Functional genomics of antibiotic susceptibility in Enterococcus faecalis from infective endocarditis

Abstract

Enterococcus faecalis is an opportunistic pathogen that causes infective endocarditis. Despite in vitro synergy of the recommended combination ampicillin and ceftriaxone (AC), E. faecalis infective endocarditis (EFIE) mortality remains high. We characterized 119 isolates from EFIE patients in our health system from 2018 to 2023 genomically and phenotypically. Three genetic lineages (ST6, ST40, and ST179) accounted for 41% of all infections. ST6 isolates were less susceptible to ceftriaxone and AC in dual-antibiotic checkerboard assays, but also exhibited decreased survival in dual-antibiotic killing assays using humanized AC exposures. ST6 isolates encoded a known genetic disruption affecting pbp4 as well as mutation of a putative hydrolase called phnP. Functional studies confirmed that both alterations contributed to altered AC susceptibility. Finally, we found that ampicillin plus daptomycin showed robust growth inhibition and killing against EFIE isolates with decreased AC susceptibility. These findings enhance our understanding of EFIE and may guide better therapeutic strategies.

Keywords: Enterococcus faecalis; antibiotic resistance; ceftriaxone; endocarditis.

Figure 1.

Core genome phylogeny of Enterococcus faecalis infective endocarditis (EFIE) isolates. A midpoint-rooted RAxML tree of 119 E faecalis isolates from patients with possible or definite EFIE at the University of Pittsburgh Medical Center was constructed from a single-copy core genome alignment containing 1882 genes identified with Roary. Three dominant lineages, including ST6, ST179, and ST40 are shaded. For each isolate, the multilocus sequence type (ST), predicted resistances to antibiotic classes, cytolysin operon genes, cytolytic activity, fsr quorum-sensing system genes, and gelatinase activity are shown.

Figure 2.

Variability in ampicillin-ceftriaxone (AC) susceptibility among Enterococcus faecalis infective endocarditis isolates. Minimum inhibitory concentration (MIC) values of ampicillin (AMP; A) and ceftriaxone (CRO; B). C, Background-subtracted area under the curve (AUC) values from AC in vitro checkerboard assays. Each dot represents the average MIC or AUC value from 2 biological replicates. Lines show the median value of each group. *P < .05, **P < .01, ****P < .0001.

Figure 3.

Mutations associated with ceftriaxone resistance in ST6 isolates. A, The locus encoding phnP in ST6 isolates (top) is disrupted by insertion of a 6-gene mobile genetic element (bottom). B, The proportion of different multilocus sequence types (STs) among Enterococcus faecalis infective endocarditis isolates collected at the University of Pittsburgh Medical Center (UPMC-EFIE), human-associated E faecalis isolate genomes in the National Center for Biotechnology Information (NCBI-human associated), and human blood E faecalis isolate genomes in NCBI (NCBI-blood). C, Prevalence of pbp4−82delT mutation and phnP disruption among 2696 human-associated E faecalis isolate genomes in NCBI. D, Prevalence of genotypes shown in (C) over time among 550 ST6 isolates.

Figure 4.

In vitro ampicillin-ceftriaxone (AC) susceptibility of Enterococcus faecalis OG1RF wild-type, pbp4 and phnP mutants, and complemented strains. A–C, Minimum inhibitory concentration (MIC) values of ampicillin (AMP; A) and ceftriaxone (CRO; B), and background-subtracted area under the curve (AUC) values from AC in vitro checkerboard assays (C). Lines show the median value of each group. *P < .05, **P < .01; ns, not significant.

Figure 5.

Survival percentage after ampicillin-ceftriaxone exposure in time-kill assay. A, Survival percentages after 4 or 6 hours for all Enterococcus faecalis infective endocarditis isolates following exposure to ampicillin = 120 μg/mL plus ceftriaxone = 25.7 μg/mL. Survival percentage = [colony-forming units (CFU)/mL_{4h or 6h}] / [CFU/mL_0h]. Isolates are grouped by multilocus sequence type (ST) and lines show the median value of each group. B, Survival percentages after 4 and 6 hours for OG1RF wild-type, pbp4 mutant, and phnP mutant strains following exposure to ampicillin = 120 μg/mL plus ceftriaxone = 25.7 μg/mL. Lines show the median value of each group. *P < .05, ****P < .0001. LOD indicates the limit of detection.

Figure 6.

In vitro activity of alternative antibiotic combinations against Enterococcus faecalis infective endocarditis (EFIE) isolates with reduced ampicillin-ceftriaxone (AC) activity. A and B, Minimum inhibitory concentration (MIC; A) and fractional inhibitory concentration index (FICI) values (B) of 20 E faecalis isolates with the highest area under the curve values in AC checkerboard assays. One EFIE isolate was resistant to both teicoplanin and vancomycin and is not shown in A because its MIC exceeded the highest antibiotic concentrations tested. C, Survival percentages after 4 and 6 hours for 20 EFIE isolates comparing ampicillin = 120 μg/mL plus ceftriaxone = 25.7 μg/mL with ampicillin = 120 μg/mL and/or daptomycin = 8 μg/mL. Lines show the median value of each group. ****P < .0001. Abbreviations: AMP, ampicillin; CRO, ceftriaxone; DAP, daptomycin; FICI, fractional inhibitory concentration index; FOS, fosfomycin; LOD, limit of detection; MERO, meropenem; MIC, minimum inhibitory concentration; TCP, teicoplanin; VAN, vancomycin.