Contribution of gene amplification to evolution of increased antibiotic resistance in Salmonella typhimurium

Abstract

The use of beta-lactam antibiotics has led to the evolution and global spread of a variety of resistance mechanisms, including beta-lactamases, a group of enzymes that degrade the beta-lactam ring. The evolution of increased beta-lactam resistance was studied by exposing independent lineages of Salmonella typhimurium to progressive increases in cephalosporin concentration. Each lineage carried a beta-lactamase gene (bla(TEM-1)) that provided very low resistance. In most lineages, the initial response to selection was an amplification of the bla(TEM-1) gene copy number. Amplification was followed in some lineages by mutations (envZ, cpxA, or nmpC) that reduced expression of the uptake functions, the OmpC, OmpD, and OmpF porins. The initial resistance provided by bla(TEM-1) amplification allowed the population to expand sufficiently to realize rare secondary point mutations. Mathematical modeling showed that amplification often is likely to be the initial response because events that duplicate or further amplify a gene are much more frequent than point mutations. These models show the importance of the population size to appearance of later point mutations. Transient gene amplification is likely to be a common initial mechanism and an intermediate in stable adaptive improvement. If later point mutations (allowed by amplification) provide sufficient adaptive improvement, the amplification may be lost.

Figure 1.—

The region containing the bla_TEM-1 gene. The structure of F′₁₂₈ was previously described (Kofoid et al. 2003).

Figure 2.—

Phylogeny of clones selected for increased resistance to cephalosporins. The cephalosporin concentration (milligrams per liter) used at each selection step is indicated in parentheses following the strain number. (A) CE selection with cephalothin. (B) CF selection with cefaclor. Boxes indicate clones that were examined in detail with respect to DNA copy number, RNA levels, and MIC (Figure 3) as well as associated mutations (Table 3). The lineage number is indicated in italics at the endpoint strain for each lineage examined.

Figure 3.—

Six lineages from the CE and two lineages from the CF evolution experiments were chosen to examine changes in DNA and RNA levels of the bla _TEM-1 gene and MIC values for all isolated clones during evolution in the presence of increasing levels of cephalothin or cefaclor. (A) Lineages with parallel increases in MIC and bla_TEM-1 gene copy number. (B) Lineages with increased MIC, initial bla _TEM-1 gene amplification, and subsequent secondary resistance mutations. The standard deviation of relative fold change in DNA and RNA levels is ∼15%.

Figure 4.—

mRNA levels of the genes encoding the porin proteins (OmpC, OmpF, and OmpD) in cpxA (DA13421) and envZ (DA13407) mutants and wild type (DA11049). The relative fold change for each omp mRNA was calculated as the expression level of the cephalosporin-resistant mutants divided by that of the parental T-BAG strain (DA11049). The standard deviation of relative fold change in mRNA levels is ∼10%.

Figure 5.—

Size and endpoints of amplified units in five clones with amplified bla_TEM-1 genes. Copy number of the amplified unit is indicated with n. Numbering and designations of Rep sequences and IS3 elements were previously described (Kofoid et al. 2003).

Figure 6.—

Expected fraction of the population that carries an amplification containing i extra gene copies as a function of the s-value per copy for i = 1, 2, 3, 5, 8 (solid curves from top to bottom). Curves are drawn between the simulated data points as indicated. Dashed curves are for a point mutation, Equation A3, assuming u = 10⁻⁷, 10⁻⁸, or 10⁻⁹, from top to bottom. k_dup = 3 × 10⁻⁴ and k_rec = 0.05 assumed in all cases.

Figure 7.—

The probability from Equation A4 that the population has at least one individual carrying an array with i extra gene copies (solid curves with i = 3, 4, 5, 8, 10 from top to bottom) or at least one with a point mutation (dashed curves for u = 10⁻⁷, 10⁻⁸, and 10⁻⁹, from top to bottom). The data from Figure 6 and N = 10⁶ were used. The two dotted curves with open symbols show the results for i = 4 and N = 10⁷ (top) or N = 10⁵ (bottom).