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Antibiotic heteroresistance is a phenotype in which a bacterial isolate contains subpopulations of cells that show a substantial reduction in antibiotic susceptibility compared with the main population. Recent work indicates that heteroresistance is very common for several different bacterial species and antibiotic classes. The resistance phenotype is often unstable, and in the absence of antibiotic pressure it rapidly reverts to susceptibility. A common mechanistic explanation for the instability is the occurrence of genetically unstable tandem amplifications of genes that cause resistance. Due to their instability, low frequency and transient character, it is challenging to detect and study these subpopulations, which often leads to difficulties in unambiguously classifying bacteria as susceptible or resistant. Finally, in vitro experiments, mathematical modelling, animal infection models and clinical studies show that the resistant subpopulations can be enriched during antibiotic exposure, and increasing evidence suggests that heteroresistance can lead to treatment failure. In this Review, Andersson and colleagues outline factors to consider when studying and defining heteroresistance, and they explore the different genetic mechanisms underlying heterogeneity in antibiotic resistance phenotypes, the impact of such mechanisms on the efficacy of treatment and the methods and difficulties in diagnosing heteroresistance in clinical bacterial isolates.

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 β-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°C than at 37°C.

ABSTRACTThirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coliAG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 μg/ml). The phenotype was linked to insertion sequence (IS) insertions in marRor acrRor unstable large tandem genomic amplifications which included acrABand which were bordered by IS3or IS5sequences. Five different lonmutations, 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 lonmutations. That the lonlocus 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 3.7 × 10−4) of lon::IS186mutants, (ii) new lonmutants appeared during the selection on antibiotic-containing agar plates, (iii) lonmutants could slowly grow in the presence of low amounts (about 2× MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lonmutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lonmutations 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.

Carbamoyl-phosphate synthetases (CPS) catalyze carbamoyl phosphate (CP) biosynthesis from glutamine, bicarbonate and ATP. CPS are formed of two subunits, a small glutaminase subunit and a large synthetase subunit. CP is a common intermediate of arginine and pyrimidine biosynthesis. CPS in prokaryotes are either arginine-regulated (CPS-A), pyrimidine-regulated (CPS-P) or regulated by both components. Two to zero CPS are present in the four lactic acid bacteria studied (Lactobacillus plantarum, Enterococcus faecalis, Lactococcus lactis and Lactobacillus delbrueckii ssp. lactis). Only L. plantarum harbours two CPS with a CPS-P providing CP for both metabolic pathways. CPS-A can only supplement CP for arginine biosynthesis in higher concentrations of CO$_2$ or bicarbonate. The CPS-P present in L. plantarum and E. faecalis is encoded by genes within the pyr operon, and genes dispersed within the chromosome in Lc. lactis. CPS is absent in L. delbrueckii ssp. lactis and the catabolism of arginine via the arginine deiminase pathway (ADI) provides the CP for pyrimidine biosynthesis. In addition to their functional CPS-P, E. faecalis and Lc. lactis also harbour an ADI pathway so that arginine catabolism may regulate CP biosynthesis in these species. Lactic acid bacteria CPS were compared to CPS of 13 Gram-positive bacteria with sequenced or partially sequenced genomes. Most organisms harbour a CPS-P. CPS-P is also found in the few organisms (L. plantarum, B. subtilis and B. stearothermophilus) which harbour a CPS-A. The number of CPS and the organization of their genes is variable in Gram-positive bacteria.