Abstract
Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427 motif I; L475Q, 7 aa upstream of the 482SDN484 motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622 motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes.
Highlights
Enterococci are the third most commonly isolated nosocomial pathogens, accounting for 12% of all hospital infections (Hollenbeck and Rice, 2012)
The aims of this study were: 1) to investigate the in vitro antibacterial and bactericidal activity of BPR alone against E. faecalis clinical isolates belonging to selected antibiotic-resistance classes; 2) to analyze the occurrence of pbp4 mutations and verify their role in influencing the activity of b-lactams and, of BPR; 3) to compare the pbp4 expression levels in all E. faecalis clinical isolates with reduced susceptibility to beta-lactams and PBP4 alterations, with the aim of evaluating which of these alterations may be involved in non-susceptibility and BPR cidal activity, and how
The Penicillin-Resistant Ampicillin-Susceptible (PRAS) phenotype was described in several countries (Metzidie et al, 2006; Guardabassi et al, 2010; Conceicão et al, 2014), but its epidemiological and clinical impact remains ambiguous, as ampicillin is the treatment of choice for enterococcal infections (Kristich et al, 2014a) and penicillin MIC values were never reported (Mendes et al, 2016)
Summary
Enterococci are the third most commonly isolated nosocomial pathogens, accounting for 12% of all hospital infections (Hollenbeck and Rice, 2012). It was assumed that ampicillin- susceptible E. faecalis was susceptible to penicillin, but E. faecalis clinical isolates have been exhibiting increasing levels of resistance to penicillin, due to the emergence of Penicillin-Resistant Ampicillin-Susceptible (PRAS) isolates, eliminating b-lactams as a treatment option. This uncommon phenotype has been reported in various hospitals worldwide but its real epidemiological impact is still unknown (Metzidie et al, 2006; Guardabassi et al, 2010; Tan et al, 2014; Cabrera et al, 2020; Conceicão et al, 2020; Gawryszewska et al, 2021)
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