Differential in vitro susceptibility to ampicillin/ceftriaxone combination therapy among Enterococcus faecalis infective endocarditis clinical isolates.

OBJECTIVES: To investigate the genomic diversity and ß-lactam susceptibilities of Enterococcus faecalis collected from patients with infective endocarditis (IE). METHODS: We collected 60 contemporary E. faecalis isolates from definite or probable IE cases identified between 2018 and 2021 at the University of Pittsburgh Medical Center. We used whole-genome sequencing to study bacterial genomic diversity and employed antibiotic checkerboard assays and a one-compartment pharmacokinetic-pharmacodynamic (PK/PD) model to investigate bacterial susceptibility to ampicillin and ceftriaxone both alone and in combination. RESULTS: Genetically diverse E. faecalis were collected, however, isolates belonging to two STs, ST6 and ST179, were collected from 21/60 (35%) IE patients. All ST6 isolates encoded a previously described mutation upstream of penicillin-binding protein 4 (pbp4) that is associated with pbp4 overexpression. ST6 isolates had higher ceftriaxone MICs and higher fractional inhibitory concentration index values for ampicillin and ceftriaxone (AC) compared to other isolates, suggesting diminished in vitro AC synergy against this lineage. Introduction of the pbp4 upstream mutation found among ST6 isolates caused increased ceftriaxone resistance in a laboratory E. faecalis isolate. PK/PD testing showed that a representative ST6 isolate exhibited attenuated efficacy of AC combination therapy at humanized antibiotic exposures. CONCLUSIONS: We find evidence for diminished in vitro AC activity among a subset of E. faecalis IE isolates with increased pbp4 expression. These findings suggest that alternate antibiotic combinations against diverse contemporary E. faecalis IE isolates should be evaluated.

Isolation and Characterization of Lytic Bacteriophages Targeting Diverse Enterobacter spp. Clinical Isolates.

Background: Enterobacter spp. are opportunistic pathogens that cause nosocomial infections. Bacteriophages could be used to treat antibiotic-resistant Enterobacter infections. Materials and Methods: We used 10 genetically diverse clinical Enterobacter spp. isolates to identify lytic bacteriophages in hospital and municipal wastewater. Comparative genomics was performed on host bacterial isolates and isolated phages. Activity of each phage against all 10 host isolates was determined. We also tested phage activity against paired isolates from two patients who developed ceftazidime-avibactam resistance. Results: Bacteria belonged to three Enterobacter species and Klebsiella aerogenes. We isolated 12 bacteriophages, most of which belonged to the Myoviridae and Autographiviridae families. Most phages were able to lyse multiple bacterial isolates, and many lysed isolates of different species. Ceftazidime-avibactam-resistant isolates were still phage susceptible, and one isolate showed increased susceptibility compared with the parent isolate. Conclusion: The phages we isolated expand the diversity of Enterobacter-targeting phages, and could be useful for treating antibiotic-resistant Enterobacter infections.

Genomic characterization of lytic bacteriophages targeting genetically diverse Pseudomonas aeruginosa clinical isolates.

Pseudomonas aeruginosa infections can be difficult to treat and new therapeutics are needed. Bacteriophage therapy is a promising alternative to traditional antibiotics, but large numbers of isolated and characterized phages are lacking. We collected 23 diverse P. aeruginosa isolates from people with cystic fibrosis (CF) and clinical infections, and used them to screen and isolate over a dozen P. aeruginosa-targeting phages from hospital wastewater. Phages were characterized with genome sequencing, comparative genomics, and lytic activity screening against all 23 bacterial host isolates. We evolved bacterial mutants that were resistant to phage infection for four different phages, and used genome sequencing and functional analysis to study them further. We also tested phages for their ability to kill P. aeruginosa grown in biofilms in vitro and ex vivo on CF airway epithelial cells. Overall, this study demonstrates how systematic genomic and phenotypic characterization can be deployed to develop bacteriophages as precision antibiotics.