Abstract
In recent years, after the emergence of a large number of multidrug-resistant bacteria, phages and phage-associated products for the prevention and control of bacterial disease have revealed prominent advantages as compared with antibiotics. However, bacteria are susceptible to becoming phage-resistant, thus severely limiting the application of phage therapy. In this study, Escherichia coli cells were incubated with lytic bacteriophages to obtain mutants that were resistant to the lytic phages. Then, bacteriophages against the phage-resistant variants were isolated and subsequently mixed with the original lytic phage to prepare a novel phage cocktail for bactericidal use. The data showed that our phage cocktail not only had notable bactericidal effects, including a widened host range and rapid lysis, but also decreased the generation and mutation frequency of phage-resistant strains in vitro. In addition, we tested our cocktail in a murine bacteremia model. The results suggested that compared with the single phage, fewer phage-resistant bacteria appeared during the treatment of phage cocktail, thus prolonging the usable time of the phage cocktail and improving its therapeutic effect in phage applications. Importantly, our preparation method of phage cocktail was proved to be generalizable. Because the bacteriophage against the phage-resistant strain is an ideal guard that promptly attacks potential phage resistance, this guard-killer dual-function phage cocktail provides a novel strategy for phage therapy that allows the natural ecology to be sustained.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Blower TR, Chai R, Przybilski R, Chindhy S, Fang X, Kidman SE, Tan H, Luisi BF, Fineran PC, Salmond GPC (2017) Evolution of Pectobacterium bacteriophage ΦM1 to escape two bifunctional type III toxin-antitoxin and abortive infection systems through mutations in a single viral gene. Appl Environ Microbiol 83:e3216–e3229. https://doi.org/10.1128/AEM.03229-16.
Chan BK, Abedon ST, Loc-Carrillo C (2013) Phage cocktails and the future of phage therapy. Future Microbiol 8:769–783. https://doi.org/10.2217/fmb.13.47
Chang HC, Chen CR, Lin JW, Shen GH, Chang KM, Tseng YH, Weng SF (2005) Isolation and characterization of novel giant Stenotrophomonas maltophilia phage SMA5. Appl Environ Microbiol 71:1387–1393. https://doi.org/10.1128/AEM.71.3.1387-1393.2005
Cheng G, Hao H, Xie S, Wang X, Dai M, Huang L, Yuan Z (2014) Antibiotic alternatives: the substitution of antibiotics in animal husbandry? Front Microbiol 5:217. https://doi.org/10.3389/fmicb.2014.00217
Christiansen RH, Madsen L, Dalsgaard I, Castillo D, Kalatzis PG, Middelboe M (2016) Effect of bacteriophages on the growth of Flavobacterium psychrophilum and development of phage-resistant strains. Microb Ecol 71:845–859. https://doi.org/10.1007/s00248-016-0737-5
Cisek AA, Dąbrowska I, Gregorczyk KP, Wyżewski Z (2017) Phage therapy in bacterial infections treatment: one hundred years after the discovery of bacteriophages. Curr Microbiol 74:277–283. https://doi.org/10.1007/s00284-016-1166-x
Datsenko KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E (2012) Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat Commun 3:945. https://doi.org/10.1038/ncomms1937
Domingo-Calap P, Georgel P, Bahram S (2016) Back to the future: bacteriophages as promising therapeutic tools. HLA 87:133–140. https://doi.org/10.1111/tan.12742
Duerkop BA, Huo W, Bhardwaj P, Palmer KL, Hooper LV (2016) Molecular basis for lytic bacteriophage resistance in Enterococci. MBio 7:e1304–e1316. https://doi.org/10.1128/mBio.01304-16.
Endersen L, O'Mahony J, Hill C, Ross RP, McAuliffe O, Coffey A (2014) Phage therapy in the food industry. Annu Rev Food Sci Technol 5:327–349. https://doi.org/10.1146/annurev-food-030713-092415
Fan J, Zeng Z, Mai K, Yang Y, Feng J, Bai Y, Sun B, Xie Q, Tong Y, Ma J (2016) Preliminary treatment of bovine mastitis caused by Staphylococcus aureus, with trx-SA1, recombinant endolysin of S. aureus bacteriophage IME-SA1. Vet Microbiol 191:65–71. https://doi.org/10.1016/j.vetmic.2016.06.001
Filippov AA, Sergueev KV, He Y, Huang X, Gnade BT, Mueller AJ, Fernandez-Prada CM, Nikolich MP (2011) Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice. PLoS One 6:e25486. https://doi.org/10.1371/journal.pone.0025486
Gu J, Liu X, Li Y, Han W, Lei L, Yang Y, Zhao H, Gao Y, Song J, Lu R, Sun C, Feng X (2012) A method for generation phage cocktail with great therapeutic potential. PLoS One 7:e31698. https://doi.org/10.1371/journal.pone.0031698
Hong Y, Schmidt K, Marks D, Hatter S, Marshall A, Albino L, Ebner P (2016) Treatment of Salmonella-contaminated eggs and pork with a broad-spectrum, single bacteriophage: assessment of efficacy and resistance development. Foodborne Pathog Dis 13:679–688. https://doi.org/10.1089/fpd.2016.2172
Hyman P, Abedon ST (2010) Bacteriophage host range and bacterial resistance. Adv Appl Microbiol 70:217–248. https://doi.org/10.1016/S0065-2164(10)70007-1
Kelly D, McAuliffe O, O Mahony J, Coffey A (2014) Development of a broad-host-range phage cocktail for biocontrol. Bioeng Bugs 2:31–37. https://doi.org/10.4161/bbug.2.1.13657
Kutateladze M, Adamia R (2010) Bacteriophages as potential new therapeutics to replace or supplement antibiotics. Trends Biotechnol 28:591–595. https://doi.org/10.1016/j.tibtech.2010.08.001
Kutter E (2009) Phage host range and efficiency of plating. Methods Mol Biol 501:141–149. https://doi.org/10.1007/978-1-60327-164-6_14
Labrie SJ, Samson JE, Moineau S (2010) Bacteriophage resistance mechanisms. Nat Rev Microbiol 8:317–327. https://doi.org/10.1038/nrmicro2315
Laxminarayan R, Duse A, Wattal C, Zaidi AKM, Wertheim HFL, Sumpradit N, Vlieghe E, Hara GL, Gould IM, Goossens H, Greko C, So AD, Bigdeli M, Tomson G, Woodhouse W, Ombaka E, Peralta AQ, Qamar FN, Mir F, Kariuki S, Bhutta ZA, Coates A, Bergstrom R, Wright GD, Brown ED, Cars O (2013) Antibiotic resistance—the need for global solutions. Lancet Infect Dis 13:1057–1098. https://doi.org/10.1016/S1473-3099(13)70318-9
Le S, Yao X, Lu S, Tan Y, Rao X, Li M, Jin X, Wang J, Zhao Y, Wu NC, Lux R, He X, Shi W, Hu F (2014) Chromosomal DNA deletion confers phage resistance to Pseudomonas aeruginosa. Sci Rep 4:4738. https://doi.org/10.1038/srep04738
Li Z, Li X, Zhang J, Wang X, Wang L, Cao Z, Xu Y (2016a) Use of phages to control Vibrio splendidus infection in the juvenile sea cucumber Apostichopus japonicus. Fish Shellfish Immunol 54:302–311. https://doi.org/10.1016/j.fsi.2016.04.026
Li E, Zhao J, Ma Y, Wei X, Li H, Lin W, Wang X, Li C, Shen Z, Zhao R, Jiang A, Yang H, Yuan J, Zhao X (2016b) Characterization of a novel Achromobacter xylosoxidans specific siphoviruse: phiAxp-1. Sci Rep 6:21943. https://doi.org/10.1038/srep21943
Makarova KS, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, Moineau S, Mojica FJM, Wolf YI, Yakunin AF, van der Oost J, Koonin EV (2011) Evolution and classification of the CRISPR–Cas systems. Nat Rev Microbiol 9:467–477. https://doi.org/10.1038/nrmicro2577
Molineux IJ, Panja D (2013) Popping the cork: mechanisms of phage genome ejection. Nat Rev Microbiol 11:194–204. https://doi.org/10.1038/nrmicro2988
Mumford R, Friman V (2017) Bacterial competition and quorum-sensing signalling shape the eco-evolutionary outcomes of model in vitro phage therapy. Evol Appl 10:161–169. https://doi.org/10.1111/eva.12435
Nale JY, Spencer J, Hargreaves KR, Buckley AM, Trzepinski P, Douce GR, Clokie MR (2015) Bacteriophage combinations significantly reduce Clostridium difficile growth in vitro and proliferation in vivo. Antimicrob Agents Chemother 60:968–981. https://doi.org/10.1128/AAC.01774-15
Sarker SA, Sultana S, Reuteler G, Moine D, Descombes P, Charton F, Bourdin G, McCallin S, Ngom-Bru C, Neville T, Akter M, Huq S, Qadri F, Talukdar K, Kassam M, Delley M, Loiseau C, Deng Y, El Aidy S, Berger B, Brüssow H (2016) Oral phage therapy of acute bacterial diarrhea with two coliphage preparations: a randomized trial in children from Bangladesh. EBioMedicine 4:124–137. https://doi.org/10.1016/j.ebiom.2015.12.023.
Shen G, Wang J, Wen F, Chang K, Kuo C, Lin C, Luo H, Hung C (2012) Isolation and characterization of φkm18p, a novel lytic phage with therapeutic potential against extensively drug resistant Acinetobacter baumannii. PLoS One 7:e46537. https://doi.org/10.1371/journal.pone.0046537
Soffer N, Abuladze T, Woolston J, Li M, Hanna LF, Heyse S, Charbonneau D, Sulakvelidze A (2016) Bacteriophages safely reduce Salmonella contamination in pet food and raw pet food ingredients. Bacteriophage 6:e1220347. https://doi.org/10.1080/21597081.2016.1220347
Speck P, Smithyman A (2016) Safety and efficacy of phage therapy via the intravenous route. FEMS Microbiol Lett 363:v242. https://doi.org/10.1093/femsle/fnv242
Stalin N, Srinivasan P (2016) Characterization of Vibrio parahaemolyticus and its specific phage from shrimp pond in Palk Strait, south east coast of India. Biologicals 44:526–533. https://doi.org/10.1016/j.biologicals.2016.08.003
van Zyl LJ, Taylor MP, Trindade M (2016) Engineering resistance to phage GVE3 in Geobacillus thermoglucosidasius. Appl Microbiol Biotechnol 100:1833–1841. https://doi.org/10.1007/s00253-015-7109-9
Wang C, Chen Q, Zhang C, Yang J, Lu Z, Lu F, Bie X (2017) Characterization of a broad host-spectrum virulent Salmonella bacteriophage fmb-p1 and its application on duck meat. Virus Res 236:14–23. https://doi.org/10.1016/j.virusres.2017.05.001
Watts J, Schreier HJ, Lanska L, Hale MS (2017) The rising tide of antimicrobial resistance in aquaculture: sources, sinks and solutions. Mar Drugs 15. https://doi.org/10.3390/md15060158
Wong CL, Sieo CC, Tan WS, Abdullah N, Hair-Bejo M, Abu J, Ho YW (2014) Evaluation of a lytic bacteriophage, Φ st1, for biocontrol of Salmonella enterica serovar Typhimurium in chickens. Int J Food Microbiol 172:92–101. https://doi.org/10.1016/j.ijfoodmicro.2013.11.034
Woolhouse M, Farrar J (2014) Policy: an intergovernmental panel on antimicrobial resistance. Nature 509:555–557
Yosef I, Goren MG, Globus R, Molshanski-Mor S, Qimron U (2017) Extending the host range of bacteriophage particles for DNA transduction. Mol Cell 66:721–728. https://doi.org/10.1016/j.molcel.2017.04.025
Zhang J, Li Z, Cao Z, Wang L, Li X, Li S, Xu Y (2015) Bacteriophages as antimicrobial agents against major pathogens in swine: a review. J Anim Sci Biotechnol 6:39. https://doi.org/10.1186/s40104-015-0039-7
Funding
The study was funded by the National Key Basic Research Program of China (No. 2013CB127205).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
All animal studies were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved through the State Council of People’s Republic of China (2017.3.1 revision) and with approval of the Animal Welfare and Research Ethics Committee at Jilin University.
Rights and permissions
About this article
Cite this article
Yu, L., Wang, S., Guo, Z. et al. A guard-killer phage cocktail effectively lyses the host and inhibits the development of phage-resistant strains of Escherichia coli . Appl Microbiol Biotechnol 102, 971–983 (2018). https://doi.org/10.1007/s00253-017-8591-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-017-8591-z