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Background Vancomycin-resistant enterococci (VRE) are a major cause of morbidity and mortality in immunocompromised patients. Tracking the dissemination of VRE strains is crucial to understand the dynamics of emergence and spread of VRE in the hospital setting. Methods Whole genome sequencing (WGS) and phylogenetic analyses were performed to identify dominant VRE strains and potential transmission networks between 35 patients with VRE-positive rectal swabs and their rooms (main rooms and bathrooms) on the leukemia (LKM) and the hematopoietic cell transplant (HCT) floors. Sequence types (STs), drug resistance genes, and patients’ outcomes were also determined. Results A total of 89 VRE strains grouped into 10 different STs, of which newly described STs were isolated from both floors (ST736, ST494, ST772, and ST1516). We observed highly genetically related strains transmitted between rooms, floors, and time periods in an average period of 39 days (ranging from 3 to 90 days). Of 5 VRE bacteremia events, 3 strains were lacking the pili operon fms14–17–13 (ST203) and the remaining 2 were resistant to daptomycin (DAP; ST736, ST664). Of 10 patients harboring DAP-resistant strains, only 2 were exposed to DAP within 4 months before strain recovery. Conclusions Our comparisons of VRE strains derived from the environment and immunocompromised patients confirmed horizontal transfer of highly related genetic lineages of multidrug-resistant (particularly to DAP) VRE strains between HCT and LKM patients and their room environment. Implementing WGS can be useful in distinguishing VRE reservoirs where interventions can be targeted to prevent and control the spread of highly resistant organisms.

Phages are naturally occurring viruses that selectively kill bacterial species without disturbing the individual’s normal flora, averting the collateral damage of antimicrobial usage. The safety and the effectiveness of phages have been mainly confirmed in the food industry as well as in animal models. In this study, we report on the successful isolation of phages specific to Vancomycin-resistant Enterococci, including Enterococcus faecium (VREfm) and Enterococcus faecalis from sewage samples, and demonstrate their efficacy and safety for VREfm infection in the greater wax moth Galleria mellonella model. No virulence-associated genes, antibiotic resistance genes or integrases were detected in the phages’ genomes, rendering them safe to be used in an in vivo model. Phages may be considered as potential agents for therapy for bacterial infections secondary to multidrug-resistant organisms such as VREfm.

Control CMV reactivation after HCT is highly dependent on CMV-specific T cells. Using 13-color flow cytometry, we studied CD8+ T cell responses to pp65 and IE-1 CMV peptide stimulation in cryopreserved PBMC from three clinically distinct subgroups (n = 10 each) of HCT patients: 1) Elite Controllers (EC): CMV seropositive (R+) recipients who never reactivated CMV based on weekly surveillance testing; 2) Spontaneous Controllers (SC): CMV R+ recipients who spontaneously resolved low-grade viremia without antiviral therapy; and 3) Non-controllers (NC): CMV R+ recipients who experienced high-grade CMV viremia (>1000 IU/mL) requiring antiviral therapy. We identified two CMV-specific CD8+ T cell cytokine signatures, measured at day +30, that were strongly associated with the risk of CMV reactivation (Figure 1): i) the non-protective signature (NPS) consisting of IL-2 neg IFNg pos TNFa neg MIP-1b pos CD8+ T cells was positively associated with CMV reactivation (4.9% of CMV-specific CD8+ T cells vs. 19.4 P = .002 for EC vs. SC/NC; 4.9 vs. 10.8 P = .02 for EC vs. SC; 4.9 vs. 22.8 P = .005 for EC vs. NC for pp65 stimulated cells; similar trends were observed in IE-1 stimulated cells); ii) the protective signature (PS) consisted of quadruple producers (IL-2 pos IFNg pos TNFa pos MIP-1b pos), and was significantly reduced 94

BackgroundVancomycin-resistant Enterococcus faecium (VRE) is a major multidrug-resistant organism which may cause infection or colonization in hematopoietic cell transplant (HCT) patients. The use of VRE-specific bacteriophages (phages) may potentially help eradicate VRE colonization and subsequent infections. To test the efficacy and safety of phages against VRE in vivo, a cocktail combining four phages was used in a VRE-infected larva model.MethodsThe pre-screening model Greater Wax Galleria mellonella larva was used in this study. Larvae were infected with VRE by injecting a VRE strain isolated from stools of a VRE-colonized HCT patient at a concentration of 107 colony-forming units/10 μL. A single phage (MDA1) or a phage cocktail (MDA1, MDA2, MDA3, and MDA4) were also injected at a concentration of 106 colony-forming units/10 μL. Two model groups were tested; a prevention group (PG) and a treatment group (TG). For the PG, phages were administered 1 hour before bacterial injection whereas the TG were injected with phages 1 hour post bacterial injection. Control groups included larvae injected with bacteria alone, phages alone (to measure toxicity due to phage administration), sterile media (to measure any lethal effects due to physical trauma from the injection), or without any manipulation. Every group was composed of 5 larvae. The insect’s health state was observed and scored after 8 hours of incubation at 37ºC using a published health index scoring system.ResultsPhages improved survival of VRE-infected larvae (table). Only 32% of the VRE-infected larvae survived after 8 hours of infection whereas more than 80% survived when adding phages, whether phages were administered before or after VRE infection. The phage cocktail was shown to be more effective than the single phage MDA1 in improving survival (66% vs. 82% survival). Injecting larvae with phages alone was safe as the same survival rate was observed when compared with those injected with sterile media or those without manipulation.ConclusionThe use of larva model G. mellonella allows for rapid and efficient screening of the bacterial virulence and phage efficacy and safety. Such results highlight the feasibility and the potential impact of phage therapy on VRE colonization and infections. DisclosuresRoy F. Chemaly, MD, MPH, FACP, FIDSA, Chimerix: Advisory Board, Research Grant; Clinigen: Advisory Board; Merck: Advisory Board, Consultant, Grant/Research Support, Research Grant, Speaker’s Bureau; Oxford immunotec: Consultant, Grant/Research Support; Shire: Research Grant, Speaker’s Bureau; Viracor: Grant/Research Support.

BackgroundVRE are a major cause of morbidity and mortality in immunocompromised patients. Tracking the dissemination of VRE strains is crucial to understand the dynamics of infections, emergence, and spread of VRE in the hospital setting.MethodsWhole-genome sequencing (WGS) and phylogenetic analyses were performed to identify dominant VRE strains and potential transmission networks between patients and their rooms on the leukemia (LKM) and the stem cell transplant (SCT) units, located on two consecutive floors. We included 35 VRE-positive rectal swabs from SCT and LKM patients, and 55 environmental swabs from the patients’ main rooms and bathrooms. Sequence types, drug resistance genes, virulence genes, and patients’ outcomes were also determined.ResultsWe identified VRE strains with newly described sequence types (ST) such as ST736, ST494, and ST772 which were isolated from both floors. One VRE genetic lineage belonged to ST494 (only previously isolated in Peru and was the only VanB-type strain). All other strains harbored the vanA gene. We observed highly genetically related strains transmitted between distinct rooms, floors, and time periods within the hospital in a period of 1 month (figure). Of five VRE bacteremia events, three strains were lacking the pili operon fms14-17-13 (ST203) and the remaining two were resistant to daptomycin (ST736, ST664) (figure). Of 10 patients harboring daptomycin-resistant strains, only 3 (30%) were exposed to daptomycin within 18 months before strain recovery.ConclusionOur findings confirmed horizontal transfer of highly related genetic lineages of multidrug resistant and invasive VRE strains between SCT and LKM patients and their room environment. New STs were identified and some correlated with bacteremia events. The use of a routine real-time WGS can characterize VRE strains and identify potential reservoirs of transmission in the healthcare setting in order to design interventions to prevent and control the spread of opportunistic and highly resistant organisms. Disclosures C. Arias, Merck & Co., Inc.: Grant Investigator, Research support. MeMed: Grant Investigator, Research support. Allergan: Grant Investigator, Research support. M. Stibich, Xenex Services: Employee, Salary. R. F. Chemaly, Xenex Services: Consultant and Grant Investigator, Research grant.

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BackgroundVancomycin-resistant Enterococci (VRE) is a well-known infectious complication among immunocompromised patients, especially hematopoietic cell transplant (HCT) recipients. VRE colonization of the gastrointestinal tract could be associated with VRE bacteremia and worse outcomes in HCT recipients. The use of systemic antibiotics to eradicate VRE colonization is highly discouraged because of the lack of efficacy, the rapid onset of antibiotic resistance, and the disruption of the normal microbiota. Bacteriophages (phages) may constitute a good alternative to antibiotics to eliminate specific pathogens without disrupting the patient’s normal microbiota.MethodsSewage samples were collected from the City of Houston for phages isolation. Samples were centrifuged, filtered and exposed to several targeted VRE host strains. After several amplification, the final filtrate was titrated and checked for the presence of VRE-specific phages. Isolated phages were observed under electron microscopy and were tested against multiple VRE strains isolated from different sources including patients’ stool samples, patients’ room environment, sewage samples, clinical isolates of daptomycin-resistant VRE strains or vancomycin-susceptible Enterococcus faecium (VSEF) and Enterococcus faecalis (VSEf) strains.ResultsFour VRE-specific phages were isolated from sewage samples (MDA1, MDA2, MDA3, and MDA4). All phages belong to the Caudovirales order. Phage MDA1 belongs to the Podoviridae family, phage MDA2 is a Siphoviridae, whereas MDA3 and MDA4 belong to the Myoviridae family (Figure 1A). All phages were lytic and were able to inhibit at least four VRE strains and only MDA1 had activity against VSEF and MDA4 against VSEf. The efficacy of these lytic phages complemented one another as the combination of these four phages inhibited all different VRE strains (Figure 1B).ConclusionOur results highlight the feasibility and the potential success of these phages in inhibiting VRE in vitro. These VRE-specific phage cocktails may be used in future studies to reduce VRE colonization and subsequent infections in HCT recipients. DisclosuresRoy F. Chemaly, MD, MPH, FACP, FIDSA, Chimerix: Advisory Board, Research Grant; Clinigen: Advisory Board; Merck: Advisory Board, Consultant, Grant/Research Support, Research Grant, Speaker’s Bureau; Oxford immunotec: Consultant, Grant/Research Support; Shire: Research Grant, Speaker’s Bureau; Viracor: Grant/Research Support.