A fitness cost associated with the antibiotic resistance enzyme SME-1 beta-lactamase

Abstract

The bla(TEM-1) beta-lactamase gene has become widespread due to the selective pressure of beta-lactam use and its stable maintenance on transferable DNA elements. In contrast, bla(SME-1) is rarely isolated and is confined to the chromosome of carbapenem-resistant Serratia marcescens strains. Dissemination of bla(SME-1) via transfer to a mobile DNA element could hinder the use of carbapenems. In this study, bla(SME-1) was determined to impart a fitness cost upon Escherichia coli in multiple genetic contexts and assays. Genetic screens and designed SME-1 mutants were utilized to identify the source of this fitness cost. These experiments established that the SME-1 protein was required for the fitness cost but also that the enzyme activity of SME-1 was not associated with the fitness cost. The genetic screens suggested that the SME-1 signal sequence was involved in the fitness cost. Consistent with these findings, exchange of the SME-1 signal sequence for the TEM-1 signal sequence alleviated the fitness cost while replacing the TEM-1 signal sequence with the SME-1 signal sequence imparted a fitness cost to TEM-1 beta-lactamase. Taken together, these results suggest that fitness costs associated with some beta-lactamases may limit their dissemination.

Figure 1.—

Effect of β-lactamase genes on E. coli growth rates. (A) Growth curves in LB broth of E. coli K12 XL1-Blue containing pTP123, pTP123-TEM-1, or pTP123-SME-1 were obtained by monitoring OD₆₀₀ over time. (B) Growth curves of a frameshifted SME-1 clone and point mutants that eliminate enzymatic activity (S70A:E166Q) or disulfide bond formation (C69V:C238V). (C) Growth curves of chimeric constructs in which signal sequences of TEM-1 and SME-1 have been exchanged. Error bars represent standard deviation of at least three independent experiments.

Figure 1.—

Effect of β-lactamase genes on E. coli growth rates. (A) Growth curves in LB broth of E. coli K12 XL1-Blue containing pTP123, pTP123-TEM-1, or pTP123-SME-1 were obtained by monitoring OD₆₀₀ over time. (B) Growth curves of a frameshifted SME-1 clone and point mutants that eliminate enzymatic activity (S70A:E166Q) or disulfide bond formation (C69V:C238V). (C) Growth curves of chimeric constructs in which signal sequences of TEM-1 and SME-1 have been exchanged. Error bars represent standard deviation of at least three independent experiments.

Figure 1.—

Effect of β-lactamase genes on E. coli growth rates. (A) Growth curves in LB broth of E. coli K12 XL1-Blue containing pTP123, pTP123-TEM-1, or pTP123-SME-1 were obtained by monitoring OD₆₀₀ over time. (B) Growth curves of a frameshifted SME-1 clone and point mutants that eliminate enzymatic activity (S70A:E166Q) or disulfide bond formation (C69V:C238V). (C) Growth curves of chimeric constructs in which signal sequences of TEM-1 and SME-1 have been exchanged. Error bars represent standard deviation of at least three independent experiments.

Figure 2.—

Effect of β-lactamase genes on culture viability. The number of colony-forming units per milliliter is indicative of culture viability. Data for E. coli K12 XL1-Blue containing the indicated plasmids were normalized to E. coli K12 XL1-Blue harboring isogenic plasmid without a β-lactamase gene. Two genetic contexts (pTP123 parent and pUC18-Kn parent plasmids) were examined. Error bars represent the standard deviation of at least six independent experiments. Significance is based on t-test against empty vector: ***P < 0.001.

Figure 3.—

Plasmid loss in the absence of antibiotic selection. Overnight cultures were spread onto LB agar plates and LB agar plates containing chloramphenicol to determine the percentage of cells harboring plasmid. Errors bars represent the standard deviation for at least two independent experiments.