RESUMO
Antimicrobial resistance (AMR) is a major problem globally. The main bacterial organisms associated with urinary tract infection (UTI) associated sepsis are E. coli and Klebsiella along with Enterobacter species. These all have AMR strains known as ESBL (Extended Spectrum Beta-Lactamase), which are featured on the WHO priority pathogens list as "critical" for research. Bacteriophages (phages), as viruses that can infect and kill bacteria, could provide an effective tool to tackle these AMR strains. There is currently no "gold standard" for developing a phage cocktail. Here we describe a novel approach to develop an effective phage cocktail against a set of ESBL-producing E. coli and Klebsiella largely isolated from patients in United Kingdom hospitals. By comparing different measures of phage efficacy, we show which are the most robust, and suggest an efficient screening cascade that could be used to develop phage cocktails to target other AMR bacterial species. A target panel of 38 ESBL-producing clinical strains isolated from urine samples was collated and used to test phage efficacy. After an initial screening of 68 phages, six were identified and tested against these 38 strains to determine their clinical coverage and killing efficiency. To achieve this, we assessed four different methods to assess phage virulence across these bacterial isolates. These were the Direct Spot Test (DST), the Efficiency of Plating (EOP) assay, the planktonic killing assay (PKA) and the biofilm assay. The final ESBL cocktail of six phages could effectively kill 23/38 strains (61%), for Klebsiella 13/19 (68%) and for E. coli 10/19 (53%) based on the PKA data. The ESBL E. coli collection had six isolates from the prevalent UTI-associated ST131 sequence type, five of which were targeted effectively by the final cocktail. Of the four methods used to assess phage virulence, the data suggests that PKAs are as effective as the much more time-consuming EOPs and data for the two assays correlates well. This suggests that planktonic killing is a good proxy to determine which phages should be used in a cocktail. This assay when combined with the virulence index also allows "phage synergy" to inform cocktail design.
RESUMO
Bacteriophages offer a viable solution to addressing the global issue of bacterial resistance to antimicrobials. Although knowledge of bacteriophages has increased greatly since their discovery in 1915, a significant amount of what is currently known is based on studies conducted in model conditions and aerobic environments. There are a variety of environments in which bacteriophages could be applied to successfully replace or supplement antimicrobials in agriculture, food production, and human medicine where the amount of oxygen is limited. There is a need to use phages in oxygen-limited environments, but few studies have examined the impact oxygen-limited environments have on the ability of phages to kill their hosts. The work that has been done is, however, insightful and will likely stimulate this area that is growing in importance as our need to use phages grows. This review summarizes the studies to date that have reported the characteristics of phages in both oxygen-rich and oxygen-limited environments. We also discuss the importance of considering the ultimate environment a phage will be applied to when designing experiments to isolate and characterize phages for use in phage-based antimicrobial products.