Author:
- ABSTRACT.
- BACKGROUND
- MATERIALS AND METHODS
- Bacteria strains and preparations.
- Isolation and enrichment of phages.
- Host range test and covering spectrum test using clinically isolated P. aeruginosa strains.
- Genome extraction and RAPD typing of phages.
- Morphology of phage virions by electron microscopy.
- Multiplicity of infection, adsorption efficiency, and one-step growth curve experiment with phages.
- Abdominal cavity infection by P. aeruginosa and the determination of the minimum lethal dose in a mouse model.
- Phage therapeutic effect in the mouse model.
- Distribution of phages and bacteria inĀ vivo.
- Statistical analysis.
- RESULTS
- Isolated phages and their spectra.
- Genomic typing and electron microscopy of phages.
- The MOI of four broad-spectrum phages, their adsorption efficiency, and one-step growth curves.
- Therapeutic effect of phages in mice with abdominal cavity P. aeruginosa infection.
- Quantity distribution of phage I and P. aeruginosa in the mouse model.
- DISCUSSION
- CONCLUSION
- Supplemental Materials
- ACKNOWLEDGMENT
- REFERENCES
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Wilson R , Aksamit T , Aliberti S , De Soyza A , Elborn JS , Goeminne P , Hill AT , Menendez R , Polverino E , 2016. Challenges in managing Pseudomonas aeruginosa in non-cystic fibrosis bronchiectasis. Respir Med 117: 179ā189.
-
2.
Chen MJ , Wang H , 2003. Continuous surveillance of antimicrobial resistance among nosocomial gram-negative bacilli from intensive care units in China. Zhonghua Yi Xue Za Zhi 83: 375ā381.
-
3.
Sanford E , Farnaes L , Batalov S , Bainbridge M , Laubach S , Worthen HM , Tokita M , Kingsmore SF , Bradley J , 2018. Concomitant diagnosis of immune deficiency and Pseudomonas sepsis in a 19 month old with ecthyma gangrenosum by host whole-genome sequencing. Cold Spring Harb Mol Case Stud 4: a003244.
-
4.
Jean SS , Harnod D , Hsueh PR , 2022. Global threat of carbapenem-resistant gram-negative bacteria. Front Cell Infect Microbiol 12: 823684.
-
5.
Hong DJ , Bae IK , Jang IH , Jeong SH , Kang HK , Lee K , 2015. Epidemiology and characteristics of metallo-Ī²-lactamase-producing Pseudomonas aeruginosa. Infect Chemother 47: 81ā97.
-
6.
Merril CR , Scholl D , Adhya SL , 2003. The prospect for bacteriophage therapy in Western medicine. Nat Rev Drug Discov 2: 489ā497.
-
7.
Sulakvelidze A , 2005. Phage therapy: an attractive option for dealing with antibiotic-resistant bacterial infections. Drug Discov Today 10: 807ā809.
-
8.
Schooley RT et al., 2017. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob Agents Chemother 61: e00954-17.
-
9.
Mahenthiralingam E , Campbell ME , Foster J , Lam JS , Speert DP , 1996. Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J Clin Microbiol 34: 1129ā1135.
-
10.
Ceyssens PJ et al., 2009. Survey of Pseudomonas aeruginosa and its phages: de novo peptide sequencing as a novel tool to assess the diversity of worldwide collected viruses. Environ Microbiol 11: 1303ā1313.
-
11.
Premamalini T , Rajyoganandh V , Vijayakumar R , Veena H , Kindo AJ , Marak RS , 2021. Strain typing of Trichosporon asahii clinical isolates by random amplification of polymorphic DNA (RAPD) analysis. J Lab Physicians 13: 245ā251.
-
12.
Qamar W , Khan MR , Arafah A , 2017. Optimization of conditions to extract high quality DNA for PCR analysis from whole blood using SDS-proteinase K method. Saudi J Biol Sci 24: 1465ā1469.
-
13.
Li L , Yang H , Lin S , Jia S , 2010. Classification of 17 newly isolated virulent bacteriophages of Pseudomonas aeruginosa. Can J Microbiol 56: 925ā933.
-
14.
Biswas B , Adhya S , Washart P , Paul B , Trostel AN , Powell B , Carlton R , Merril CR , 2002. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infect Immun 70: 204ā210.
-
15.
Wang J et al., 2006. Use of bacteriophage in the treatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosa. Int J Mol Med 17: 309ā317.
-
16.
Shivshetty N , Hosamani R , Ahmed L , Oli AK , Sannauallah S , Sharanbassappa S , Patil SA , Kelmani CR , 2014. Experimental protection of diabetic mice against lethal P. aeruginosa infection by bacteriophage. BioMed Res Int 2014: 793242.
-
17.
National Institutes of Health , 2011. NIH Guide for the Care and Use of Laboratory Animals. Available at: https://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf. Accessed March 27, 2023.
-
18.
Kilkenny C , Brown W , Cuthill IC , Emerson M , Altman DG , NC3Rs Reporting Guidelines Working Group, 2010. Animal research: reporting in vivo experiments: the ARRIVE guidelines. Available at: https://doi.org/10.1111/j.1476-5381.2010.00872.x. Accessed March 27, 2023.
-
19.
Carter CD , Parks A , Abuladze T , Li M , Woolston J , Magnone J , Senecal A , Kropinski AM , Sulakvelidze A , 2012. Bacteriophage cocktail significantly reduces Escherichia coli O157:H7 contamination of lettuce and beef, but does not protect against recontamination. Bacteriophage 2: 178ā185.
-
20.
Jaiswal A , Koley H , Ghosh A , Palit A , Sarkar B , 2013. Efficacy of cocktail phage therapy in treating Vibrio cholerae infection in rabbit model. Microbes Infect 15: 152ā156.
-
21.
Ziyong S , Li L , Xuhui Z , Yue M , Jingyun L , Zhengyi S , Shaohong J , 2006. Analysis on antimicrobial resistance of clinical bacteria isolated from county hospitals and a teaching hospital. J Huazhong Univ Sci Technol (Med Sci) 26: 386ā388.
-
22.
China Antimicrobial Resistance Surveillance System , 2018. National Bacterial Resistance Monitoring Report 2018 (Brief Edition). Available at: http://www.carss.cn/Report/Details?aId=648. Accessed March 27, 2023.
-
23.
Betts JW , Hornsey M , Higgins PG , Lucassen K , Wille J , Salguero FJ , Seifert H , La Ragione RM , 2019. Restoring the activity of the antibiotic aztreonam using the polyphenol epigallocatechin gallate (EGCG) against multidrug-resistant clinical isolates of Pseudomonas aeruginosa. J Med Microbiol 68: 1552ā1559.
-
24.
Yusuf E , Van Herendael B , Verbrugghe W , Ieven M , Goovaerts E , Bergs K , Wouters K , Jorens PG , Goossens H , 2017. Emergence of antimicrobial resistance to Pseudomonas aeruginosa in the intensive care unit: association with the duration of antibiotic exposure and mode of administration. Ann Intensive Care 7: 72.
-
25.
Pachori P , Gothalwal R , Gandhi P , 2019. Emergence of antibiotic resistance Pseudomonas aeruginosa in intensive care unit; a critical review. Genes Dis 6: 109ā119.
-
26.
Tang C , Deng C , Zhang Y , Xiao C , Wang J , Rao X , Hu F , Lu S , 2018. Characterization and genomic analyses of Pseudomonas aeruginosa podovirus TC6: establishment of genus Pa11virus. Front Microbiol 9: 2561.
-
27.
de Melo ACC , da Mata Gomes A , Melo FL , Ardisson-AraĆŗjo DMP , de Vargas APC , Ely VL , Kitajima EW , Ribeiro BM , Wolff JLC , 2019. Characterization of a bacteriophage with broad host range against strains of Pseudomonas aeruginosa isolated from domestic animals. BMC Microbiol 19: 134.
-
28.
Jeon J , Yong D , 2019. Two novel bacteriophages improve survival in Galleria mellonella infection and mouse acute pneumonia models infected with extensively drug-resistant Pseudomonas aeruginosa. Appl Environ Microbiol 85: e02900ā18.
-
29.
Cisek AA , Dabrowska I , Gregorczyk KP , Wyzewski Z , 2017. Phage therapy in bacterial infections treatment: one hundred years after the discovery of bacteriophages. Curr Microbiol 74: 277ā283.
-
30.
El Haddad L , Harb CP , Gebara MA , Stibich MA , Chemaly RF , 2019. A systematic and critical review of bacteriophage therapy against multidrug-resistant ESKAPE organisms in humans. Nephrol Dial Transplant 69: 167ā178.
-
31.
Wright RCT , Friman VP , Smith MCM , Brockhurst MA , 2019. Resistance evolution against phage combinations depends on the timing and order of exposure. mBio 10: e01652ā19.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 329 | 329 | 62 |
| Full Text Views | 958 | 958 | 2 |
| PDF Downloads | 116 | 116 | 3 |
Isolation and Characterization of Pseudomonas aeruginosa Phages with a Broad Host Spectrum from Hospital Sewage Systems and Their Therapeutic Effect in a Mouse Model
Fangfang Dai
Fangfang Dai
Department of Clinical Laboratory, Beijing Youan Hospital, Capital Medical University, Beijing, Peopleās Republic of China;
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Gengxia Yang
Gengxia Yang
General Surgical Center, Beijing Youan Hospital, Capital Medical University, Beijing, Peopleās Republic of China;
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Jinli Lou
Jinli Lou
Department of Clinical Laboratory, Beijing Youan Hospital, Capital Medical University, Beijing, Peopleās Republic of China;
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Xiuying Zhao
Xiuying Zhao
Department of Clinical Laboratory, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing, Peopleās Republic of China
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Ming Chen
Ming Chen
Department of Clinical Laboratory, Beijing Youan Hospital, Capital Medical University, Beijing, Peopleās Republic of China;
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Guizhen Sun
Guizhen Sun
Department of Clinical Laboratory, Beijing Youan Hospital, Capital Medical University, Beijing, Peopleās Republic of China;
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Yanhua Yu
Yanhua Yu
Department of Clinical Laboratory, Beijing Youan Hospital, Capital Medical University, Beijing, Peopleās Republic of China;
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ABSTRACT.
This study aimed to isolate and characterize phages as an alternative treatment of multidrug- or pan-drug-resistant Pseudomonas aeruginosa. Phage titers and bacterial densities correlated, with the phages disappearing after bacteria were eliminated. We isolated phages in filtered sewage water by a double-layered agar spot test. Fifty-eight P. aeruginosa strains were used to screen the host spectrum of the 14 phages isolated. Random amplification of polymorphic DNA-typing polymerase chain reaction was used to analyze the genomic homologies of the 58 host bacteria strains and four phages with a broad host spectrum. Transmission electron microscopy was used to observe the morphology of the four phages with a broad host spectrum. Mice with intraabdominal P. aeruginosa infection were used as an inĀ vivo animal model to investigate the therapeutic effect of the selected phage. Four virulent phages with a broad host spectrum specific to P. aeruginosa strains were isolated. They were all double-stranded DNA viruses and belonged to four different genotypes. The test curve showed that phage I had the highest adsorption rate, the shortest latent period, and the largest burst size. The infected mouse model indicated that small doses of phage I could prevent the death of infected mice. Phage titers and bacterial densities correlated, with phages disappearing after bacteria were eliminated. Phage I was the most effective and promising treatment of drug-resistant P. aeruginosa.
- Supplemental Materials (PDF 69.529 KB)
Author Notes
These authors contributed equally to this work.
Financial support: This study was supported by the
Authorsā addresses: Fangfang Dai, Jinli Lou, Ming Chen, Guizhen Sun, and Yanhua Yu, Department of Clinical Laboratory, Beijing Youan Hospital, Capital Medical University, Beijing, Peopleās Republic of China, E-mails: daifangfang@126.com, loujinli@163.com, chenming@126.com, sunguizhen@126.com, and yyhs2005186@163.com. Gengxia Yang, General Surgical Center, Beijing Youan Hospital, Capital Medical University, Beijing, Peopleās Republic of China, E-mail: yanggengxia@126.com. Xiuying Zhao, Department of Clinical Laboratory, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing, Peopleās Republic of China, E-mail: zxya00340@btch.edu.cn.
- ABSTRACT.
- BACKGROUND
- MATERIALS AND METHODS
- Bacteria strains and preparations.
- Isolation and enrichment of phages.
- Host range test and covering spectrum test using clinically isolated P. aeruginosa strains.
- Genome extraction and RAPD typing of phages.
- Morphology of phage virions by electron microscopy.
- Multiplicity of infection, adsorption efficiency, and one-step growth curve experiment with phages.
- Abdominal cavity infection by P. aeruginosa and the determination of the minimum lethal dose in a mouse model.
- Phage therapeutic effect in the mouse model.
- Distribution of phages and bacteria inĀ vivo.
- Statistical analysis.
- RESULTS
- Isolated phages and their spectra.
- Genomic typing and electron microscopy of phages.
- The MOI of four broad-spectrum phages, their adsorption efficiency, and one-step growth curves.
- Therapeutic effect of phages in mice with abdominal cavity P. aeruginosa infection.
- Quantity distribution of phage I and P. aeruginosa in the mouse model.
- DISCUSSION
- CONCLUSION
- Supplemental Materials
- ACKNOWLEDGMENT
- REFERENCES
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Wilson R , Aksamit T , Aliberti S , De Soyza A , Elborn JS , Goeminne P , Hill AT , Menendez R , Polverino E , 2016. Challenges in managing Pseudomonas aeruginosa in non-cystic fibrosis bronchiectasis. Respir Med 117: 179ā189.
-
2.
Chen MJ , Wang H , 2003. Continuous surveillance of antimicrobial resistance among nosocomial gram-negative bacilli from intensive care units in China. Zhonghua Yi Xue Za Zhi 83: 375ā381.
-
3.
Sanford E , Farnaes L , Batalov S , Bainbridge M , Laubach S , Worthen HM , Tokita M , Kingsmore SF , Bradley J , 2018. Concomitant diagnosis of immune deficiency and Pseudomonas sepsis in a 19 month old with ecthyma gangrenosum by host whole-genome sequencing. Cold Spring Harb Mol Case Stud 4: a003244.
-
4.
Jean SS , Harnod D , Hsueh PR , 2022. Global threat of carbapenem-resistant gram-negative bacteria. Front Cell Infect Microbiol 12: 823684.
-
5.
Hong DJ , Bae IK , Jang IH , Jeong SH , Kang HK , Lee K , 2015. Epidemiology and characteristics of metallo-Ī²-lactamase-producing Pseudomonas aeruginosa. Infect Chemother 47: 81ā97.
-
6.
Merril CR , Scholl D , Adhya SL , 2003. The prospect for bacteriophage therapy in Western medicine. Nat Rev Drug Discov 2: 489ā497.
-
7.
Sulakvelidze A , 2005. Phage therapy: an attractive option for dealing with antibiotic-resistant bacterial infections. Drug Discov Today 10: 807ā809.
-
8.
Schooley RT et al., 2017. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob Agents Chemother 61: e00954-17.
-
9.
Mahenthiralingam E , Campbell ME , Foster J , Lam JS , Speert DP , 1996. Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J Clin Microbiol 34: 1129ā1135.
-
10.
Ceyssens PJ et al., 2009. Survey of Pseudomonas aeruginosa and its phages: de novo peptide sequencing as a novel tool to assess the diversity of worldwide collected viruses. Environ Microbiol 11: 1303ā1313.
-
11.
Premamalini T , Rajyoganandh V , Vijayakumar R , Veena H , Kindo AJ , Marak RS , 2021. Strain typing of Trichosporon asahii clinical isolates by random amplification of polymorphic DNA (RAPD) analysis. J Lab Physicians 13: 245ā251.
-
12.
Qamar W , Khan MR , Arafah A , 2017. Optimization of conditions to extract high quality DNA for PCR analysis from whole blood using SDS-proteinase K method. Saudi J Biol Sci 24: 1465ā1469.
-
13.
Li L , Yang H , Lin S , Jia S , 2010. Classification of 17 newly isolated virulent bacteriophages of Pseudomonas aeruginosa. Can J Microbiol 56: 925ā933.
-
14.
Biswas B , Adhya S , Washart P , Paul B , Trostel AN , Powell B , Carlton R , Merril CR , 2002. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infect Immun 70: 204ā210.
-
15.
Wang J et al., 2006. Use of bacteriophage in the treatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosa. Int J Mol Med 17: 309ā317.
-
16.
Shivshetty N , Hosamani R , Ahmed L , Oli AK , Sannauallah S , Sharanbassappa S , Patil SA , Kelmani CR , 2014. Experimental protection of diabetic mice against lethal P. aeruginosa infection by bacteriophage. BioMed Res Int 2014: 793242.
-
17.
National Institutes of Health , 2011. NIH Guide for the Care and Use of Laboratory Animals. Available at: https://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf. Accessed March 27, 2023.
-
18.
Kilkenny C , Brown W , Cuthill IC , Emerson M , Altman DG , NC3Rs Reporting Guidelines Working Group, 2010. Animal research: reporting in vivo experiments: the ARRIVE guidelines. Available at: https://doi.org/10.1111/j.1476-5381.2010.00872.x. Accessed March 27, 2023.
-
19.
Carter CD , Parks A , Abuladze T , Li M , Woolston J , Magnone J , Senecal A , Kropinski AM , Sulakvelidze A , 2012. Bacteriophage cocktail significantly reduces Escherichia coli O157:H7 contamination of lettuce and beef, but does not protect against recontamination. Bacteriophage 2: 178ā185.
-
20.
Jaiswal A , Koley H , Ghosh A , Palit A , Sarkar B , 2013. Efficacy of cocktail phage therapy in treating Vibrio cholerae infection in rabbit model. Microbes Infect 15: 152ā156.
-
21.
Ziyong S , Li L , Xuhui Z , Yue M , Jingyun L , Zhengyi S , Shaohong J , 2006. Analysis on antimicrobial resistance of clinical bacteria isolated from county hospitals and a teaching hospital. J Huazhong Univ Sci Technol (Med Sci) 26: 386ā388.
-
22.
China Antimicrobial Resistance Surveillance System , 2018. National Bacterial Resistance Monitoring Report 2018 (Brief Edition). Available at: http://www.carss.cn/Report/Details?aId=648. Accessed March 27, 2023.
-
23.
Betts JW , Hornsey M , Higgins PG , Lucassen K , Wille J , Salguero FJ , Seifert H , La Ragione RM , 2019. Restoring the activity of the antibiotic aztreonam using the polyphenol epigallocatechin gallate (EGCG) against multidrug-resistant clinical isolates of Pseudomonas aeruginosa. J Med Microbiol 68: 1552ā1559.
-
24.
Yusuf E , Van Herendael B , Verbrugghe W , Ieven M , Goovaerts E , Bergs K , Wouters K , Jorens PG , Goossens H , 2017. Emergence of antimicrobial resistance to Pseudomonas aeruginosa in the intensive care unit: association with the duration of antibiotic exposure and mode of administration. Ann Intensive Care 7: 72.
-
25.
Pachori P , Gothalwal R , Gandhi P , 2019. Emergence of antibiotic resistance Pseudomonas aeruginosa in intensive care unit; a critical review. Genes Dis 6: 109ā119.
-
26.
Tang C , Deng C , Zhang Y , Xiao C , Wang J , Rao X , Hu F , Lu S , 2018. Characterization and genomic analyses of Pseudomonas aeruginosa podovirus TC6: establishment of genus Pa11virus. Front Microbiol 9: 2561.
-
27.
de Melo ACC , da Mata Gomes A , Melo FL , Ardisson-AraĆŗjo DMP , de Vargas APC , Ely VL , Kitajima EW , Ribeiro BM , Wolff JLC , 2019. Characterization of a bacteriophage with broad host range against strains of Pseudomonas aeruginosa isolated from domestic animals. BMC Microbiol 19: 134.
-
28.
Jeon J , Yong D , 2019. Two novel bacteriophages improve survival in Galleria mellonella infection and mouse acute pneumonia models infected with extensively drug-resistant Pseudomonas aeruginosa. Appl Environ Microbiol 85: e02900ā18.
-
29.
Cisek AA , Dabrowska I , Gregorczyk KP , Wyzewski Z , 2017. Phage therapy in bacterial infections treatment: one hundred years after the discovery of bacteriophages. Curr Microbiol 74: 277ā283.
-
30.
El Haddad L , Harb CP , Gebara MA , Stibich MA , Chemaly RF , 2019. A systematic and critical review of bacteriophage therapy against multidrug-resistant ESKAPE organisms in humans. Nephrol Dial Transplant 69: 167ā178.
-
31.
Wright RCT , Friman VP , Smith MCM , Brockhurst MA , 2019. Resistance evolution against phage combinations depends on the timing and order of exposure. mBio 10: e01652ā19.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 329 | 329 | 62 |
| Full Text Views | 958 | 958 | 2 |
| PDF Downloads | 116 | 116 | 3 |