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Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model.

ABSTRACT: Bacteriophage therapy is a promising approach to address antimicrobial infections though questions remain regarding the impact of the immune response on clinical effectiveness. Here, we develop a mouse model to assess phage treatment using a cocktail of five phages from the Myoviridae and Siphoviridae families that target Vancomycin-Resistant Enterococcus gut colonization. Phage treatment significantly reduces fecal bacterial loads of Vancomycin-Resistant Enterococcus. We also characterize immune responses elicited following administration of the phage cocktail. While minimal innate responses are observed after phage administration, two rounds of treatment induces phage-specific neutralizing antibodies and accelerate phage clearance from tissues. Interestingly, the myophages in our cocktail induce a more robust neutralizing antibody response than the siphophages. This anti-phage immunity reduces the effectiveness of the phage cocktail in our murine model. Collectively, this study shows phage-specific immune responses may be an important consideration in the development of phage cocktails for therapeutic use.

SUBMITTER: Berkson JD

PROVIDER: S-EPMC10998888 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model.

Berkson Julia D JD Wate Claire E CE Allen Garrison B GB Schubert Alyxandria M AM Dunbar Kristin E KE Coryell Michael P MP Sava Rosa L RL Gao Yamei Y Hastie Jessica L JL Smith Emily M EM Kenneally Charlotte R CR Zimmermann Sally K SK Carlson Paul E PE

Nature communications 20240406 1

Bacteriophage therapy is a promising approach to address antimicrobial infections though questions remain regarding the impact of the immune response on clinical effectiveness. Here, we develop a mouse model to assess phage treatment using a cocktail of five phages from the Myoviridae and Siphoviridae families that target Vancomycin-Resistant Enterococcus gut colonization. Phage treatment significantly reduces fecal bacterial loads of Vancomycin-Resistant Enterococcus. We also characterize immun ...[more]

PMID: 38582763

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Project description:IntroductionVancomycin-resistant Enterococcus faecalis (VRE) poses a significant challenge in clinical settings due to its resistance to multiple antibiotics. Phage therapy offers a promising alternative to address this resistance crisis. However, critical gaps remain regarding optimal dosing, therapeutic design, and treatment timing for phage therapy targeting VRE-induced bacteremia.MethodsThe biological and genomic characteristics of a novel lytic phage specific to VRE were investigated. Its in vitro bactericidal and antibiofilm activities were evaluated, along with its synergy with antimicrobial agents. In vitro safety and protective efficacy were assessed using a mouse bacteremia model. The impact of phage therapy on gut microbiota was examined through 16S rDNA gene sequencing.ResultsWe isolated and characterized a novel lytic phage, vB_EfaS-1017, specific to vancomycin-resistant E. faecalis. This phage features a circular, double-stranded DNA genome (40,766 bp), sharing 91.19% identity and 79% coverage with Enterococcus phage vB_EfaS_SRH2. vB_EfaS-1017 exhibited robust bactericidal and antibiofilm activity in vitro and demonstrated synergy with levofloxacin. Safety assessments confirmed its non-toxicity to mammalian cells and lack of hemolytic activity. In a mouse bacteremia model, phage treatment alone rescued 60% of infected mice, while combining phage with levofloxacin increased survival to 80%. Prophylactic administration of phage 24 hours prior to infection failed to prevent mortality. However, a combination of prophylactic phage administration and delayed treatment rescued 60% of mice, compared to 100% mortality in the delayed treatment alone group. Additionally, phage therapy helped maintain or restore gut microbiota balance.DiscussionThese findings underscore the potential of phage-antibiotic combinations as a superior therapeutic strategy against VRE infections. The observed synergy between phages and antibiotics highlights a promising approach to overcoming bacterial resistance and improving clinical outcomes. Furthermore, prophylactic phage administration may provide a critical time window for effective delayed treatment. Further preclinical research is essential to refine phage therapy protocols for clinical application.

Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Project description:Vancomycin-resistant Enterococcus (VRE) is a leading cause of hospital-acquired infections and continues to spread despite widespread implementation of pathogen-targeted control guidelines. Commensal gut microbiota provide colonization resistance to VRE, but the role of gut microbiota in VRE acquisition in at-risk patients is unknown. To address this gap in our understanding, we performed a case-control study of gut microbiota in hospitalized patients who did (cases) and did not (controls) acquire VRE. We matched case subjects to control subjects by known risk factors and "time at risk," defined as the time elapsed between admission until positive VRE screen. We characterized gut bacterial communities using 16S rRNA gene amplicon sequencing of rectal swab specimens. We analyzed 236 samples from 59 matched case-control pairs. At baseline, case and control subjects did not differ in gut microbiota when measured by community diversity (P = 0.33) or composition (P = 0.30). After hospitalization, gut communities of cases and controls differed only in the abundance of the Enterococcus-containing operational taxonomic unit (OTU), with the gut microbiota of case subjects having more of this OTU than time-matched control subjects (P = 0.01). Otherwise, case and control communities after the time at risk did not differ in diversity (P = 0.33) or community structure (P = 0.12). Among patients who became VRE colonized, those having the Blautia-containing OTU on admission had lower Enterococcus relative abundance once colonized (P = 0.004). Our results demonstrate that the 16S profile of the gut microbiome does not predict VRE acquisition in hospitalized patients, likely due to rapid and profound microbiota change. The gut microbiome does not predict VRE acquisition, but it may be associated with Enterococcus expansion, suggesting that these should be considered two distinct processes.IMPORTANCE The Centers for Disease Control and Prevention estimates that VRE causes an estimated 54,000 infections and 539 million dollars in attributable health care costs annually. Despite improvements in hand washing, environmental cleaning, and antibiotic use, VRE is still prevalent in many hospitals. There is a pressing need to better understand the processes by which patients acquire VRE. Multiple lines of evidence suggest that intestinal microbiota may help some patients resist VRE acquisition. In this large case-control study, we compared the 16S profile of intestinal microbiota on admission in patients that did and did not subsequently acquire VRE. The 16S profile did not predict subsequent VRE acquisition, in part due to rapid and dramatic change in the gut microbiome following hospitalization. However, Blautia spp. present on admission predicted decreased Enterococcus abundance after VRE acquisition, and Lactobacillus spp. present on admission predicted Enterococcus dominance after VRE acquisition. Thus, VRE acquisition and domination may be distinct processes.

Dataset Information

Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model.

Publications

Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model.

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