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Heteroresistance is a medically relevant phenotype where small antibiotic-resistant subpopulations coexist within predominantly susceptible bacterial populations. Heteroresistance reduces treatment efficacy across diverse bacterial species and antibiotic classes, yet its genetic and physiological mechanisms remain poorly understood. Here, we investigated a multi-resistant Klebsiella pneumoniae isolate and identified three primary drivers of gene dosage-dependent heteroresistance for several antibiotic classes: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms imposed fitness costs and were genetically unstable, leading to fast reversion to susceptibility in the absence of antibiotics. We used a mouse gut colonization model to show that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. Importantly, we observed that the three mechanisms are prevalent among Escherichia coli bloodstream isolates. Our findings underscore the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations., (© 2024. The Author(s).)

In a collection of Escherichia coli isolates, we discovered a new mechanism leading to frequent and high-level tigecycline resistance involving tandem gene amplifications of an efflux pump encoded by the tet(A) determinant. Some isolates, despite carrying a functional tet(A), could not evolve high-level tigecycline resistance by amplification due to the presence of a deletion in the TetR(A) repressor. This mutation impaired induction of tetA(A) (encoding the TetA(A) efflux pump) in presence of tetracyclines, with the strongest effect observed for tigecycline, subsequently preventing the development of tet(A) amplification-dependent high-level tigecycline resistance. We found that this mutated tet(A) determinant was common among tet(A)-carrying E. coli isolates and analysed possible explanations for this high frequency. First, while the mutated tet(A) was found in several ST-groups, we found evidence of clonal spread among ST131 isolates, which increases its frequency within E. coli databases. Second, evolution and competition experiments revealed that the mutation in tetR(A) could be positively selected over the wild-type allele at sub-inhibitory concentrations of tetracyclines. Our work demonstrates how low concentrations of tetracyclines, such as those found in contaminated environments, can enrich and select for a mutation that generates an evolutionary dead-end that precludes the evolution towards high-level, clinically relevant tigecycline resistance., Competing Interests: The authors have declared that no competing interests exist.

The alternative sigma factor σ E is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of σ E increases during entry into stationary phase, suggesting an important role for σ E when nutrients are limiting. Elevated σ E activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand σ E -directed cell lysis and the role of σ E in stationary phase, we investigated the effects of elevated σ E activity in cultures grown for 10 days. We demonstrate that high σ E activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced σ E activity, due primarily to point mutations in the region of σ E that binds the -35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High σ E activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg 2+ and buffering the growth medium or by deleting the σ E -dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of σ E activity. Our results demonstrate that appropriate regulation of σ E activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability. IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The σ E response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of σ E is tightly regulated to match the production of σ E regulon members to the needs of the cell. In E. coli , loss of σ E results in lethality. Here we demonstrate that excessive σ E activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent., (Copyright © 2017 American Society for Microbiology.)

Previous work demonstrated that selection for Escherichia coli mutants with low antibiotic resistance frequently resulted in co-selection of lon mutations and that lon(-) mutants evolved higher-level resistance faster than a lon(+) strain. Here we show that lon mutation causes a very low multidrug resistance by inducing the AcrAB-TolC pump via stabilization of the acrAB transcriptional activators MarA and SoxS, which are substrates of the Lon protease. Fast evolution of lon(-) mutants towards higher resistance involves selection of frequent next-step mutations consisting of large duplications including acrAB and the mutated lon gene. Resistance results from the combined effects of acrAB duplication and lon mutation increasing dosage of efflux pump. In contrast, when acrAB duplication occurs as the first step mutation, increased Lon activity caused by lon(+) co-duplication mitigates the effect of acrAB duplication on resistance, and faster evolution towards higher resistance is not observed. As predicted, when the functional lon gene is relocated far from acrAB to prevent their co-duplication, first-step acrAB duplication confers higher resistance, which then allows selection of frequent next-step mutations and results in faster evolution towards higher resistance. Our results demonstrate how order of appearance of mutations and gene location can influence the rate of resistance evolution., (© 2013 John Wiley & Sons Ltd.)

Transposon inactivation of ycgE, a gene encoding a putative transcriptional regulator, led to decreased multidrug susceptibility in an Escherichia coli lon mutant. The multidrug susceptibility phenotype (e.g., to tetracycline and beta-lactam antibiotics) required the inactivation of both lon and ycgE. In this mutant, a decreased amount of OmpF porin contributes to the lowered drug susceptibility, with a greater effect at 26 degrees C than at 37 degrees C.

Thirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coli AG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 microg/ml). The phenotype was linked to insertion sequence (IS) insertions in marR or acrR or unstable large tandem genomic amplifications which included acrAB and which were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lon mutations. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7x10(-4)) of lon::IS186 mutants, (ii) new lon mutants appeared during the selection on antibiotic-containing agar plates, (iii) lon mutants could slowly grow in the presence of low amounts (about 2x MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.

A spontaneous mutant (M113) of Escherichia coli AG100 with an unstable multiple antibiotic resistance (Mar) phenotype was isolated in the presence of tetracycline. Two mutations were found: an insertion in the promoter of lon (lon3::IS186) that occurred first and a subsequent large tandem duplication, dupIS186, bearing the genes acrAB and extending from the lon3::IS186 to another IS186 present 149 kb away from lon. The decreased amount of Lon protease increased the amount of MarA by stabilization of the basal quantities of MarA produced, which in turn increased the amount of multidrug effux pump AcrAB-TolC. However, in a mutant carrying only a lon mutation, the overproduced pump mediated little, if any, increased multidrug resistance, indicating that the Lon protease was required for the function of the pump. This requirement was only partial since resistance was mediated when amounts of AcrAB in a lon mutant were further increased by a second mutation. In M113, amplification of acrAB on the duplication led to increased amounts of AcrAB and multidrug resistance. Spontaneous gene duplication represents a new mechanism for mediating multidrug resistance in E. coli through AcrAB-TolC.

Heteroresistance, a phenomenon where subpopulations of a bacterial isolate exhibit different susceptibilities to an antibiotic, is a growing clinical problem where the underlying genetic mechanisms in most cases remain unknown. We isolated colistin resistant mutants in Escherichia coli and Salmonella enterica serovar Typhimurium at different concentrations of colistin. Genetic analysis showed that genetically stable pmrAB point mutations were responsible for colistin resistance during selection at high drug concentrations for both species and at low concentrations for E. coli. In contrast, for S. Typhimurium mutants selected at low colistin concentrations, amplification of different large chromosomal regions conferred a heteroresistant phenotype. All amplifications included the pmrD gene, which encodes a positive regulator that up-regulates proteins that modify lipid A, and as a result increase colistin resistance. Inactivation and over-expression of the pmrD gene prevented and conferred resistance, respectively, demonstrating that the PmrD protein is required and sufficient to confer resistance. The heteroresistance phenotype is explained by the variable gene dosage of pmrD in a population, where sub-populations with different copy number of the pmrD gene show different levels of colistin resistance. We propose that variability in gene copy number of resistance genes can explain the heteroresistance observed in clinically isolated pathogenic bacteria. [ABSTRACT FROM AUTHOR]