Comparison of culture and PCR-DGGE methods to evaluate the airways of cystic fibrosis patients and determination of their antibiotic resistance profile
-
Somayeh Moazami Goudarzi
,
Yasamin Shahpouri Arani
,
Ahya Abdi Ali
,
Parisa Mohammadi
,
Nassim Ghorbanmehr
,
Mohammadreza Modaresi
,
Mahtab Ghorban Movahed
,
Tooba Ghazanfari
Abstract
Background and Objectives: Respiratory infections are the most serious condition in cystic fibrosis (CF) patients; therefore, a thorough comprehension of the diversity and dominant microbial species in CF airways has a crucial role in treatment. Our objective was to determine the antibiotic resistance profile of CF airways microbiota and compare culture methods and PCR-DGGE to evaluate bacterial diversity.
Materials and Methods: Pharyngeal swabs from 121 CF patients were collected. The samples were then cultured, identified and antibiotic resistance testing was performed. Thirty samples were subjected to further molecular surveys. DNA contents of these samples were extracted and amplified using nested-PCR technique and their bacterial diversity was assessed by DGGE. The DGGE patterns were visualized and certain bands were excised and purified. Next, the DNA was amplified by another round of PCR and sent out for sequencing.
Results: Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae were the most prevalent species isolated using culture methods. S. aureus was the most common bacteria among 6 years and younger patients; while, P. aeruginosa had more prevalence among older ones. The PCR-DGGE results showed more diversity than culture methods, particularly in younger patients who exhibited more bacterial diversity than the older groups. Sequencing results unveiled the presence of certain bacterial species including Haemophilus parainfluenzae and Stenotrophomonas maltophilia which were completely missed in culture.
Conclusion: Even though culture-dependent methods are cost-effective, PCR-DGGE appeared to be more efficient to determine bacterial diversity. PCR-DGGE detects less abundant species, though their viability could not be determined using this method.
1. Bhagirath AY, Li Y, Somayajula D, Dadashi M, Badr S, Duan K. Cystic fibrosis lung environment and Pseudomonas aeruginosa infection. BMC Pulm Med 2016; 16: 174.
2. Charman S, McClenaghan E, Cosgriff R, Lee A, Carr S. UK Cystic Fibrosis Registry annual data report, 2018. Cyst Fibros Trust London, United Kingdom. 2019. https://www.cysticfibrosis.org.uk/sites/default/files/2020-12/2018%20Registry%20Annual%20Data%20Report.pdf
3. Vandeplassche E, Tavernier S, Coenye T, Crabbé A. Influence of the lung microbiome on antibiotic susceptibility of cystic fibrosis pathogens. Eur Respir Rev 2019; 28: 190041.
4. Huang YJ, LiPuma JJ. The microbiome in cystic fibrosis. Clin Chest Med 2016; 37: 59-67.
5. Cuthbertson L, Walker AW, Oliver AE, Rogers GB, Rivett DW, Hampton TH, et al. Lung function and microbiota diversity in cystic fibrosis. Microbiome 2020; 8: 45.
6. Vandeplassche E, Sass A, Ostyn L, Burmølle M, Kragh KN, Bjarnsholt T, et al. Antibiotic susceptibility of cystic fibrosis lung microbiome members in a multispecies biofilm. Biofilm 2020; 2: 100031.
7. Garcia-Nuñez M, Garcia-Gonzalez M, Pomares X, Montón C, Millares L, Quero S, et al. The respiratory microbiome in cystic fibrosis: compartment patterns and clinical relationships in early stage disease. Front Microbiol 2020; 11: 1463.
8. O’Brien TJ, Welch M. A continuous-flow model for in vitro cultivation of mixed microbial populations associated with cystic fibrosis airway infections. Front Microbiol 2019; 10: 2713.
9. Liu T, Jia T, Chen J, Liu X, Zhao M, Liu P. Analysis of microbial diversity in Shenqu with different fermentation times by PCR-DGGE. Braz J Microbiol 2017; 48: 246-250.
10. Siqueira JF, Sakamoto M, Rosado AS. Microbial community profiling using terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE). Methods Mol Biol 2017; 1537: 139-152.
11. Bluth MJ, Bluth MH. Molecular pathology techniques: advances in 2018. Clin Lab Med 2018; 38: 215-236.
12. Phadke S, Salvador AF, Alves JI, Bretschger O, Alves MM, Pereira MA. Harnessing the power of PCR molecular fingerprinting methods and next generation sequencing for understanding structure and function in microbial communities. Methods Mol Biol 2017; 1620: 225-248.
13. Singh, Akhlash. (2021). Genomic Techniques Used to Investigate the Human Gut Microbiota. 10.5772/intechopen.91808.
14. Moazami-Goudarzi S, Eftekhar F. Multidrug resistance and integron carriage in clinical isolates of Pseudomonas aeruginosa in Tehran, Iran. Turk J Med Sci 2015;45:789-793.
15. Clinical and Laboratory Standards Institute (2016) CLSI Document M100- S21. Performances Standards for Antimicrobial Susceptibility Testing. 26th Edition Informational Supplement, Wayne.
16. Willner D, Daly J, Whiley D, Grimwood K, Wainwright CE, Hugenholtz P. Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples. PLoS One 2012; 7(4): e34605.
17. Sundström K, Mishra PP, Pyysalo MJ, Lehtimäki T, Karhunen PJ, Pessi T. Similarity of salivary microbiome in parents and adult children. PeerJ 2020; 8: e8799.
18. Fasoli S, Marzotto M, Rizzotti L, Rossi F, Dellaglio F, Torriani S. Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. Int J Food Microbiol 2003; 82: 59-70.
19. Hong SW, Lee JS, Chung KS. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community structure in the food, intestines, and feces of earthworms. J Microbiol 2011; 49: 544-550.
20. Nelson A, De Soyza A, Bourke SJ, Perry JD, Cummings SP. Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis. Lett Appl Microbiol 2010; 51: 272-277.
21. Ding C, Adrian L, Peng Y, He J. 16S rRNA gene-based primer pair showed high specificity and quantification accuracy in detecting freshwater Brocadiales anammox bacteria. FEMS Microbiol Ecol 2020; 96: fiaa013.
22. Hahn A, Burrell A, Fanous H, Chaney H, Sami I, Perez GF, et al. Antibiotic multidrug resistance in the cystic fibrosis airway microbiome is associated with decreased diversity. Heliyon 2018; 4(9): e00795.
23. Kodori M, Nikmanesh B, Hakimi H, Ghalavand Z. Antibiotic susceptibility and biofilm formation of bacterial isolates derived from pediatric patients with cystic fibrosis from Tehran, Iran. Arch Razi Inst 2021;76:397-406.
24. Emaneini M, Kalantar-Neyestanaki D, Jabalameli L, Hashemi M, Beigverdi R, Jabalameli F. Molecular analysis and antimicrobial resistance pattern of distinct strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Iran. Iran J Microbiol 2019; 11: 98-107.
25. Pournajaf A, Razavi S, Irajian G, Ardebili A, Erfani Y, Solgi S, et al. Integron types, antimicrobial resistance genes, virulence gene profile, alginate production and biofilm formation in Iranian cystic fibrosis Pseudomonas aeruginosa isolates. Infez Med 2018; 26: 226-236.
26. Kordes A, Preusse M, Willger SD, Braubach P, Jonigk D, Haverich A, et al. Genetically diverse Pseudomonas aeruginosa populations display similar transcriptomic profiles in a cystic fibrosis explanted lung. Nat Commun 2019; 10: 3397.
27. Mahboubi MA, Carmody LA, Foster BK, Kalikin LM, VanDevanter DR, LiPuma JJ. Culture-based and culture-independent bacteriologic analysis of cystic fibrosis respiratory specimens. J Clin Microbiol 2016; 54: 613-619.
28. Muhlebach MS, Zorn BT, Esther CR, Hatch JE, Murray CP, Turkovic L, et al. Initial acquisition and succession of the cystic fibrosis lung microbiome is associated with disease progression in infants and preschool children. PLoS Pathog 2018; 14(1): e1006798.
29. Acosta N, Heirali A, Somayaji R, Surette MG, Workentine ML, Sibley CD, et al. Sputum microbiota is predictive of long-term clinical outcomes in young adults with cystic fibrosis. Thorax 2018; 73: 1016-1025.
30. Parkins MD, Somayaji R, Waters VJ. Epidemiology, biology, and impact of clonal Pseudomonas aeruginosa infections in cystic fibrosis. Clin Microbiol Rev 2018; 31(4): e00019-18.
31. Planet PJ. Adaptation and evolution of pathogens in the Cystic Fibrosis Lung. J Pediatric Infect Dis Soc 2022; 11(Supplement_2): S23-S31.
32. Brooks JP, Edwards DJ, Harwich MD, Rivera MC, Fettweis JM, Serrano MG, et al. The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies. BMC Microbiol 2015; 15: 66.
33. Scott JE, O’Toole GA. The yin and yang of Streptococcus lung infections in cystic fibrosis: a model for studying polymicrobial interactions. J Bacteriol 2019; 201(11): e00115-19.
34. Zachariah P, Ryan C, Nadimpalli S, Coscia G, Kolb M, Smith H, et al. Culture-independent analysis of pediatric bronchoalveolar lavage specimens. Ann Am Thorac Soc 2018; 15: 1047-1056.
35. Ahmed B, Cox MJ, Cuthbertson L, James P, Cookson WOC, Davies JC, et al. Longitudinal development of the airway microbiota in infants with cystic fibrosis. Sci Rep 2019; 9: 5143.
36. Law DKS, Shuel M, Bekal S, Bryce E, Tsang RSW. Genetic detection of quinolone resistance in Haemophilus parainfluenzae: mutations in the quinolone resistance-determining regions of gyrA and parC. Can J Infect Dis Med Microbiol 2010; 21(1): e20-22.
37. Barsky EE, Williams KA, Priebe GP, Sawicki GS. Incident Stenotrophomonas maltophilia infection and lung function decline in cystic fibrosis. Pediatr Pulmonol 2017; 52: 1276-1282.
38. Berdah L, Taytard J, Leyronnas S, Clement A, Boelle P-Y, Corvol H. Stenotrophomonas maltophilia: a marker of lung disease severity. Pediatr Pulmonol 2018; 53: 426-430.
2. Charman S, McClenaghan E, Cosgriff R, Lee A, Carr S. UK Cystic Fibrosis Registry annual data report, 2018. Cyst Fibros Trust London, United Kingdom. 2019. https://www.cysticfibrosis.org.uk/sites/default/files/2020-12/2018%20Registry%20Annual%20Data%20Report.pdf
3. Vandeplassche E, Tavernier S, Coenye T, Crabbé A. Influence of the lung microbiome on antibiotic susceptibility of cystic fibrosis pathogens. Eur Respir Rev 2019; 28: 190041.
4. Huang YJ, LiPuma JJ. The microbiome in cystic fibrosis. Clin Chest Med 2016; 37: 59-67.
5. Cuthbertson L, Walker AW, Oliver AE, Rogers GB, Rivett DW, Hampton TH, et al. Lung function and microbiota diversity in cystic fibrosis. Microbiome 2020; 8: 45.
6. Vandeplassche E, Sass A, Ostyn L, Burmølle M, Kragh KN, Bjarnsholt T, et al. Antibiotic susceptibility of cystic fibrosis lung microbiome members in a multispecies biofilm. Biofilm 2020; 2: 100031.
7. Garcia-Nuñez M, Garcia-Gonzalez M, Pomares X, Montón C, Millares L, Quero S, et al. The respiratory microbiome in cystic fibrosis: compartment patterns and clinical relationships in early stage disease. Front Microbiol 2020; 11: 1463.
8. O’Brien TJ, Welch M. A continuous-flow model for in vitro cultivation of mixed microbial populations associated with cystic fibrosis airway infections. Front Microbiol 2019; 10: 2713.
9. Liu T, Jia T, Chen J, Liu X, Zhao M, Liu P. Analysis of microbial diversity in Shenqu with different fermentation times by PCR-DGGE. Braz J Microbiol 2017; 48: 246-250.
10. Siqueira JF, Sakamoto M, Rosado AS. Microbial community profiling using terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE). Methods Mol Biol 2017; 1537: 139-152.
11. Bluth MJ, Bluth MH. Molecular pathology techniques: advances in 2018. Clin Lab Med 2018; 38: 215-236.
12. Phadke S, Salvador AF, Alves JI, Bretschger O, Alves MM, Pereira MA. Harnessing the power of PCR molecular fingerprinting methods and next generation sequencing for understanding structure and function in microbial communities. Methods Mol Biol 2017; 1620: 225-248.
13. Singh, Akhlash. (2021). Genomic Techniques Used to Investigate the Human Gut Microbiota. 10.5772/intechopen.91808.
14. Moazami-Goudarzi S, Eftekhar F. Multidrug resistance and integron carriage in clinical isolates of Pseudomonas aeruginosa in Tehran, Iran. Turk J Med Sci 2015;45:789-793.
15. Clinical and Laboratory Standards Institute (2016) CLSI Document M100- S21. Performances Standards for Antimicrobial Susceptibility Testing. 26th Edition Informational Supplement, Wayne.
16. Willner D, Daly J, Whiley D, Grimwood K, Wainwright CE, Hugenholtz P. Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples. PLoS One 2012; 7(4): e34605.
17. Sundström K, Mishra PP, Pyysalo MJ, Lehtimäki T, Karhunen PJ, Pessi T. Similarity of salivary microbiome in parents and adult children. PeerJ 2020; 8: e8799.
18. Fasoli S, Marzotto M, Rizzotti L, Rossi F, Dellaglio F, Torriani S. Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. Int J Food Microbiol 2003; 82: 59-70.
19. Hong SW, Lee JS, Chung KS. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community structure in the food, intestines, and feces of earthworms. J Microbiol 2011; 49: 544-550.
20. Nelson A, De Soyza A, Bourke SJ, Perry JD, Cummings SP. Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis. Lett Appl Microbiol 2010; 51: 272-277.
21. Ding C, Adrian L, Peng Y, He J. 16S rRNA gene-based primer pair showed high specificity and quantification accuracy in detecting freshwater Brocadiales anammox bacteria. FEMS Microbiol Ecol 2020; 96: fiaa013.
22. Hahn A, Burrell A, Fanous H, Chaney H, Sami I, Perez GF, et al. Antibiotic multidrug resistance in the cystic fibrosis airway microbiome is associated with decreased diversity. Heliyon 2018; 4(9): e00795.
23. Kodori M, Nikmanesh B, Hakimi H, Ghalavand Z. Antibiotic susceptibility and biofilm formation of bacterial isolates derived from pediatric patients with cystic fibrosis from Tehran, Iran. Arch Razi Inst 2021;76:397-406.
24. Emaneini M, Kalantar-Neyestanaki D, Jabalameli L, Hashemi M, Beigverdi R, Jabalameli F. Molecular analysis and antimicrobial resistance pattern of distinct strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Iran. Iran J Microbiol 2019; 11: 98-107.
25. Pournajaf A, Razavi S, Irajian G, Ardebili A, Erfani Y, Solgi S, et al. Integron types, antimicrobial resistance genes, virulence gene profile, alginate production and biofilm formation in Iranian cystic fibrosis Pseudomonas aeruginosa isolates. Infez Med 2018; 26: 226-236.
26. Kordes A, Preusse M, Willger SD, Braubach P, Jonigk D, Haverich A, et al. Genetically diverse Pseudomonas aeruginosa populations display similar transcriptomic profiles in a cystic fibrosis explanted lung. Nat Commun 2019; 10: 3397.
27. Mahboubi MA, Carmody LA, Foster BK, Kalikin LM, VanDevanter DR, LiPuma JJ. Culture-based and culture-independent bacteriologic analysis of cystic fibrosis respiratory specimens. J Clin Microbiol 2016; 54: 613-619.
28. Muhlebach MS, Zorn BT, Esther CR, Hatch JE, Murray CP, Turkovic L, et al. Initial acquisition and succession of the cystic fibrosis lung microbiome is associated with disease progression in infants and preschool children. PLoS Pathog 2018; 14(1): e1006798.
29. Acosta N, Heirali A, Somayaji R, Surette MG, Workentine ML, Sibley CD, et al. Sputum microbiota is predictive of long-term clinical outcomes in young adults with cystic fibrosis. Thorax 2018; 73: 1016-1025.
30. Parkins MD, Somayaji R, Waters VJ. Epidemiology, biology, and impact of clonal Pseudomonas aeruginosa infections in cystic fibrosis. Clin Microbiol Rev 2018; 31(4): e00019-18.
31. Planet PJ. Adaptation and evolution of pathogens in the Cystic Fibrosis Lung. J Pediatric Infect Dis Soc 2022; 11(Supplement_2): S23-S31.
32. Brooks JP, Edwards DJ, Harwich MD, Rivera MC, Fettweis JM, Serrano MG, et al. The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies. BMC Microbiol 2015; 15: 66.
33. Scott JE, O’Toole GA. The yin and yang of Streptococcus lung infections in cystic fibrosis: a model for studying polymicrobial interactions. J Bacteriol 2019; 201(11): e00115-19.
34. Zachariah P, Ryan C, Nadimpalli S, Coscia G, Kolb M, Smith H, et al. Culture-independent analysis of pediatric bronchoalveolar lavage specimens. Ann Am Thorac Soc 2018; 15: 1047-1056.
35. Ahmed B, Cox MJ, Cuthbertson L, James P, Cookson WOC, Davies JC, et al. Longitudinal development of the airway microbiota in infants with cystic fibrosis. Sci Rep 2019; 9: 5143.
36. Law DKS, Shuel M, Bekal S, Bryce E, Tsang RSW. Genetic detection of quinolone resistance in Haemophilus parainfluenzae: mutations in the quinolone resistance-determining regions of gyrA and parC. Can J Infect Dis Med Microbiol 2010; 21(1): e20-22.
37. Barsky EE, Williams KA, Priebe GP, Sawicki GS. Incident Stenotrophomonas maltophilia infection and lung function decline in cystic fibrosis. Pediatr Pulmonol 2017; 52: 1276-1282.
38. Berdah L, Taytard J, Leyronnas S, Clement A, Boelle P-Y, Corvol H. Stenotrophomonas maltophilia: a marker of lung disease severity. Pediatr Pulmonol 2018; 53: 426-430.
| Files | ||
| Issue | Vol 15 No 6 (2023) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v15i6.14135 | |
| Keywords | ||
| Cystic fibrosis; Respiratory tract infections; Antibiotic resistance; Denaturing gradient gel electrophoresis; Microbiota | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Moazami Goudarzi S, Shahpouri Arani Y, Abdi Ali A, Mohammadi P, Ghorbanmehr N, Modaresi M, Ghorban Movahed M, Ghazanfari T. Comparison of culture and PCR-DGGE methods to evaluate the airways of cystic fibrosis patients and determination of their antibiotic resistance profile. Iran J Microbiol. 2023;15(6):750-758.
