Environmental and genetic modulation of the phenotypic expression of antibiotic resistance

Abstract

Antibiotic resistance can be acquired by mutation or horizontal transfer of a resistance gene, and generally an acquired mechanism results in a predictable increase in phenotypic resistance. However, recent findings suggest that the environment and/or the genetic context can modify the phenotypic expression of specific resistance genes/mutations. An important implication from these findings is that a given genotype does not always result in the expected phenotype. This dissociation of genotype and phenotype has important consequences for clinical bacteriology and for our ability to predict resistance phenotypes from genetics and DNA sequences. A related problem concerns the degree to which the genes/mutations currently identified in vitro can fully explain the in vivo resistance phenotype, or whether there is a significant additional amount of presently unknown mutations/genes (genetic 'dark matter') that could contribute to resistance in clinical isolates. Finally, a very important question is whether/how we can identify the genetic features that contribute to making a successful pathogen, and predict why some resistant clones are very successful and spread globally? In this review, we describe different environmental and genetic factors that influence phenotypic expression of antibiotic resistance genes/mutations and how this information is needed to understand why particular resistant clones spread worldwide and to what extent we can use DNA sequences to predict evolutionary success.

Keywords: epistasis; heteroresistance; pan genome; persisters; successful clones; virulence.

Figure 1.

Collective resistance to antibiotics. (A) Bacteria in biofilms have greater resistance to antibiotics (due to the combined effects of physical protection and altered growth physiology) than planktonic bacteria. Dead bacteria are shown with an crosses; live bacteria are shown as green circles. (B) Indirect resistance can occur when some bacteria in an environment reduce the active antibiotic concentration. Antibiotic is shown as light brown colour, bacteria causing antibiotic inactivation shown as blue, zone of antibiotic inactivation shown as white circles. Antibiotic inactivation provides protection to susceptible bacteria in the antibiotic-free environment (grey bacteria within the white zones). Dead bacteria are indicated by grey circles with a red X.

Figure 2.

Antibiotic resistance is influenced by growth physiology. Bacteria growing faster are usually more susceptible to antibiotic inhibition than bacteria that are slow growing or in stationary phase. The figure illustrates relative susceptibility of E. coli during chemostat growth to killing by a cephalosporin as a function of bacterial generation time (1.5–12 h). Slower growing cultures are less susceptible to the antibiotic than faster growing cultures. The data are adapted from Tuomanen et al. (1986). As described in the text, many different physiological and environmental factors associated with slow growth/stationary phase could, individually or in combination, make bacteria more refractory to antibiotics.

Figure 3.

Antibiotic- or metabolite-induced resistance. Inhibition of translation or transcription (x) by an antibiotic (e.g. a macrolide) can cause production of proteins/enzymes (y, z), which increase resistance to the antibiotic by one of several mechanisms: (a) modification of the polymerase to make it resistant to the antibiotic, (b) inactivation of the antibiotic, (c) increased expression of an efflux pump to reduce the antibiotic concentration. Metabolites (environmental or produced as a consequence of antibiotic-associated disruption of normal metabolism) could also increase resistance by various mechanisms, including by causing increased expression of efflux pumps.

Figure 4.

Genetic resistance mechanisms as a function of drug and bug. The relative importance of different resistance mechanisms depends on both the bacterial species and the drug class. Mutations are indicated by a red star (one mutations is sufficient to confer resistance to rifampicin in M. tuberculosis, whereas multiple mutations are required to confer resistance to fluoroquinolones in E. coli). Plasmid-borne resistance (indicated by a red circle) can also contribute to fluoroquinolone resistance, and is the major mechanism of resistance to β-lactam antibiotics in most species, including K. pneumoniae. In contrast, in some species (Streptococcal spp., and Neisseria spp.), the creation of mosaic PBP genes by HGT (indicated by a red section on the chromosome) is the major mechanism of resistance to β-lactam antibiotics.

Figure 5.

Epistasis can influence antibiotic resistance phenotype. Epistatic interactions between resistance and/or other genes can influence the phenotypic expression of antibiotic resistance (or affect other aspects of bacterial fitness). In this example, E. coli MIC for ciprofloxacin (0.015) is increased by a mutation in gyrA (red star) but not by a mutation in parC (blue star). The double mutant shows clear evidence of epistasis, with an increase in MIC much greater than predicted by additivity. Data from Huseby et al. (2017).

Figure 6.

Heteroresistance causes phenotypic instability. Heteroresistance is a phenomenon of unstable (reversible) antibiotic resistance, reflecting the appearance of subpopulations of bacteria with different levels of susceptibilities in apparently isogenic populations. A disc diffusion assay with a zone of clearing (A). Heteroresistance in a disc diffusion assay (B), with colonies of resistant bacteria growing in the zone of clearing. A possible genetic explanation for heteroresistance (C and D). A gene responsible for the resistance phenotype (AbR) is subject to genetic amplification (a relatively frequent and reversible genetic phenomenon) that alters the level of drug susceptibility as a function of gene copy number.

Figure 7.

Globally successful resistant clones. Under selection the frequencies of some clonal lineages within a species will expand while others shrink and may go extinct. Clonal expansion may be favoured by mutation or HGT events improve transmission and resistance to antibiotics. Mechanisms and events associated with the global expansion and success of, for example, E. coli ST131 and K. pneumoniae ST258, are described in detail in the text.