Progress toward a vaccine for extraintestinal pathogenic E. coli (ExPEC) II: efficacy of a toxin-autotransporter dual antigen approach.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of worldwide morbidity and mortality, the top cause of antimicrobial-resistant (AMR) infections, and the most frequent cause of life-threatening sepsis and urinary tract infections (UTI) in adults. The development of an effective and universal vaccine is complicated by this pathogen's pan-genome, its ability to mix and match virulence factors and AMR genes via horizontal gene transfer, an inability to decipher commensal from pathogens, and its intimate association and co-evolution with mammals. Using a pan virulome analysis of >20,000 sequenced E. coli strains, we identified the secreted cytolysin α-hemolysin (HlyA) as a high priority target for vaccine exploration studies. We demonstrate that a catalytically inactive pure form of HlyA, expressed in an autologous host using its own secretion system, is highly immunogenic in a murine host, protects against several forms of ExPEC infection (including lethal bacteremia), and significantly lowers bacterial burdens in multiple organ systems. Interestingly, the combination of a previously reported autotransporter (SinH) with HlyA was notably effective, inducing near complete protection against lethal challenge, including commonly used infection strains ST73 (CFT073) and ST95 (UTI89), as well as a mixture of 10 of the most highly virulent sequence types and strains from our clinical collection. Both HlyA and HlyA-SinH combinations also afforded some protection against UTI89 colonization in a murine UTI model. These findings suggest recombinant, inactive hemolysin and/or its combination with SinH warrant investigation in the development of an E. coli vaccine against invasive disease.

Draft genome sequences of Pseudomonas strains zfem001-005 isolated from the intestine of larval zebrafish Danio rerio.

Here, we report the draft genome sequences of Pseudomonas strains zfem001-005, five isolates from the intestinal microbiota of healthy larval zebrafish Danio rerio at a developmental age of 7 days post fertilization. The isolates have been identified as Pseudomonas sediminis, Pseudomonas japonica, Pseudomonas otitidis, Pseudomonas sichuanensis, and Pseudomonas tohonis, respectively.

Assessment of a SARS-CoV-2 wastewater monitoring program in El Paso, Texas, from November 2020 to June 2022.

The border city of El Paso, Texas, and its water utility, El Paso Water, initiated a SARS-CoV-2 wastewater monitoring program to assess virus trends and the appropriateness of a wastewater monitoring program for the community. Nearly weekly sample collection at four wastewater treatment facilities (WWTFs), serving distinct regions of the city, was analyzed for SARS-CoV-2 genes using the CDC 2019-Novel coronavirus Real-Time RT-PCR diagnostic panel. Virus concentrations ranged from 86.7 to 268,000 gc/L, varying across time and at each WWTF. The lag time between virus concentrations in wastewater and reported COVID-19 case rates (per 100,00 population) ranged from 4-24 days for the four WWTFs, with the strongest trend occurring from November 2021 - June 2022. This study is an assessment of the utility of a geographically refined SARS-CoV-2 wastewater monitoring program to supplement public health efforts that will manage the virus as it becomes endemic in El Paso.

Appelmans protocol - A directed in vitro evolution enables induction and recombination of prophages with expanded host range.

Infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) present significant healthcare challenges due to limited treatment options. Bacteriophage (phage) therapy offers potential as an alternative treatment. However, the high host specificity of phages poses challenges for their therapeutic application. To broaden the phage spectrum, laboratory-based phage training using the Appelmans protocol was employed in this study. As a result, the protocol successfully expanded the host range of a phage cocktail targeting CRAB. Further analysis revealed that the expanded host range phages isolated from the output cocktail were identified as recombinant derivatives originating from prophages induced from encountered bacterial strains. These findings provide valuable genetic insights into the protocol's mechanism when applied to phages infecting A. baumannii strains that have never been investigated before. However, it is noteworthy that the expanded host range phages obtained from this protocol exhibited limited stability, raising concerns about their suitability for therapeutic purposes.

Wastewater analysis of Mpox virus in a city with low prevalence of Mpox disease: an environmental surveillance study.

Background: Tracking infectious diseases at the community level is challenging due to asymptomatic infections and the logistical complexities of mass surveillance. Wastewater surveillance has emerged as a valuable tool for monitoring infectious disease agents including SARS-CoV-2 and Mpox virus. However, detecting the Mpox virus in wastewater is particularly challenging due to its relatively low prevalence in the community. In this study, we aim to characterize three molecular assays for detecting and tracking the Mpox virus in wastewater from El Paso, Texas, during February and March 2023. Methods: In this study, a combined approach utilizing three real-time PCR assays targeting the C22L, F3L, and F8L genes and sequencing was employed to detect and track the Mpox virus in wastewater samples. The samples were collected from four sewersheds in the City of El Paso, Texas, during February and March 2023. Wastewater data was compared with reported clinical case data in the city. Findings: Mpox virus DNA was detected in wastewater from all the four sewersheds, whereas only one Mpox case was reported during the sampling period. Positive signals were still observed in multiple sewersheds after the Mpox case was identified. Higher viral concentrations were found in the pellet than in the supernatant of wastewater. Notably, an increasing trend in viral concentration was observed approximately 1-2 weeks before the reporting of the Mpox case. Further sequencing and epidemiological analysis provided supporting evidence for unreported Mpox infections in the city. Interpretation: Our analysis suggests that the Mpox cases in the community is underestimated. The findings emphasize the value of wastewater surveillance as a public health tool for monitoring infectious diseases even in low-prevalence areas, and the need for heightened vigilance to mitigate the spread of Mpox disease for safeguarding global health. Funding: Center of Infectious Diseases at UTHealth, the University of Texas System, and the Texas Epidemic Public Health Institute. The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of these funding organizations.

Wastewater sequencing reveals community and variant dynamics of the collective human virome.

Wastewater is a discarded human by-product, but its analysis may help us understand the health of populations. Epidemiologists first analyzed wastewater to track outbreaks of poliovirus decades ago, but so-called wastewater-based epidemiology was reinvigorated to monitor SARS-CoV-2 levels while bypassing the difficulties and pit falls of individual testing. Current approaches overlook the activity of most human viruses and preclude a deeper understanding of human virome community dynamics. Here, we conduct a comprehensive sequencing-based analysis of 363 longitudinal wastewater samples from ten distinct sites in two major cities. Critical to detection is the use of a viral probe capture set targeting thousands of viral species or variants. Over 450 distinct pathogenic viruses from 28 viral families are observed, most of which have never been detected in such samples. Sequencing reads of established pathogens and emerging viruses correlate to clinical data sets of SARS-CoV-2, influenza virus, and monkeypox viruses, outlining the public health utility of this approach. Viral communities are tightly organized by space and time. Finally, the most abundant human viruses yield sequence variant information consistent with regional spread and evolution. We reveal the viral landscape of human wastewater and its potential to improve our understanding of outbreaks, transmission, and its effects on overall population health.

Functional Genomics of Gastrointestinal Escherichia coli Isolated from Patients with Cancer and Diarrhea.

We describe the epidemiology and clinical characteristics of 29 patients with cancer and diarrhea in whom Enteroaggregative Escherichia coli (EAEC) was initially identified by GI BioFire panel multiplex. E. coli strains were successfully isolated from fecal cultures in 14 of 29 patients. Six of the 14 strains were identified as EAEC and 8 belonged to other diverse E. coli groups of unknown pathogenesis. We investigated these strains by their adherence to human intestinal organoids, cytotoxic responses, antibiotic resistance profile, full sequencing of their genomes, and annotation of their functional virulome. Interestingly, we discovered novel and enhanced adherence and aggregative patterns for several diarrheagenic pathotypes that were not previously seen when co-cultured with immortalized cell lines. EAEC isolates displayed exceptional adherence and aggregation to human colonoids compared not only to diverse GI E. coli , but also compared to prototype strains of other diarrheagenic E. coli . Some of the diverse E. coli strains that could not be classified as a conventional pathotype also showed an enhanced aggregative and cytotoxic response. Notably, we found a high carriage rate of antibiotic resistance genes in both EAEC strains and diverse GI E. coli isolates and observed a positive correlation between adherence to colonoids and the number of metal acquisition genes carried in both EAEC and the diverse E. coli strains. This work indicates that E. coli from cancer patients constitute strains of remarkable pathotypic and genomic divergence, including strains of unknown disease etiology with unique virulomes. Future studies will allow for the opportunity to re-define E. coli pathotypes with greater diagnostic accuracy and into more clinically relevant groupings.

Class-Driven Synergy and Antagonism between a Pseudomonas Phage and Antibiotics.

The ubiquitous bacterial pathogen Pseudomonas aeruginosa is responsible for severe infections in patients with burns, cystic fibrosis, and neutropenia. Biofilm formation gives physical refuge and a protected microenvironment for sessile cells, rendering cure by antibiotics a challenge. Bacteriophages have evolved to prey on these biofilms over millions of years, using hydrolases and depolymerases to penetrate biofilms and reach cellular targets. Here, we assessed how a newly discovered KMV-like phage (ΦJB10) interacts with antibiotics to treat P. aeruginosa more effectively in both planktonic and biofilm forms. By testing representatives of four classes of antibiotics (cephalosporins, aminoglycosides, fluoroquinolones, and carbapenems), we demonstrated class-dependent interactions between ΦJB10 and antibiotics in both biofilm clearance and P. aeruginosa killing. Despite identifying antagonism between some antibiotic classes and ΦJB10 at early time points, all classes showed neutral to favorable interactions with the phage at later time points. In one notable example where the antibiotic alone had poor activity against both biofilm and high-density planktonic cells, we found that addition of ΦJB10 demonstrated synergy and resulted in effective treatment of both. Further, ΦJB10 seemed to act as an adjuvant to several antibiotics, reducing the concentration of antibiotics required to ablate the biofilm. This report shows that phages such as ΦJB10 may be valuable additions to the armamentarium against difficult-to-treat biofilm-based infections.

A Retrospective, Observational Study of 12 Cases of Expanded-Access Customized Phage Therapy: Production, Characteristics, and Clinical Outcomes.

BACKGROUND: Antimicrobial resistance (AMR) is undermining modern medicine, a problem compounded by bacterial adaptation to antibiotic pressures. Phages are viruses that infect bacteria. Their diversity and evolvability offer the prospect of their use as a therapeutic solution. Reported are outcomes of customized phage therapy for patients with difficult-to-treat antimicrobial resistant infections. METHODS: We retrospectively assessed 12 cases of customized phage therapy from a phage production center. Phages were screened, purified, sequenced, characterized, and Food and Drug Administration-approved via the IND (investigational new drug) compassionate-care route. Outcomes were assessed as favorable or unfavorable by microbiologic and clinical standards. Infections were device-related or systemic. Other experiences such as time to treatment, antibiotic synergy, and immune responses were recorded. RESULTS: Fifty requests for phage therapy were received. Customized phages were generated for 12 patients. After treatment, 42% (5/12) of cases showed bacterial eradication and 58% (7/12) showed clinical improvement, with two-thirds of all cases (66%) showing favorable responses. No major adverse reactions were observed. Antibiotic-phage synergy in vitro was observed in most cases. Immunological neutralization of phages was reported in 5 cases. Several cases were complicated by secondary infections. Complete characterization of the phages (morphology, genomics, and activity) and their production (methods, sterility, and endotoxin tests) are reported. CONCLUSIONS: Customized phage production and therapy was safe and yielded favorable clinical or microbiological outcomes in two-thirds of cases. A center or pipeline dedicated to tailoring the phages against a patient's specific AMR bacterial infection may be a viable option where standard treatment has failed.

Effect of various types of extracellular DNA on V. hyugaensis biofilm formation.

Marine bacteria face a constant influx of new extracellular DNA (exDNA) due to the massive viral lysis that occurs in the ocean on a daily basis. Generally, biofilms have shown to be induced by self-secreted exDNA. However, the effect of various types of exDNA with varying lengths, self vs non-self, as well as guanine-cytosine content (GC) content on biofilm formation has not been explored, despite being a critical component of the extracellular polymeric substance. To test the effect of such exDNA on biofilms, a marine bioluminescent bacterium (Vibrio hyugaensis) was isolated from the Sippewissett Salt Marsh, USA, and treated with various types of exDNA. We observed rapid pellicle formation with distinct morphologies only in cultures treated with herring sperm gDNA, another Vibrio spp. gDNA, and an oligomer of 61-80% GC content. With pH measurements before and after the treatment, we observed a positive correlation between biofilm formation and the change to a more neutral pH. Our study highlights the importance of studying DNA-biofilm interaction by carefully examining the physical properties of the DNA and by varying its content, length, and source. Our observation may serve as the basis for future studies that seek to interrogate the molecular explanation for the various types of exDNA and their effects on biofilm formation. IMPORTANCE Bacteria mostly exist as biofilm, a protective niche that promotes protection from the environment and nutrient uptake. By forming these structures, bacteria have caused recalcitrant antibiotic-resistant infections, contamination of dairy and seafood, and fouling equipment in the industry. A critical component that makes up the extracellular polymeric substances, the structural component of a biofilm, is the extracellular DNA secreted by the bacteria found in the biofilm. However, previous studies on DNA and biofilm formation have neglected the unique properties of nucleic acid and its high diversity. Our study aims at disentangling these DNA properties by monitoring their effect at inducing biofilm formation. By varying length, self vs non-self, and GC percentage, we used various microscopy techniques to visualize the structural composition of a Vibrio hyugaensis biofilm. We observed DNA-dependent biofilm stimulation in this organism, a novel function of DNA in biofilm biology.

Wastewater pandemic preparedness: Toward an end-to-end pathogen monitoring program.

Molecular analysis of public wastewater has great potential as a harbinger for community health and health threats. Long-used to monitor the presence of enteric viruses, in particular polio, recent successes of wastewater as a reliable lead indicator for trends in SARS-CoV-2 levels and hospital admissions has generated optimism and emerging evidence that similar science can be applied to other pathogens of pandemic potential (PPPs), especially respiratory viruses and their variants of concern (VOC). However, there are substantial challenges associated with implementation of this ideal, namely that multiple and distinct fields of inquiry must be bridged and coordinated. These include engineering, molecular sciences, temporal-geospatial analytics, epidemiology and medical, and governmental and public health messaging, all of which present their own caveats. Here, we outline a framework for an integrated, state-wide, end-to-end human pathogen monitoring program using wastewater to track viral PPPs.

Broad protective vaccination against systemic Escherichia coli with autotransporter antigens.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of adult life-threatening sepsis and urinary tract infections (UTI). The emergence and spread of multidrug-resistant (MDR) ExPEC strains result in a considerable amount of treatment failure and hospitalization costs, and contribute to the spread of drug resistance amongst the human microbiome. Thus, an effective vaccine against ExPEC would reduce morbidity and mortality and possibly decrease carriage in healthy or diseased populations. A comparative genomic analysis demonstrated a gene encoding an invasin-like protein, termed sinH, annotated as an autotransporter protein, shows high prevalence in various invasive ExPEC phylogroups, especially those associated with systemic bacteremia and UTI. Here, we evaluated the protective efficacy and immunogenicity of a recombinant SinH-based vaccine consisting of either domain-3 or domains-1,2, and 3 of the putative extracellular region of surface-localized SinH. Immunization of a murine host with SinH-based antigens elicited significant protection against various strains of the pandemic ExPEC sequence type 131 (ST131) as well as multiple sequence types in two distinct models of infection (colonization and bacteremia). SinH immunization also provided significant protection against ExPEC colonization in the bladder in an acute UTI model. Immunized cohorts produced significantly higher levels of vaccine-specific serum IgG and urinary IgG and IgA, findings consistent with mucosal protection. Collectively, these results demonstrate that autotransporter antigens such as SinH may constitute promising ExPEC phylogroup-specific and sequence-type effective vaccine targets that reduce E. coli colonization and virulence.

Discovery of the First Lytic Staphylococcus pseudintermedius/Staphylococcus aureus Polyvalent Bacteriophages.

Background: There are no verified lytic Staphylococcus pseudintermedius phages in the literature and few temperate phage genomes in databases. S. pseudintermedius is an opportunistic zoonotic pathogen of great importance in veterinary and human medicine. Materials and Methods: We discovered phages against canine-derived S. pseudintermedius isolates by screening dog feces, hair, and skin swabs. Fourteen new phages were isolated and characterized by genomic analysis, transmission electron microscopy, and host range determination. Results: Three phages-DH2, DH5, and DS10, a phage K variant-were predicted lytic by sequencing, a designation supported by mitomycin C induction. All three are S. pseudintermedius and Staphylococcus aureus polyvalent phages, with DH2 and DS10 being strong killers of both species. Conclusions: We report discovery of the first verified lytic S. pseudintermedius phages and suggest dog hair as a novel reservoir. DH2, DH5, and DS10 are promising candidates toward developing an anti-Staphylococcal phage cocktail.

Genomic and Functional Characterization of Vancomycin-Resistant Enterococci-Specific Bacteriophages in the Galleria mellonella Wax Moth Larvae Model.

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.

Corrigendum: Development of phage cocktails to treat E. coli catheter-associated urinary tract infection and associated biofilms.

[This corrects the article DOI: 10.3389/fmicb.2022.796132.].

Phage Resistance Accompanies Reduced Fitness of Uropathogenic Escherichia coli in the Urinary Environment.

Urinary tract infection (UTI) is among the most common infections treated worldwide each year and is caused primarily by uropathogenic Escherichia coli (UPEC). Rising rates of antibiotic resistance among uropathogens have spurred a consideration of alternative treatment strategies, such as bacteriophage (phage) therapy; however, phage-bacterial interactions within the urinary environment are poorly defined. Here, we assess the activity of two phages, namely, HP3 and ES17, against clinical UPEC isolates using in vitro and in vivo models of UTI. In both bacteriologic medium and pooled human urine, we identified phage resistance arising within the first 6 to 8 h of coincubation. Whole-genome sequencing revealed that UPEC strains resistant to HP3 and ES17 harbored mutations in genes involved in lipopolysaccharide (LPS) biosynthesis. Phage-resistant strains displayed several in vitro phenotypes, including alterations to adherence to and invasion of human bladder epithelial HTB-9 cells and increased biofilm formation in some isolates. Interestingly, these phage-resistant UPEC isolates demonstrated reduced growth in pooled human urine, which could be partially rescued by nutrient supplementation and were more sensitive to several outer membrane-targeting antibiotics than parental strains. Additionally, phage-resistant UPEC isolates were attenuated in bladder colonization in a murine UTI model. In total, our findings suggest that while resistance to phages, such as HP3 and ES17, may arise readily in the urinary environment, phage resistance is accompanied by fitness costs which may render UPEC more susceptible to host immunity or antibiotics. IMPORTANCE UTI is one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control UTI unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest; however, much like with antibiotics, bacteria can readily become resistant to phages. For successful UTI treatment, we must predict how bacteria will evade killing by phage and identify the downstream consequences of phage resistance during bacterial infection. In our current study, we found that while phage-resistant bacteria quickly emerged in vitro, these bacteria were less capable of growing in human urine and colonizing the murine bladder. These results suggest that phage therapy poses a viable UTI treatment if phage resistance confers fitness costs for the uropathogen. These results have implications for developing cocktails of phage with multiple different bacterial targets, of which each is evaded only at the cost of bacterial fitness.

Development of Phage Cocktails to Treat E. coli Catheter-Associated Urinary Tract Infection and Associated Biofilms.

High rates of antimicrobial resistance and formation of biofilms makes treatment of Escherichia coli catheter-associated urinary tract infections (CAUTI) particularly challenging. CAUTI affect 1 million patients per year in the United States and are associated with morbidity and mortality, particularly as an etiology for sepsis. Phage have been proposed as a potential therapeutic option. Here, we report the development of phage cocktails that lyse contemporary E. coli strains isolated from the urine of patients with spinal cord injury (SCI) and display strong biofilm-forming properties. We characterized E. coli phage against biofilms in two in vitro CAUTI models. Biofilm viability was measured by an MTT assay that determines cell metabolic activity and by quantification of colony forming units. Nine phage decreased cell viability by >80% when added individually to biofilms of two E. coli strains in human urine. A phage cocktail comprising six phage lyses 82% of the strains in our E. coli library and is highly effective against young and old biofilms and against biofilms on silicon catheter materials. Using antibiotics together with our phage cocktail prevented or decreased emergence of E. coli resistant to phage in human urine. We created an anti-biofilm phage cocktail with broad host range against E. coli strains isolated from urine. These phage cocktails may have therapeutic potential against CAUTI.

Interactions between Enterohemorrhagic Escherichia coli (EHEC) and Gut Commensals at the Interface of Human Colonoids.

The interactions between the gut microbiota and pathogens are complex and can determine the outcome of an infection. Enterohemorrhagic Escherichia coli (EHEC) is a major human enteric pathogen that colonizes the colon through attaching and effacing (AE) lesions and uses microbiota-derived molecules as cues to control its virulence. Different gut commensals can modulate EHEC virulence. However, the lack of an animal model that recapitulates the human pathophysiology of EHEC infection makes it challenging to investigate how variations in microbiota composition could affect host susceptibility to this pathogen. Here, we addressed these interactions building from simple to more complex in vitro systems, culminating with the use of the physiological relevant human colonoids as a model to study the interactions between EHEC and different gut commensals. We demonstrated that Bacteroides thetaiotaomicron and Enterococcus faecalis enhance virulence expression and AE lesion formation in cultured epithelial cells, as well as on the colonic epithelium, while commensal E. coli did not affect these phenotypes. Importantly, in the presence of these three commensals together, virulence and AE lesion are enhanced. Moreover, we identified specific changes in the metabolic landscape promoted by different members of the gut microbiota and showed that soluble factors released by E. faecalis can increase EHEC virulence gene expression. Our study highlights the importance of interspecies bacterial interactions and chemical exchange in the modulation of EHEC virulence. IMPORTANCE Enterohemorrhagic E. coli (EHEC) is a natural human pathogen that poorly colonizes mice. Hence, the use of murine models to understand features of EHEC infection is a challenge. In this study, we use human colonoids as a physiologically relevant model to study interactions between EHEC and gut commensals. We demonstrate that the ability of EHEC to form AE lesions on the intestinal epithelium is enhanced by the presence of certain gut commensals, such as B. thetaiotaomicron and E. faecalis, while it is not affected by commensal E. coli. Furthermore, we show that commensal bacteria differently impact the metabolic landscape. These data suggest that microbiota compositions can differentially modulate EHEC-mediated disease.

Considerations for the Use of Phage Therapy in Clinical Practice.

Increasing antimicrobial resistance and medical device-related infections have led to a renewed interest in phage therapy as an alternative or adjunct to conventional antimicrobials. Expanded access and compassionate use cases have risen exponentially but have varied widely in approach, methodology, and clinical situations in which phage therapy might be considered. Large gaps in knowledge contribute to heterogeneity in approach and lack of consensus in many important clinical areas. The Antibacterial Resistance Leadership Group (ARLG) has convened a panel of experts in phage therapy, clinical microbiology, infectious diseases, and pharmacology, who worked with regulatory experts and a funding agency to identify questions based on a clinical framework and divided them into three themes: potential clinical situations in which phage therapy might be considered, laboratory testing, and pharmacokinetic considerations. Suggestions are provided as answers to a series of questions intended to inform clinicians considering experimental phage therapy for patients in their clinical practices.

Identifying Causative Microorganisms in Left Ventricular Assist Device Infections as a Guide for Developing Bacteriophage Therapy.

BACKGROUND: As more left ventricular-assist devices (LVADs) are implanted, multidrug-resistant LVAD infections are becoming increasingly common, partly due to bacterial biofilm production. To aid in developing bacteriophage therapy for LVAD infections, we have identified the most common bacterial pathogens that cause LVAD driveline infections (DLIs) in our heart transplant referral center. MATERIALS AND METHODS: We studied a retrospective cohort of patients who received LVADs from November 2003 to August 2017 to identify the common causative organisms of LVAD infection. We also studied a prospective cohort of patients diagnosed with DLIs from October 2018 to May 2019 to collect bacterial strains from DLIs for developing bacteriophages to lyse causative pathogens. LVAD infections were classified as DLI, bacteremia, and pump/device infections in the retrospective cohort. RESULTS: In the retrospective cohort of 582 patients, 186 (32.0%) developed an LVAD infection, with 372 microbial isolates identified. In the prospective cohort, 96 bacterial strains were isolated from 54 DLIs. The microorganisms causing DLIs were similar in the two cohorts; the most common isolate was Staphylococcus aureus. We identified 6 prospective S. aureus strains capable of biofilm formation. We developed 3 bacteriophages that were able to lyse 5 of 6 of the biofilm-forming S. aureus strains. CONCLUSIONS: Similar pathogens caused LVAD DLIs in our retrospective and prospective cohorts, indicating our bacterial strain bank will be representative of future DLIs. Our banked bacterial strains will be useful in developing phage cocktails that can lyse ≥80% of the bacteria causing LVAD infections at our institution.