Excreted Antibiotics May Be Key to Emergence of Increasingly Efficient Antibiotic Resistance in Food Animal Production

Abstract

At a time when antibiotic resistance is seemingly ubiquitous worldwide, understanding the mechanisms responsible for successful emergence of new resistance genes may provide insights into the persistence and pathways of dissemination for antibiotic-resistant organisms in general. For example, Escherichia coli strains harboring a class A β-lactamase-encoding gene (bla_CTX-M-15) appear to be displacing strains that harbor a class C β-lactamase gene (bla_CMY-2) in Washington State dairy cattle. We cloned these genes with native promoters into low-copy-number plasmids that were then transformed into isogenic strains of E. coli, and growth curves were generated for two commonly administered antibiotics (ampicillin and ceftiofur). Both strains met the definition of resistance for ampicillin (≥32 μg/mL) and ceftiofur (≥16 μg/mL). Growth of the CMY-2-producing strain was compromised at 1,000 μg/mL ampicillin, whereas the CTX-M-15-producing strain was not inhibited in the presence of 3,000 μg/mL ampicillin or with most concentrations of ceftiofur, although there were mixed outcomes with ceftiofur metabolites. Consequently, in the absence of competing genes, E. coli harboring either gene would experience a selective advantage if exposed to these antibiotics. Successful emergence of CTX-M-15-producing strains where CMY-2-producing strains are already established, however, requires high concentrations of antibiotics that can only be found in the urine of treated animals (e.g., >2,000 μg/mL for ampicillin, based on literature). This ex vivo selection pressure may be important for the emergence of new and more efficient antibiotic resistance genes and likely for persistence of antibiotic-resistant bacteria in food animal populations. IMPORTANCE We studied the relative fitness benefits of a cephalosporin resistance enzyme (CTX-M-15) that is displacing a similar enzyme (CMY-2), which is extant in E. coli from dairy cattle in Washington State. In vitro experiments demonstrated that CTX-M-15 provides a significant fitness advantage, but only in the presence of very high concentrations of antibiotic that are only found when the antibiotic ampicillin, and to a lesser extent ceftiofur, is excreted in urine from treated animals. As such, the increasing prevalence of bacteria with bla_CTX-M-15 is likely occurring ex vivo. Interventions should focus on controlling waste from treated animals and, when possible, selecting antibiotics that are less likely to impact the proximal environment of treated animals.

Keywords: antibiotic; antibiotic resistance; antimicrobial; blaCMY-2; blaCTX-M-15; blaKPC-3; competition; fitness; hydrolysis; resistance; β-lactamase.

FIG 1

Relative protein production of CMY-2 (blue), CTX-M-15 (green) and KPC-3 (purple) by isogenic strains of E. coli with or without exposure to ceftiofur. Average normalized densitometry values (3 independent replicates; ± SEM) for Flag-tagged recombinant CMY-2, CTX-M, and KPC-3 after 8, 10, 12, 14, or 16 h of culture with no antibiotic (A) or with 64 μg/mL of ceftiofur (B). Significant differences were found, based on two-way ANOVAs and Tukey multiple-comparison test: *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001.

FIG 2

Average area under the curve (AUC) values for E. coli cultures exposed to ampicillin. Heatmap depicts average AUC for growth curves from CTX-M-15-, CMY-2-, and KPC-3-producing strains of E. coli and for the plasmid-only negative-control strain (p207) in the presence of no antibiotic (3 independent replicates) or 8 to 3,000 μg/mL (3 independent replicates). See Fig. S2 in the supplemental material for original growth curves and P values for multiple comparison tests.

FIG 3

Average area under the curve (AUC) values for E. coli cultures exposed to ceftiofur and its metabolites. Heatmap depicts average AUC for growth curves from CTX-M-15-, CMY-2-, and KPC-3-producing strains of E. coli and for the plasmid-only negative-control strain (p207) in the presence of 0 to 256 μg/mL ceftiofur (A), DFC (B), DFC-cysteine (C), or DFC-dimer (D). Values represent averages (3 independent replicates). See Fig. S3 to S6 in the supplemental material for original growth curves and P values for multiple-comparison tests.

FIG 4

Average k_cat/K_m values for hydrolysis of antibiotics. Values were estimated for hydrolysis of ampicillin (A), ceftiofur (B), DFC (C), DFC-cysteine (D), and DFC-dimer (E) using recombinant CMY-2, CTX-M-15, and KPC-3 enzymes. See Materials and Methods for additional details and Table 1 and Fig. S7 and S8 for supporting information.

FIG 5

Real-time PCR results for coculture competition assays. (A) Coculture of CMY-2- and CTX-M-15-producing strains. (B) Coculture of CMY-2- and KPC-3-producing strains. (C) Coculture of CTX-M-15- and KPC-3-producing strains. Each time point is the average ± SEM for three independent replicates.

FIG 6

E. coli cultures with ceftiofur (64 or 256 μg/mL) added at 5 or 8 h postinoculation. Cultures were initiated with no antibiotic for the first 5 h (A and C) or for the first 8 h (B and D), before adding 64 μg/mL ceftiofur (A and B) or 256 μg/mL ceftiofur (C and D). Each time point is the average ± SEM for three independent replicates. See Fig. S9 in the supplemental material for additional results.