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Analysis of Selection Methods to Develop Novel Phage Therapy Cocktails Against Antimicrobial Resistant Clinical Isolates of Bacteria.

ABSTRACT: 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.

SUBMITTER: Haines MEK

PROVIDER: S-EPMC8044893 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The rapid antiphage mutation of pathogens is a big challenge often encountered in the application of phages in aquaculture, animal husbandry, and human disease prevention. A cocktail composed of phages with different infection strategies can better suppress the antiphage resistance of pathogens. However, randomly selecting phages with different infection strategies is time-consuming and labor intensive. Here, we verified that using a resistant pathogen quickly evolved under single phage infection, as the new host can easily obtain phages with different infection strategies. We randomly isolated two lytic phages (i.e., Va1 and Va2) that infect the opportunistic pathogen Vibrio alginolyticus. Whether they were used alone or in combination, the pathogen easily gained resistance. Using a mutated pathogen resistant to Va1 as a new host, a third lytic phage Va3 was isolated. These three phages have a similar infection cycle and lytic ability but quite different morphologies and genome information. Notably, phage Va3 is a jumbo phage containing a larger and more complex genome (240 kb) than Va1 and Va2. Furthermore, the 34 tRNAs and multiple genes encoding receptor binding proteins and NAD+ synthesis proteins in the Va3 genome implicated its quite different infection strategy from Va1 and Va2. Although the wild-type pathogen could still readily evolve resistance under single phage infection by Va3, when Va3 was used in combination with Va1 and Va2, pathogen resistance was strongly suppressed. This study provides a novel approach for rapid isolation of phages with different infection strategies, which will be highly beneficial when designing effective phage cocktails. IMPORTANCE The rapid antiphage mutation of pathogens is a big challenge often encountered in phage therapy. Using a cocktail composed of phages with different infection strategies can better overcome this problem. However, randomly selecting phages with different infection strategies is time-consuming and labor intensive. To address this problem, we developed a method to efficiently obtain phages with disparate infection strategies. The trick is to use the characteristics of the pathogenic bacteria that are prone to develop resistance to single phage infection to rapidly obtain the antiphage variant of the pathogen. Using this antiphage variant as the host results in other phages with different infection strategies being efficiently isolated. We also verified the reliability of this method by demonstrating the ideal phage control effects on two pathogens and thus revealed its potential importance in the development of phage therapies.

Project description:Temperate phages integrated with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems have been gaining attention as potential strategies for combating bacteria resistant to antimicrobials. To further advance this technology, phage recombination procedure should be improved, and the bactericidal effect should be examined in detail and compared with conventional lytic phage strategy. The possibility of the emergence of mutational resistance, a phenomenon commonly observed with lytic phage therapy, should be illustrated. Methods: Here, we developed a novel one-step cloning method to fulfil the recombination of CRISPR/Cas9 system within the genome of a new isolated lysogenic Escherichia coli phage. Then, we proposed and developed a phage-delivered resistance eradication with subsequent antibiotic treatment (PRESA) strategy. The removal efficiency and antimicrobial effect of the plasmids were analysed. Long-term antimicrobial effect was evaluated by continued OD600 monitoring for 240 hours to illustrate the potential mutational resistance, compared with the lytic phage strategy. The treatment effect of PRESA was evaluated in vivo by determining bacterial loads in the skin and intestine of infected mice, in contrast with lytic phage therapy. Genome sequencing was performed to identify mutations in bacterial cells treated with phage strategies. Results: Phage-delivered CRISPR targeting efficiently eradicated and blocked the transfer of the antibiotic resistance plasmid. PRESA decreased the bacterial load by over 6- and 5-logs in vitro and in vivo, respectively. Importantly, while lytic phages induced mutational phage resistance at 24 h in vitro and 48 hours in vivo, PRESA demonstrated a constant effect and revealed no resistant mutants. Genes involved in DNA mismatch repair were upregulated in cells undergoing Cas9-based plasmid cleavage, which may reduce the development of mutations. Conclusion: The PRESA strategy for eradicating resistant bacteria showed high bactericidal efficacy and a sustained inhibition effect against resistant bacteria. By restoring the efficacy of low-cost antibiotics, PRESA could be developed as an efficient and economical therapy for infections of antibiotic resistant bacteria.

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Analysis of Selection Methods to Develop Novel Phage Therapy Cocktails Against Antimicrobial Resistant Clinical Isolates of Bacteria.

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