Development of Phage Cocktails to Treat E. coli Catheter-Associated Urinary Tract Infection and Associated Biofilms
Belkys C. Sanchez1, 
Emmaline R. Heckmann1, 
Sabrina I. Green1, 
Justin R. Clark1, 
Heidi B. Kaplan2, 
Robert F. Ramig1, 
Casey Hines-Munson3, 
Felicia Skelton3,4, 
Barbara W. Trautner3,5,6† and 
Anthony W. Maresso1*†
- 1Tailored Antibacterials and Innovative Laboratories for Phage (Φ) Research, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, United States
- 2Department of Microbiology and Molecular Genetics, McGovern Medical School, UTHealth Houston, Houston, TX, United States
- 3Center for Innovations in Quality, Effectiveness and Safety, Michael E. DeBakey VA Medical Center, Houston, TX, United States
- 4H. Ben Taub Department of Physical Medicine and Rehabilitation, Baylor College of Medicine, Houston, TX, United States
- 5Department of Medicine and Surgery, Baylor College of Medicine, Houston, TX, United States
- 6Center for Translational Research on Inflammatory Diseases, Michael E. DeBakey VA Medical Center, Houston, TX, United States
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. coliresistant 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.
Introduction
Urinary tract infections (UTI) are among the most common community and nosocomial bacterial infections (Flores-Mireles et al., 2019; Medina and Castillo-Pino, 2019) affecting 150 million people worldwide each year (Stamm, 2002), and resulting in a high economic burden on society (Lo et al., 2014; Skelton et al., 2019). UTI cause serious complications, including pyelonephritis, sepsis and frequent recurrences, resulting in repetitive antimicrobial administration and selection of multidrug-resistant uropathogens (Flores-Mireles et al., 2019). The presence of a urinary catheter facilitates entrance and colonization of pathogens to the urinary tract, increasing the risk of infection (Jacobsen et al., 2008). Although efforts have been made to improve prevention and management of catheter-associated urinary tract infections (CAUTI), almost all long-term catheterized patients develop bacteriuria, and 10–30% of patients with catheter-associated bacteriuria develop UTI-related symptoms (Warren, 1991; Trautner and Darouiche, 2004). CAUTI is one of the most common infections acquired in hospitals, accounting for 40% of all nosocomial infections and 1 million cases in the United States each year (Tambyah and Maki, 2000; Stamm and Norrby, 2001; Foxman, 2010). Persons with spinal cord injury (SCI) suffer disproportionately from CAUTI, given the secondary complication of neurogenic bladder and the need for chronic urinary catheterization (Manack et al., 2011; Skelton et al., 2015; Skelton-Dudley et al., 2019). Persons with SCI also experience a higher proportion of multidrug-resistant infections due to frequent healthcare exposure and courses of antibiotics over their lifetime (Kang et al., 2015; Suda et al., 2016; Evans et al., 2017). CAUTI is associated with increased morbidity and mortality in persons with SCI and the management of this condition presents unique challenges in this patient population (Skelton-Dudley et al., 2019).
Escherichia coli is the main causative agent of UTI, causing 80% of acute UTI and 33% of CAUTI (Stamm, 2002; Stickler, 2008; Foxman, 2010). Similarly, E. coli is one of the most commonly isolated pathogens from the urine of persons with SCI (Kang et al., 2015). E. coli can form biofilms on urinary catheters which complicates management of CAUTI (Stickler, 2008; Niveditha et al., 2012). Biofilms are surface-associated multicellular bacterial communities that protect individual cells from host defenses and environmental stresses, and mediate bacterial persistence and recurrent infections in the urinary tract (Trautner and Darouiche, 2004; Soto et al., 2006). Due to their structural and metabolic properties, biofilms are recalcitrant to antimicrobial therapy (Hall and Mah, 2017). Many antibiotics do not easily kill cells within biofilms (Singh et al., 2016; Ciofu et al., 2022), and E. coli biofilms have been observed on urinary catheters recovered from patients that received antibiotic therapy (Walker et al., 2020). Furthermore, persister cells within the biofilm can reemerge once antibiotic therapy is discontinued (Gollan et al., 2019). Thus, not only do antibiotics often fail to eradicate biofilms, but repetitive therapy required to treat recurrent UTI can select for resistant microorganisms.
The current approach for treatment of CAUTI includes targeted antibiotic therapy and replacement of the indwelling catheter (Hooton et al., 2010; Fekete, 2021), which may contain biofilms of the infecting organism (Trautner and Darouiche, 2004). Because multidrug-resistant UTI represent a threat to the health and quality of life of persons with SCI, a new management approach is needed. To address this unmet need, here we have characterized bacteriophage (phage or Φ) with specificity toward contemporary E. colistrains isolated from the urine of patients with SCI. Phage are ubiquitous viruses that infect and kill bacteria irrespective of their antibiotic sensitivity (Chan et al., 2013). Phage have been successfully used to treat biofilm-associated infections recalcitrant to antibiotics (Wright et al., 2009; Chan et al., 2018; Aslam et al., 2019, 2020; Cano et al., 2020). Additionally, phage may self-dose (Terwilliger et al., 2020), be evolved to re-target phage-resistant strains (Salazar et al., 2021) and have features that enhance their activity in the complex microenvironments of the mammalian host, especially at mucosal surfaces (Green et al., 2021). Our group previously characterized a library of phage that lyse multidrug-resistant extra-intestinal pathogenic E. coli strains (Gibson et al., 2019). Some of these phage have been shown efficacious in several murine models of infection and in a case of compassionate use of phage to treat a recurrent UTI (Green et al., 2017; Terwilliger et al., 2021). Here, we screened and characterized this phage library and additional novel phage for their ability to reduce the viability of bacterial cells in biofilms of E. coli clinical strains. Our data reveals that it is possible to generate highly lytic cocktails with anti-biofilm activity against most E. coli isolates from our population of patients with SCI, and that these cocktails are active against biofilms grown in human urine and on silicone catheter materials.