Bacteriophage and antibiotic combination therapy for recurrent Enterococcus faecium bacteremia.

Enterococcus faecium is a member of the human gastrointestinal (GI) microbiota but can also cause invasive infections, especially in immunocompromised hosts. Enterococci display intrinsic resistance to many antibiotics, and most clinical E. faecium isolates have acquired vancomycin resistance, leaving clinicians with a limited repertoire of effective antibiotics. As such, vancomycin-resistant E. faecium (VREfm) has become an increasingly difficult to treat nosocomial pathogen that is often associated with treatment failure and recurrent infections. We followed a patient with recurrent E. faecium bloodstream infections (BSIs) of increasing severity, which ultimately became unresponsive to antibiotic combination therapy over the course of 7 years. Whole-genome sequencing (WGS) showed that the patient was colonized with closely related E. faecium strains for at least 2 years and that invasive isolates likely emerged from a large E. faecium population in the patient's gastrointestinal (GI) tract. The addition of bacteriophage (phage) therapy to the patient's antimicrobial regimen was associated with several months of clinical improvement and reduced intestinal burden of VRE and E. faecium. In vitro analysis showed that antibiotic and phage combination therapy improved bacterial growth suppression compared to therapy with either alone. Eventual E. faecium BSI recurrence was not associated with the development of antibiotic or phage resistance in post-treatment isolates. However, an anti-phage-neutralizing antibody response occurred that coincided with an increased relative abundance of VRE in the GI tract, both of which may have contributed to clinical failure. Taken together, these findings highlight the potential utility and limitations of phage therapy to treat antibiotic-resistant enterococcal infections. IMPORTANCE: Phage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patient's quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patient's stool. Eventually, an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit.

Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium.

Enterococcus faecium, a commensal of the human intestine, has emerged as a hospital-adapted, multi-drug resistant (MDR) pathogen. Bacteriophages (phages), natural predators of bacteria, have regained attention as therapeutics to stem the rise of MDR bacteria. Despite their potential to curtail MDR E. faecium infections, the molecular events governing E. faecium-phage interactions remain largely unknown. Such interactions are important to delineate because phage selective pressure imposed on E. faecium will undoubtedly result in phage resistance phenotypes that could threaten the efficacy of phage therapy. In an effort to understand the emergence of phage resistance in E. faecium, three newly isolated lytic phages were used to demonstrate that E. faecium phage resistance is conferred through an array of cell wall-associated molecules, including secreted antigen A (SagA), enterococcal polysaccharide antigen (Epa), wall teichoic acids, capsule, and an arginine-aspartate-aspartate (RDD) protein of unknown function. We find that capsule and Epa are important for robust phage adsorption and that phage resistance mutations in sagA, epaR, and epaX enhance E. faecium susceptibility to ceftriaxone, an antibiotic normally ineffective due to its low affinity for enterococcal penicillin binding proteins. Consistent with these findings, we provide evidence that phages potently synergize with cell wall (ceftriaxone and ampicillin) and membrane-acting (daptomycin) antimicrobials to slow or completely inhibit the growth of E. faecium Our work demonstrates that the evolution of phage resistance comes with fitness defects resulting in drug sensitization and that lytic phages could serve as effective antimicrobials for the treatment of E. faecium infections.

Phage Cocktails with Daptomycin and Ampicillin Eradicates Biofilm-Embedded Multidrug-Resistant Enterococcus faecium with Preserved Phage Susceptibility.

Multidrug-resistant (MDR) Enterococcus faecium is a challenging nosocomial pathogen known to colonize medical device surfaces and form biofilms. Bacterio (phages) may constitute an emerging anti-infective option for refractory, biofilm-mediated infections. This study evaluates eight MDR E. faecium strains for biofilm production and phage susceptibility against nine phages. Two E. faecium strains isolated from patients with bacteremia and identified to be biofilm producers, R497 (daptomycin (DAP)-resistant) and HOU503 (DAP-susceptible dose-dependent (SDD), in addition to four phages with the broadest host ranges (ATCC 113, NV-497, NV-503-01, NV-503-02) were selected for further experiments. Preliminary phage-antibiotic screening was performed with modified checkerboard minimum biofilm inhibitory concentration (MBIC) assays to efficiently screen for bacterial killing and phage-antibiotic synergy (PAS). Data were compared by one-way ANOVA and Tukey (HSD) tests. Time kill analyses (TKA) were performed against R497 and HOU503 with DAP at 0.5× MBIC, ampicillin (AMP) at free peak = 72 µg/mL, and phage at a multiplicity of infection (MOI) of 0.01. In 24 h TKA against R497, phage-antibiotic combinations (PAC) with DAP, AMP, or DAP + AMP combined with 3- or 4-phage cocktails demonstrated significant killing compared to the most effective double combination (ANOVA range of mean differences 2.998 to 3.102 log10 colony forming units (CFU)/mL; p = 0.011, 2.548 to 2.868 log10 colony forming units (CFU)/mL; p = 0.023, and 2.006 to 2.329 log10 colony forming units (CFU)/mL; p = 0.039, respectively), with preserved phage susceptibility identified in regimens with 3-phage cocktails containing NV-497 and the 4-phage cocktail. Against HOU503, AMP combined with any 3- or 4-phage cocktail and DAP + AMP combined with the 3-phage cocktail ATCC 113 + NV-497 + NV-503-01 demonstrated significant PAS and bactericidal activity (ANOVA range of mean differences 2.251 to 2.466 log10 colony forming units (CFU)/mL; p = 0.044 and 2.119 to 2.350 log10 colony forming units (CFU)/mL; p = 0.028, respectively), however, only PAC with DAP + AMP maintained phage susceptibility at the end of 24 h TKA. R497 and HOU503 exposure to DAP, AMP, or DAP + AMP in the presence of single phage or phage cocktail resulted in antibiotic resistance stabilization (i.e., no antibiotic MBIC elevation compared to baseline) without identified antibiotic MBIC reversion (i.e., lowering of antibiotic MBIC compared to baseline in DAP-resistant and DAP-SDD isolates) at the end of 24 h TKA. In conclusion, against DAP-resistant R497 and DAP-SDD HOU503 E. faecium clinical blood isolates, the use of DAP + AMP combined with 3- and 4-phage cocktails effectively eradicated biofilm-embedded MDR E. faecium without altering antibiotic MBIC or phage susceptibility compared to baseline.

Evaluation of Bacteriophage-Antibiotic Combination Therapy for Biofilm-Embedded MDR Enterococcus faecium.

Multidrug-resistant (MDR) Enterococcus faecium is a challenging pathogen known to cause biofilm-mediated infections with limited effective therapeutic options. Lytic bacteriophages target, infect, and lyse specific bacterial cells and have anti-biofilm activity, making them a possible treatment option. Here, we examine two biofilm-producing clinical E. faecium strains, daptomycin (DAP)-resistant R497 and DAP-susceptible dose-dependent (SDD) HOU503, with initial susceptibility to E. faecium bacteriophage 113 (ATCC 19950-B1). An initial synergy screening was performed with modified checkerboard MIC assays developed by our laboratory to efficiently screen for antibiotic and phage synergy, including at very low phage multiplicity of infection (MOI). The data were compared by one-way ANOVA and Tukey (HSD) tests. In 24 h time kill analyses (TKA), combinations with phage-DAP-ampicillin (AMP), phage-DAP-ceftaroline (CPT), and phage-DAP-ertapenem (ERT) were synergistic and bactericidal compared to any single agent (ANOVA range of mean differences 3.34 to 3.84 log10 CFU/mL; p < 0.001). Furthermore, phage-DAP-AMP and phage-DAP-CPT prevented the emergence of DAP and phage resistance. With HOU503, the combination of phage-DAP-AMP showed the best killing effect, followed closely by phage-DAP-CPT; both showed bactericidal and synergistic effects compared to any single agent (ANOVA range of mean differences 3.99 to 4.08 log10 CFU/mL; p < 0.001).

Evaluation of Bacteriophage Cocktails Alone and in Combination with Daptomycin against Daptomycin-Nonsusceptible Enterococcus faecium.

Enterococcus faecium is a significant multidrug-resistant pathogen. Bacteriophage cocktails are being proposed to complement antibiotic therapy. After a screen of 8 E. faecium strains against 4 phages, 2 phages (113 and 9184) with the broadest host ranges were chosen for further experiments. Transmission electron microscopy, whole-genome sequencing, comparative genome analyses, and time-kill analyses were performed. Daptomycin (DAP) plus the phage cocktail (113 [myophage] and 9184 [siphopage]) showed bactericidal activity in most regimens, while DAP addition prevented phage 9184 resistance against daptomycin-nonsusceptible E. faecium.

AIDS-related Cryptococcus neoformans choroiditis.

We describe a case of Cryptococcal choroiditis in a person with advanced HIV/AIDS. A 29-year-old male with AIDS presented with fever, photophobia, and ataxia secondary to cryptococcal and toxoplasma meningoencephalitis. Dilated fundoscopic examination revealed bilateral and multifocal posterior infiltrates consistent with cryptococcal choroiditis. Treatment with parenteral and intravitreal liposomal amphotericin B, oral flucytosine, and oral trimethoprim-sulfamethoxazole led to resolution of his symptoms and improvement in his vision. Our case documents a rare, intraocular opportunistic infection and highlights the importance of ophthalmologic examination in immunocompromised hosts with visual symptoms and invasive fungal infection. We discuss diagnostic and treatment considerations in cryptococcal choroiditis.

Increased cryptococcal meningitis mortality among HIV negative, non-transplant patients: a single US center cohort study.

Cryptococcal meningitis (CM) is an opportunistic fungal infection associated with human immunodeficiency virus (HIV) and other forms of immunosuppression. We lack a clear understanding of CM associated mortality among HIV-negative, non-transplant patients in the United States (US). This article compares clinical features and outcomes across HIV status in patients with laboratory-confirmed CM. METHODS: A retrospective cohort study was performed that included adult patients with laboratory-confirmed CM treated at an academic tertiary hospital between January 2000 and September 2018. Those with a history of organ transplant or non-meningeal infections were excluded. Data were gathered on demographics, HIV status, clinical presentation, cerebrospinal fluid (CSF) profiles, neurological outcomes, hospital course, and mortality. RESULTS: A total of 70 patients with cryptococcal disease were identified. Our final sample included 36 CM patients, mean age was 48.8 ± 13.2 years; of this group, 66.7% (n = 24) had HIV. Median [interquartile range (IQR)] absolute CD4 count for the HIV group was 35 cells/µl (10-80 cells/µl). Non-HIV/non-transplant patients were significantly older (p < 0.001) and had higher rates of altered mental status (AMS) on presentation (58.3% versus 25%, p = 0.05). Non-HIV patients/non-transplant patients had significantly higher CSF white blood cell (WBC) count (p = 0.02), lower CSF glucose (p = 0.005), and higher CSF protein (p < 0.001) compared with HIV patients. There was no significant variation in temperature, blood pressure, WBC count, serum sodium, CSF opening pressure, length of stay, intensive care unit admission, or neurological outcomes. Overall, 90-day all-cause mortality was 19.4%: mortality rates were significantly higher in non-HIV/non-transplant patients at both 90 days (41.7% versus 8.3%, p = 0.017) and 1 year (41.7% versus 12.5%, p = 0.047). CONCLUSION: Compared with HIV-infected individuals, non-HIV/non-transplant CM patients have a higher CSF WBC count at the time of diagnosis, higher rates of AMS on presentation, and higher rates of 90-day and 1-year all-cause mortality. Further prospective research is needed to identify the hallmarks of CM in non-HIV/non-transplant patients to facilitate early identification and intervention.

Molecular mechanisms of enterococcal-bacteriophage interactions and implications for human health.

Once overlooked as passive bystanders of the human intestinal microbiota, new evidence is shedding light on the importance of enterococci and their bacteriophages (phages) in shaping human health. Natural predators of enterococci, phages represent a narrow spectrum, precision targeting modality for the eradication of problematic enterococci within the microbiota or infected tissue. The identification of enterococcal phage receptors, absorption factors, and transcriptional responses following phage infection reveals a complex predator-prey relationship that modulates enterococcal cell surface architecture, susceptibility to antibiotics, and adaptation to host associated environments. Considering the dry up of contemporary antibiotic discovery pipelines in the pharmaceutical industry and a continued emergence of multidrug-resistant enterococci, enterococcal phages may serve as bonafide therapeutics. We highlight current advances in enterococcal phage biology with emphasis on recent breakthroughs in potential therapeutic applications that place enterococcal phages at the forefront of next-generation biologics.

Bacteriophage-Antibiotic Combinations for Enterococcus faecium with Varying Bacteriophage and Daptomycin Susceptibilities.

Concerns regarding increased prevalence of daptomycin (DAP)-resistant strains necessitate novel therapies for Enterococcus faecium infections. Obligately lytic bacteriophages are viruses that target, infect, and kill bacterial cells. Limited studies have evaluated phage-antibiotic combinations against E. faecium After an initial screen of eight E. faecium strains, three strains with varying DAP/phage susceptibilities were selected for further experiments. Phage-to-strain specificity contributed to synergy with antibiotics by time-kill analyses and was associated with lower development of phage resistance.

Corticosteroids for Posttransplant Immune Reconstitution Syndrome in Cryptococcus gattii Meningoencephalitis: Case Report and Literature Review.

Cryptococcus gattii represents an emerging fungal pathogen of immunocompromised and immunocompetent hosts in the United States. To our knowledge, this is the first case of posttransplant immune reconstitution syndrome due to C. gattii meningoencephalitis successfully treated with corticosteroids. We also report successful maintenance phase treatment with isavuconazole, a novel triazole, following fluconazole-induced prolonged QT syndrome.

Evolution in fast forward: a potential role for mutators in accelerating Staphylococcus aureus pathoadaptation.

Pathogen evolution and subsequent phenotypic heterogeneity during chronic infection are proposed to enhance Staphylococcus aureus survival during human infection. We tested this theory by genetically and phenotypically characterizing strains with mutations constructed in the mismatch repair (MMR) and oxidized guanine (GO) system, termed mutators, which exhibit increased spontaneous-mutation frequencies. Analysis of these mutators revealed not only strain-dependent increases in the spontaneous-mutation frequency but also shifts in mutational type and hot spots consistent with loss of GO or MMR functions. Although the GO and MMR systems are relied upon in some bacterial species to prevent reactive oxygen species-induced DNA damage, no deficit in hydrogen peroxide sensitivity was found when either of these DNA repair pathways was lost in S. aureus. To gain insight into the contribution of increased mutation supply to S. aureus pathoadaptation, we measured the rate of α-hemolysin and staphyloxanthin inactivation during serial passage. Detection of increased rates of α-hemolysin and staphyloxanthin inactivation in GO and MMR mutants suggests that these strains are capable of modifying virulence phenotypes implicated in mediating infection. Accelerated derivation of altered virulence phenotypes, combined with the absence of increased ROS sensitivity, highlights the potential of mutators to drive pathoadaptation in the host and serve as catalysts for persistent infections.