Interaction between Mutations and Regulation of Gene Expression during Development of De Novo Antibiotic Resistance

Abstract

bBacteria can become resistant not only by horizontal gene transfer or other forms of exchange of genetic information but also by de novo by adaptation at the gene expression level and through DNA mutations. The interrelationship between changes in gene expression and DNA mutations during acquisition of resistance is not well documented. In addition, it is not known whether the DNA mutations leading to resistance always occur in the same order and whether the final result is always identical. The expression of >4,000 genes in Escherichia coli was compared upon adaptation to amoxicillin, tetracycline, and enrofloxacin. During adaptation, known resistance genes were sequenced for mutations that cause resistance. The order of mutations varied within two sets of strains adapted in parallel to amoxicillin and enrofloxacin, respectively, whereas the buildup of resistance was very similar. No specific mutations were related to the rather modest increase in tetracycline resistance. Ribosome-sensed induction and efflux pump activation initially protected the cell through induction of expression and allowed it to survive low levels of antibiotics. Subsequently, mutations were promoted by the stress-induced SOS response that stimulated modulation of genetic instability, and these mutations resulted in resistance to even higher antibiotic concentrations. The initial adaptation at the expression level enabled a subsequent trial and error search for the optimal mutations. The quantitative adjustment of cellular processes at different levels accelerated the acquisition of antibiotic resistance. The de novo acquisition of resistance against antibiotics is known to be accompanied by certain mutations and differential expression of specific genes (1-5). The "radical-based" theory (6, 7) proposes that bactericidal antibiotics cause cell death by a single mechanism, driven by the accumulation of oxygen radicals in the cells. In that case, the cellular response to sublethal concentrations of antibiotics should be similar even for compounds belonging to different classes of bactericidal drugs, such as beta-lactams or fluoroquinolones. The outcome might differ for bacteriostatic drugs, for example, tetracycline. The radical-based theory, however, is the subject of debate (8). The revelation of a common denominator for the adaptation processes to different antibiotics might illuminate the question of a single mechanism from a different angle.Resistance can easily be induced in Escherichia coli by exposure to stepwise increasing sublethal antibiotic concentrations (9). The effects of the acquisition of resistance to amoxicillin on the overall physiology is a complex set of adaptations at the gene expression level, preventing metabolic costs at the expense of the ecological range (10). After the initial stage, the prolonged exposure to antibiotics modulates the SOS response, leading in turn to mutations that cause resistance (11). The mutations generate more permanent resistance, which remains long after the antibiotic pressure has been ...

Effects of Stress, Reactive Oxygen Species, and the SOS Response on De Novo Acquisition of Antibiotic Resistance in Escherichia coli

“…In less than 100 generations, bacterial cells developed genetic mutations and permanent transcriptional changes (2). On the one hand, these cellular modifications allow the population to grow in the presence of high antibiotic concentrations, but on the other hand, they may decrease fitness or cause a metabolic burden (1)(2)(3). This metabolic cost does not necessarily come in the form of an increased energy requirement.…”

“…Resistance to antibiotics is rapidly induced as a result of exposure to stepwise increasing sublethal drug concentrations (1). In less than 100 generations, bacterial cells developed genetic mutations and permanent transcriptional changes (2). On the one hand, these cellular modifications allow the population to grow in the presence of high antibiotic concentrations, but on the other hand, they may decrease fitness or cause a metabolic burden (1)(2)(3).…”

“…Genes that were permanently differentially regulated in E. coli cells made resistant to amoxicillin, enrofloxacin, or tetracycline compared to their regulation in their sensitive ancestor (2) are likely to play a role in the development of resistance. For example, gadABC and hdeA, which confer resistance to acidic conditions, were differentially expressed in cells made permanently resistant to amoxicillin, enrofloxacin, or tetracycline by exposure to stepwise increasing antibiotic levels.…”

“…The acquisition of antibiotic resistance under optimized culture conditions and the underlying molecular mechanisms causing the cellular response are well documented (2,(10)(11)(12). The second aim of this study was to investigate whether additional stress factors, such as low pH or increased NaCl concentrations, influence the ability of the cell to acquire antibiotic resistance de novo.…”

“…gadE is a common regulator of gadABC and hdeA and may therefore influence the de novo acquisition of antibiotic resistance (5). Other genes that have been documented to affect antibiotic resistance are ompF, which codes for an outer membrane porin (6,7); the transcriptional regulator soxS (8), which is involved in superoxide removal; and recA, which codes for a nucleoprotein that is involved in the SOS response (2,7,9). The first aim of this study was to establish whether these genes, in addition to influencing resistance itself, also affect the acquisition of resistance.…”