Sensitizing and control of colistin-resistant E. coli O157:H7 with bacteriophage application

Abstract

In these days that we are drifting into the post-antibiotic era, antibiotics called "last-resort" are begun to be used more frequently. Colistin is one of the last-resort antibiotics that act on Gram-negative bacteria. The aim of the study was to investigate antibiotic re-sensitization effect of lytic bacteriophages on colistin resistant E. coli O157:H7 in-vitro. In the study, four E. coli O157:H7 isolates (encoded 25KA, 44RA, 120RA and 168KA) were included. These isolates had different features such as harboring some of the mcr genes but not showing resistance to colistin, or demonstrating resistance to colistin without carrying any mcr genes. A lytic bacteriophage cocktail was prepared with three Myoviridae family member phages. In order to determine the effect of lytic bacteriophage application on the colistin resistance of E. coli O157:H7 strains before, during and after bacteriophage treatment, minimum inhibitory concentrations (MIC) of the isolates were determined by broth microdilution method. The results were interpreted according to EUCAST. According to the results, up to 3.6 log cfu/ml reductions in colistin resistant E. coli O157:H7 were detected within 6h incubation at 23°C. Colistin and phage combination showed synergistic effect. While strains 25KA and 168KA became susceptible to colistin, 44GA and 120RA were totally eliminated. The survivors of the phage treatment were also become sensitive to colistin. Phage-resistant mutants of 25KA and 168KA showed susceptibility to colistin (1 µg/ml and 0.5 µg/ml, respectively). In addition, 44GA and 120GA remained susceptible. The findings of this study highlight that in addition to taking advantage of the lytic activity of phages in biocontrol area, phages also play a major role in re-sensitization to a last-resort antibiotic like colistin. The results show the synergy between phage–antibiotic combination treatment and give the promising idea that this approach has the potential to extend the effective lifetime of antibiotics.

Keywords

• bacteriophage
• colistin
• E. coli O157:H7
• re-sensitization
• MIC

References

1. Referans1. de Kraker ME, Stewardson AJ, Harbarth S. Will 10 million people die a year due to antimicrobial resistance by 2050? PLoS Med. 2016; 13(11): e1002184.
2. Referans2. Mohamed YF, Abou-Shleib HM, Khalil AM, et al. Membrane permeabilization of colistin toward pan-drug resistant Gram-negative isolates. Braz J Microbiol. 2016; 47(2): 381–388.
3. Referans3. Zavascki AP, Goldani LZ, Li J, Nation RL. Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review. J Antimicrob Chemother. 2007; 60: 1206–1215.
4. Referans4. Aris P, Robatjazi S, Nikkhahi F, Marashi SMA. Molecular mechanisms and prevalence of colistin resistance of Klebsiella pneumoniae in the Middle East region: a review over the last 5 years. J Glob Antimicrob Resist. 2020; 22: 625-630.
5. Referans5. Wang C, Feng Y, Liu L, et al. Identification of novel mobile colistin resistance gene mcr-10. Emerg Microbes Infect. 2020; 9: 508–516.
6. Referans6. Tagliaferri TL, Jansen M, Horz HP. Fighting pathogenic bacteria on two fronts: phages and antibiotics as combined strategy. Front Cell Infect Microbiol. 2019; 9: 22.
7. Referans7. Endersen L, Coffey A. The use of bacteriophages for food safety, Curr Opin Food Sci. 2020; 36: 1-8.
8. Referans8. Abedon ST. Phage-antibiotic combination treatments: antagonistic impacts of antibiotics on the pharmacodynamics of phage therapy?. Antibiotics (Basel). 2019; 8(4): 182.

9. Referans9. Ayaz ND, Gencay YE, Erol I. Prevalence and molecular characterization of sorbitol fermenting and non-fermenting Escherichia coli O157:H7+/H7– isolated from cattle at slaughterhouse and slaughterhouse wastewater. Int J Food Microbiol. 2014; 174: 31-38.
10. Referans10. Ayaz ND, Cufaoglu G, Yonsul Y, et al. Plasmid-mediated colistin resistance in Escherichia coli O157:H7 cattle and sheep isolates and whole-genome sequence of a colistin-resistant sorbitol fermentative Escherichia coli O157:H7. Microb Drug Resist. 2019; 25(10): 1497-1506.
11. Referans11. Gencay YE, Ayaz ND, Copuroglu G, et al. Biocontrol of Shiga toxigenic Escherichia coli O157:H7 in turkish raw meatball by bacteriophage. J Food Saf. 2016; 36: 120-131.
12. Referans12. Gunn JS. The Salmonella PmrAB regulon: lipopolysaccharide modifications, antimicrobial peptide resistance and more. Trends Microbiol, 2008; 16; 284-290.
13. Referans13. Cannatelli A, D’Andrea MM, Giani T, et al. In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator. Antimicrob Agents Chemother. 2013; 57: 5521-5526.
14. Referans14. Torres-Barcelo C, Hochberg ME. Evolutionary rationale for phages as complements of antibiotics. Trends Microbiol. 2016; 24: 249-256.

15. Referans15. Capparelli R, Nocerino N, Iannaccone M, et al. Bacteriophage therapy of Salmonella enterica: a fresh appraisal of bacteriophage therapy. J Infect Dis. 2010; 201; 52-61.
16. Referans16. Altamirano FG, Forsyth JH, Patwa R, et al. Bacteriophages targeting Acinetobacter baumannii capsule induce antimicrobial resensitization. Nat Microbiol. 2021; 6: 157–161.
17. Referans17. Capparelli R, Nocerino N, Lanzetta R, et al. Bacteriophage-resistant Staphylococcus aureus mutant confers broad immunity against staphylococcal infection in mice. PLoS One. 2010; 5: e11720.
18. Referans18. Chan BK, Sistrom M, Wertz JE, et al. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa. Sci Rep. 2016; 6: 26717.
19. Referans19. Engeman E, Freyberger HR, Corey BW, et al. Synergistic killing and re-sensitization of Pseudomonas aeruginosa to antibiotics by phage-antibiotic combination treatment. Pharmaceuticals. 2021; 14(3): 184.
20. Referans20. Mazurek J, Pusz P, Bok E, Stosik M, et al. The phenotypic and genotypic characteristics of antibiotic resistance in Escherichia coli populations isolated from farm animals with different exposure to antimicrobial agents. Pol J Microbiol. 2013; 62(2): 173-179.
21. Referans21. Davis MA, Besser TE, Orfe LH, et al. Genotypic-phenotypic discrepancies between antibiotic resistance characteristics of Escherichia coli isolates from calves in management settings with high and low antibiotic use. Appl Environ Microbiol. 2011; 77(10): 3293–3299.