comparison to the type 1 FimH of strain AJB3, attributing for the first time a function to the type 2 fimbriae of S. enterica. Consequently, our data show that allelic variation of the S. enterica FimH adhesin directs not only host-cell-specific recognition, but also distinctive binding to mammalian or avian receptors. It is most relevant that this allele-specific binding profile parallels the host specificity of the respective FimH-expressing pathogen.

Conflict of Interest SNH is a co-founder of Juvabis AG, a startup biotech company with an interest in aminoglycoside therapeutics. All other authors declared no competing interests for this work. Funding Some of the research leading to these results was conducted as part of the ND4BB European Gram-Negative Antibacterial Engine (ENABLE) Consortium (www.nd4bb-enable.eu) and has received funding from the Innovative Medicines Initiative Joint Undertaking under grant agreement n°115583, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme

Staphylococcus aureus is known to generate small colony variants (SCVs) that are resistant to aminoglycoside antibiotics and can cause persistent and recurrent infections. The SCV phenotype is unstable, and compensatory mutations lead to restored growth, usually with loss of resistance. However, the evolution of improved growth, by mechanisms that avoid loss of antibiotic resistance, is very poorly understood. By selection with serial passaging, we isolated and characterized different classes of extragenic suppressor mutations that compensate for the slow growth of small colony variants. Compensation occurs by two distinct bypass mechanisms: (i) translational suppression of the initial SCV mutation by mutant tRNAs, ribosomal protein S5, or release factor 2 and (ii) mutations that cause the constitutive activation of the SrrAB global transcriptional regulation system. Although compensation by translational suppression increases growth rate, it also reduces antibiotic susceptibility, thus restoring a pseudo-wild-type phenotype. In contrast, an evolutionary pathway that compensates for the SCV phenotype by activation of SrrAB increases growth rate without loss of antibiotic resistance. RNA sequence analysis revealed that mutations activating the SrrAB pathway cause upregulation of genes involved in peptide transport and in the fermentation pathways of pyruvate to generate ATP and NAD+, thus explaining the increased growth. By increasing the growth rate of SCVs without the loss of aminoglycoside resistance, compensatory evolution via the SrrAB activation pathway represents a threat to effective antibiotic therapy of staphylococcal infections.