SELECTIVE PRESSURE

3. SELECTIVE PRESSURE

The ideal environment for the selection of antibiotic-resistant bacteria is one in which the concentration of the antibiotic is too low to kill all the organisms but high enough to present an opportunity for a slightly more resistant organism to outgrow the rest of the population. Over the last 40 years, since the introduc- tion of ampicillin, more than 60 variants of the TEM-1 ␤-lactamase have been characterised. Genetic experiments have shown that many more variants can be isolated in the laboratory, but that selection in the patient involves more com- plex parameters than simply the maximum activity against a particular ␤-lactam (Blazquez et al., 2000). Similar observations have been made for

372 Ian R. Booth ciprofloxacin resistance determinants (Ruiz, 2003). In particular the “patient-

environment” represents a spatially- and temporally-variable context within which organisms must evolve to survive. The reason for the observation of a narrower spectrum of mutations in the patient isolates than in the laboratory represents the more varied challenges that are posed in the former, particularly with respect to variations in antibiotic exposure. A patient who is failing treat- ment with one ␤-lactam will be rapidly switched to another—the microorgan- ism that specialises in survival of the first antimicrobial at the expense of dealing with a related molecule will be short-lived!

3.1. The principle that not all change is good

A demonstration of this principle was achieved through the analysis of the evolution of TEM-1 ␤-lactamase in vitro (Figure 2). Escherichia coli cells

L169R D179Y D179G R164S R164H

R164S R164H

[Ceftazidime] Figure 2. Fluctuating ␤-lactam pressure alters the pattern of evolution. Blazquez and col-

leagues (2000) devised an experiment in which cells were serially sub-cultured at increasing concentrations of ceftazidime and in a parallel experiment the cells were also subjected to an overnight incubation with a fixed concentration of amoxicillin. The outcome of the evolution of a TEM-1 ␤-lactamase was investigated by sequencing the resulting plasmids when the cultures had achieved growth in the presence of 32 mg/L ceftazidime.

expressing TEM-1 were subjected to serial passage through medium containing increasing, doubling concentrations of ceftazidime until growth occurred at

32 mg/L. In parallel, a second series of similar serial passages on ceftazidime was conducted but an overnight challenge with amoxicillin was performed between each growth cycle. The protein sequences of the resulting TEM enzymes were deduced by sequencing the respective genes, and the outcome of the continuous direct selection and the dual selection compared. It was found that continuous single selection selected five different mutations, three of which had not been observed in natural variants of TEM-1. All three of the unusual amino acid changes were found to diminish the MIC for amoxicillin, suggesting that they altered enzyme activity so that this antibiotic was no longer so readily hydrolysed. As expected from the MIC data for the mutants isolated by continuous single selection, only two mutant types were isolated by dual selection, both of which had been encountered previously in natural iso- lates. These mutations were also dominant among the isolates selected by con- tinuous exposure to ceftazidime and also conferred higher MIC values for this antibiotic suggesting that these mutations create a better ␤-lactamase than did either of the rarer changes (Blazquez et al., 2000).

3.2. Small concentration differences do matter

Recent work has pointed to the small differences in ␤-lactamase enzymes that might be sufficient to lead to selection. Negri and colleagues (Negri et al., 2000) noted that many TEM variants possess multiple amino acid changes that could only arise by several rounds of mutagenesis and selection. Although

mutators increase the mutation frequency by up to 10 3 -fold (see below), they are unlikely to introduce several mutations into a single gene in any one gener- ation. Thus, selection of each mutant is mandatory. It has been pointed out that TEM-12 differs from TEM-1 by a single amino acid change, which produces a small change in growth inhibition by cefotaxime: E. coli cells expressing TEM-1 are inhibited by 0.008 mg/L compared with 0.015 mg/L for those expressing TEM-12. TEM-12-producing strains have been selected by cefo- taxime, suggesting that this small change in MIC is sufficient growth advan- tage to select for this mutation (Negri et al., 2000). In a series of elegant experiments, it was demonstrated that cells expressing TEM-12 were selected over TEM-1 when the antibiotic concentrations were very low, but that TEM-1 was selected at higher cefotaxime concentrations. The authors concluded that this confirmed the idea of selective windows—a concentration range over which a particular mutation confers a specific growth advantage to the cells expressing it. The duration of the exposure of the cultures to the antibiotic, at concentrations in the selective window range, increased the selection for the TEM-12-bearing cells.

Evolution of Antibiotic Resistance within Patients 373

374 Ian R. Booth In these studies, it was noted that there was an unpredicted second, higher,

concentration range in which the TEM-12 enzyme was more advantageous to cells than TEM-1. This proved to be due to the selection of an OmpF ⫺ muta- tion that conferred the higher MIC when combined with TEM-12 rather than with TEM-1 (Negri et al., 2000). The experiment was repeated in mice inocu- lated with a mixed population of cells expressing either TEM-1 or TEM-12, by varying the dose of cefotaxime. As predicted from the in vitro experiments, TEM-12 cells were selected over TEM-1 cells at low concentrations of cefo- taxime but not at higher concentrations. The selective window appeared to

be larger than in the in vitro experiments but the overall effect was similar (Negri et al., 2000). These data show that apparently small differences in cell performance can be selected given the appropriate antibiotic concentra- tion. Moreover, it demonstrates that spatial heterogeneity within the human body, giving rise to protected compartments with lowered antibiotic penetra- tion, is likely to provide a selective environment for a range of resistance mutations.