Antimicrobials as promoters of genetic variation

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The main causes of antibiotic resistance are the selection of naturally occurring resistant variants and horizontal gene transfer processes. In recent years, the implications of antibiotic contact or treatment in drug resistance acquisition by bacteria have been gradually more evident. The ultimate source of bacterial genetic alterations to face antibiotic toxicity is mutation. All evidence points to antibiotics, especially when present at sublethal concentrations, as responsible for increasing genetic variation and therefore participating in the emergence of antibiotic resistance. Antibiotics may cause genetic changes by means of different pathways involving an increase of free radicals inside the cell or oxidative stress, by inducing error-prone polymerases mediated by SOS response, misbalancing nucleotide metabolism or acting directly on DNA. In addition, the concerted action of certain environmental conditions with subinhibitory concentrations of antimicrobials may contribute to increasing the mutagenic effect of antibiotics even more. Here we review and discuss in detail the recent advances concerning these issues and their relevance in the field of antibiotic resistance.

Highlights

► Sublethal concentrations of antibiotics promote genetic variation in bacteria. ► Very low antibiotic concentrations select for high-level resistance. ► Subinhibitory antibiotic concentrations are present in body compartments and the environment.

Introduction

“Against necessity, against its strength, no one can fight and win”. Aeschylus (525–456 BC).

After antibiotics were first introduced in medicine, microbiologists and physicians thought that the development of antibiotic resistance was unlikely because the frequency of mutation to resistance in bacteria was negligible [1]. However, the spread of single and multiple drug resistance in clinically relevant pathogens has produced an alarming situation. Bacteria can evolve antibiotic resistance through several mechanisms, including alteration by mutations of the antibiotic target, changes in cell permeability and efflux, and horizontal transfer of resistance genes [1, 2, 3]. The amazing adaptive capacity of microbes is the reason why, despite the success of antibiotics in curing and preventing a great deal of infectious diseases, the emergence of multidrug-resistant bacteria is currently a major worldwide concern, as it is the major cause of treatment failure against many pathogens [4]. The evolution of antibiotic resistance is, obviously, based on genetic variation and selection of the succeeding genotypes generated by this variation, and it is now clear that the extended use of antibiotics over the past six decades has led to the selection and spread of resistant bacteria.

Here we review the current knowledge as to the effects of antibiotics on bacterial variability and development of antibiotic resistance. These effects may influence the evolution and spread of resistance determinants by means of different mechanisms, including selecting mutator clones, enhancing mutation and/or recombination, misbalancing nucleotide metabolism and horizontal gene transfer.

Section snippets

The classical view: antibiotics select for pre-existing resistant clones

The classical view holds that the exposure of bacteria to antibacterial agents results in the selection of pre-existing resistant variants that survive the challenge, that is, selecting agents do not influence the appearance of resistant strains [5••, 6, 7]. This view, accepted as a dogma by most biologists and clinicians for six decades, needs to include some small modifications to understand the phenomenon of antibiotic resistance development.

For example, bacteria can survive antibiotic

Selection by sublethal doses of antimicrobials

The classic view is based on the assumption that the selective force, antimicrobials in this case, is present at sufficient concentrations to kill or stop the growth of the susceptible population. However, there is growing evidence pointing to the selective capacity of low, and even very low, antimicrobial concentrations, which when maintained, can select not only for low-level [9] but also for high-level resistance [10, 11••].

In addition, sublethal concentrations of many antibiotics can

Second order selection: antibiotics can select for hypervariable strains

Bacteria with an elevated mutation rate, called hypermutable strains or mutators, can increase in frequency among laboratory bacterial populations [15, 16]. Since these alleles increase the possibility of favourable mutations, they can accelerate the evolutionary rate under some conditions. During this process, mutators can be fixed in the population by ‘hitchhiking’ with the favourable mutations they have originated [17]. This heritable hypermutation in bacteria is mainly produced by

Direct mutagenic effects caused by antibiotics

Many antibiotics can increase the mutation rate in different ways, including oxidative damage [25••], SOS response [26••, 27•, 28, 29, 30, 31], nucleotide-pool misbalancing [32], and general stress responses (for a review see [33••]).

The production of reactive oxygen species (ROS) is a common step in antibiotic mediated lethality [34]. This common pathway to cell death is mediated by an increased respiration rate, a transient depletion of NADH and the destabilization of iron–sulfur clusters,

Antibiotic-induced lateral transfer and recombination

Apart from mutation, other mechanisms generating heritable variation in bacteria are intragenomic reshuffling of genomic sequences (intrachromosomal recombination) and the acquisition of DNA sequences from other organisms via horizontal gene transfer (HGT). Both mechanisms play a major role in pathogen evolution allowing bacteria to evade the immune response, distributing genes that increase virulence or providing increased resistance to antibiotics [50, 51, 52, 53]. The majority of antibiotic

Environmental mutagenesis and its interaction with the subinhibitory antibiotic world

Environmental conditions may have a strong influence on the spontaneous mutation rate of bacteria. For example, increases of one order of magnitude in mutant frequency can be achieved by starvation [73] or simply by changing the nutritional composition of the media [74].

A very illustrative situation where antibiotic resistant mutant selection is favoured by a particular environment is the case of P. aeruginosa in chronic respiratory infections, especially in cystic fibrosis (CF). We have

‘Stress-induced mutagenesis’ or ‘amplification-reversion’

It has been known for more than three decades that certain growth-limiting stressors can apparently induce adaptive changes in bacteria [83]. Intense research revealed that this phenomenon, commonly referred to as adaptive mutation is, in fact, formed of a myriad of distinct genetic mechanisms [33••, 84, 85]. One of them is the so-called stress-induced mutagenesis (SIM), whereby bacteria become transient hypermutators after the induction of different stress response pathways. Several reports in

Conclusions

“Revolutions occur in dead ends”. Bertolt Brecht (1898–1956).

In this review, we have provided data on the activity of antibiotics as promoters of genetic variation and, consequently, true promoters of antibiotic resistance. Low concentrations of certain antibiotics can fuel mutagenesis and recombination, increasing the risk of emergence of resistance. These antibiotics increase the formation of reactive oxygen species, the misbalance of nucleotide metabolism or act directly on DNA inducing, in

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported by the Instituto de Salud Carlos III, grants PI10/00105 and RD06/0008 (Spanish Network for Research in Infectious Diseases co-financed by the European Development Regional Fund ‘A way to achieve Europe’ ERDF) and by the PAR project (Ref 241476) from the EU 7th Framework Programme.

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