Molecular analysis and antimicrobial resistance pattern of distinct strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Iran
-
Mohammad Emaneini
,
Davood Kalantar-Neyestanaki
,
Leila Jabalameli
,
Mojtaba Hashemi
,
Reza Beigverdi
,
Fereshteh Jabalameli
Abstract
Background and Objectives: Colonization of Pseudomonas aeruginosa in Cystic Fibrosis (CF) patients may lead to severe pulmonary disease and death. Different characteristics of P. aeruginosa from these patients were determined in the present study.
Materials and Methods: Antimicrobial susceptibility and AmpC-overproduction were determined. The β-lactamase genes were detected by PCR and the oprD gene was sequenced in some of the carbapenem resistance isolates. Distribution of exo genes was determined by PCR. Cytotoxicity of Exo effector proteins was measured using A549 cells. Biofilm production was determined by microtiter plate assay. Random amplified polymorphic DNA (RAPD) –PCR was performed for molecular analysis.
Results: Polymyxin B, piperacillin/tazobactam and meropenem were the most active antibiotics and 9.6% of isolates were ampC overproducers. The prevalence of blaVEB, blaOXA, blaVIM, and blaPER genes were as follow: 22.7%, 3.75%, 6.25% and 3.75%, respectively. A high proportion (83.5%) of isolates was able to produce biofilm. The exoT gene was present in all isolates while exoU was present in about 35% of them. RAPD-PCR revealed 49 patterns among 78 tested isolates in which 34 patterns were detected once.
Conclusion: Biofilm formation ability and relatively high frequency of exoS may contribute to the persistence of bacteria within lungs of CF patients. Some characteristics of isolates recovered from a single patient after several sampling procedures were similar, while others lacked resemblance.
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3. Engel J, Balachandran P. Role of Pseudomonas aeruginosa type III effectors in disease. Curr Opin Microbiol 2009;12:61-66.
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5. Yahr TL, Goranson J, Frank DW. Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway. Mol Microbiol 1996; 22:991-1003.
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7. Barbieri JT, Sun J. Pseudomonas aeruginosa ExoS and ExoT. Rev Physiol Biochem Pharmacol 2004;152:79-92.
8. Ochoa CD, Alexeyev M, Pastukh V, Balczon R, Stevens T. Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial tau phosphorylation and permeability. J Biol Chem 2012;287:25407-25418.
9. Sato H, Frank DW. ExoU is a potent intracellular phospholipase. Mol Microbiol 2004;53:1279-1290.
10. Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2000;2:1051-1060.
11. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318-1322.
12. Fuqua C, Parsek MR, Greenberg EP. Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu Rev Genet 2001;35:439-468.
13. Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ, Greenberg EP. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 2000;407:762-764.
14. Hengzhuang W, Ciofu O, Yang L, Wu H, Song Z, Oliver A, et al. High β-lactamase levels change the pharmacodynamics of β-lactam antibiotics in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 2013;57:196-204.
15. Döring G, Flume P, Heijerman H, Elborn JS. Treatment of lung infection in patients with cystic fibrosis: Current and future strategies. J Cyst Fibros 2012;11:461-479.
16. Kalantar-Neyestanaki D, Mirsalehian A, Rezagholizadeh F, Jabalameli F, Taherikalani M, Emaneini M. Determination of extended spectrum beta-lactamases, metallo-β-lactamases and AmpC-β-lactamases among carbapenem resistant Pseudomonas aeruginosa isolated from burn patients. Burns 2014;40:1556-1561.
17. Mahon CR, Lehman DC, Manuselis G. Text book of diagnostic microbiology.3rd edn. Philadelphia, PA, USA 2007; p:564-586.
18. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. M100-S25. 2016;35: 52-54.
19. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18:268-281.
20. Mirsalehian A, Feizabadi M, Nakhjavani FA, Jabalameli F, Goli H, Kalantari N. Detection of VEB-1, OXA-10 and PER-1 genotypes in extended-spectrum beta-lactamase-producing Pseudomonas aeruginosa strains isolated from burn patients. Burns 2010;36:70-74.
21. Rodríguez-Martínez JM, Poirel L, Nordmann P. Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2009;53:4783-4788.
22. Feltman H, Schulert G, Khan S, Jain M, Peterson M, Hauser AR. Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa. Microbiology 2001;147:2659-2669.
23. Jabalameli F, Mirsalehian A, Khoramian B, Aligholi M, Khoramrooz SS, Asadollahi P, et al. Evaluation of biofilm production and characterization of genes encoding type III secretion system among Pseudomonas aeruginosa isolated from burn patients. Burns 2012;38:1192-1197.
24. Lee B, Schjerling CK, Kirkby N, Hoffmann N, Borup R, Molin S, et al. Mucoid Pseudomonas aeruginosa isolates maintain the biofilm formation capacity and the gene expression profiles during the chronic lung infection of CF patients. APMIS 2011;119:263-274.
25. Bukanov N, Nathan Ravi V, Miller D, Srivastava K, Berg DE. Pseudomonas aeruginosa corneal ulcer isolates distinguished using the arbitrarily primed PCR DNA fingerprinting method. Curr Eye Res 1994;13:783-790.
26. Mahenthiralingam E, Campbell ME, Foster J, Lam JS, Speert DP. Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J Clin Microbiol 1996;34:1129-1135.
27. Ruimy R, Genauzeau E, Barnabe C, Beaulieu A, Tibayrenc M, Andremont A. Genetic diversity of Pseudomonas aeruginosa strains isolated from ventilated patients with nosocomial pneumonia, cancer patients with bacteremia, and environmental water. Infect Immun 2001;69:584-588.
28. Rao P, McCaughan J, McCalmont M, Goldsmith CE, Hall V, Millar BC, et al. Comparison of antibiotic susceptibility patterns in Pseudomonas aeruginosa isolated from adult patients with cystic fibrosis (CF) with invasive Pseudomonas aeruginosa from non-CF patients. J Cyst Fibros 2012;11:349-352.
29. Lee VT, Smith RS, Tu¨ mmler B, Lory S. Activities of Pseudomonas aeruginosa effectors secreted by the Type III secretion system in vitro and during infection. Infect Immun 2005;73: 1695-1705.
30. Werner E, Roe F, Bugnicourt A, Franklin MJ, Heydorn A, Molin S, et al. Stratified growth in Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 2004;70:6188-6196.
31. Folkesson A, Jelsbak L, Yang L, Krogh Johansen H, Ciofu O, Høiby N, et al. Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective. Nat Rev Microbiol 2012;10:841-851.
32. Perez LRR, Costa MCN, Freitas ALP, Barth AL. Evaluation of biofilm production by Pseudomonas aeruginosa isolates recovered from Cystic fibrosis and non-cystic fibrosis patients. Braz J Microbiol 2011;42:476-479 .
33. Horrevortz AM, Borst J, Puyk RJT, de Ridder R, Dzoljic-Danilovic G, Degener JE, et al. Ecology of Pseudomonas aeruginosa in patients with cystic fibrosis. J Med Microbiol 1990;31:119-124.
34. Ortiz-Herrera M, Gerónimo-Gallegos A, Cuevas-Schacht F, Pérez-Fernández L, Coria-Jiménez R. [RAPD-PCR characterization of Pseudomonas aeruginosa strains obtained from cystic fibrosis patients].Salud Publica Mex 2004;46:149-157.
35. Cohen-Cymberknoh M, Gilead N, Gartner S, Rovira S, Blau H, Mussaffi H, et al. Eradication failure of newly acquired Pseudomonas aeruginosa isolates in cystic fibrosis. J Cyst Fibros 2016;15:776-782.
2. Valadbeigi H, Tabatabaei RR, Malek A, Sekawi Z, Raftari M, Parvaneh K, et al. Genomic diversity and virulence genes among clinical isolates of Pseudomonas aeruginosa. Clin Lab 2014;60:363-367.
3. Engel J, Balachandran P. Role of Pseudomonas aeruginosa type III effectors in disease. Curr Opin Microbiol 2009;12:61-66.
4. Iglewski BH, Sadoff J, Bjorn MJ, Maxwell ES. Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. Proc Natl Acad Sci U S A 1978;75:3211-3215.
5. Yahr TL, Goranson J, Frank DW. Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway. Mol Microbiol 1996; 22:991-1003.
6. Yahr TL, Vallis AJ, Hancock MK, Barbieri JT, Frank DW. ExoY, a novel adenylate cyclase secreted by the Pseudomonas aeruginosa type III system. Proc Natl Acad Sci U S A 1998;95:13899-13904.
7. Barbieri JT, Sun J. Pseudomonas aeruginosa ExoS and ExoT. Rev Physiol Biochem Pharmacol 2004;152:79-92.
8. Ochoa CD, Alexeyev M, Pastukh V, Balczon R, Stevens T. Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial tau phosphorylation and permeability. J Biol Chem 2012;287:25407-25418.
9. Sato H, Frank DW. ExoU is a potent intracellular phospholipase. Mol Microbiol 2004;53:1279-1290.
10. Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2000;2:1051-1060.
11. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318-1322.
12. Fuqua C, Parsek MR, Greenberg EP. Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu Rev Genet 2001;35:439-468.
13. Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ, Greenberg EP. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 2000;407:762-764.
14. Hengzhuang W, Ciofu O, Yang L, Wu H, Song Z, Oliver A, et al. High β-lactamase levels change the pharmacodynamics of β-lactam antibiotics in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 2013;57:196-204.
15. Döring G, Flume P, Heijerman H, Elborn JS. Treatment of lung infection in patients with cystic fibrosis: Current and future strategies. J Cyst Fibros 2012;11:461-479.
16. Kalantar-Neyestanaki D, Mirsalehian A, Rezagholizadeh F, Jabalameli F, Taherikalani M, Emaneini M. Determination of extended spectrum beta-lactamases, metallo-β-lactamases and AmpC-β-lactamases among carbapenem resistant Pseudomonas aeruginosa isolated from burn patients. Burns 2014;40:1556-1561.
17. Mahon CR, Lehman DC, Manuselis G. Text book of diagnostic microbiology.3rd edn. Philadelphia, PA, USA 2007; p:564-586.
18. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. M100-S25. 2016;35: 52-54.
19. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18:268-281.
20. Mirsalehian A, Feizabadi M, Nakhjavani FA, Jabalameli F, Goli H, Kalantari N. Detection of VEB-1, OXA-10 and PER-1 genotypes in extended-spectrum beta-lactamase-producing Pseudomonas aeruginosa strains isolated from burn patients. Burns 2010;36:70-74.
21. Rodríguez-Martínez JM, Poirel L, Nordmann P. Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2009;53:4783-4788.
22. Feltman H, Schulert G, Khan S, Jain M, Peterson M, Hauser AR. Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa. Microbiology 2001;147:2659-2669.
23. Jabalameli F, Mirsalehian A, Khoramian B, Aligholi M, Khoramrooz SS, Asadollahi P, et al. Evaluation of biofilm production and characterization of genes encoding type III secretion system among Pseudomonas aeruginosa isolated from burn patients. Burns 2012;38:1192-1197.
24. Lee B, Schjerling CK, Kirkby N, Hoffmann N, Borup R, Molin S, et al. Mucoid Pseudomonas aeruginosa isolates maintain the biofilm formation capacity and the gene expression profiles during the chronic lung infection of CF patients. APMIS 2011;119:263-274.
25. Bukanov N, Nathan Ravi V, Miller D, Srivastava K, Berg DE. Pseudomonas aeruginosa corneal ulcer isolates distinguished using the arbitrarily primed PCR DNA fingerprinting method. Curr Eye Res 1994;13:783-790.
26. Mahenthiralingam E, Campbell ME, Foster J, Lam JS, Speert DP. Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J Clin Microbiol 1996;34:1129-1135.
27. Ruimy R, Genauzeau E, Barnabe C, Beaulieu A, Tibayrenc M, Andremont A. Genetic diversity of Pseudomonas aeruginosa strains isolated from ventilated patients with nosocomial pneumonia, cancer patients with bacteremia, and environmental water. Infect Immun 2001;69:584-588.
28. Rao P, McCaughan J, McCalmont M, Goldsmith CE, Hall V, Millar BC, et al. Comparison of antibiotic susceptibility patterns in Pseudomonas aeruginosa isolated from adult patients with cystic fibrosis (CF) with invasive Pseudomonas aeruginosa from non-CF patients. J Cyst Fibros 2012;11:349-352.
29. Lee VT, Smith RS, Tu¨ mmler B, Lory S. Activities of Pseudomonas aeruginosa effectors secreted by the Type III secretion system in vitro and during infection. Infect Immun 2005;73: 1695-1705.
30. Werner E, Roe F, Bugnicourt A, Franklin MJ, Heydorn A, Molin S, et al. Stratified growth in Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 2004;70:6188-6196.
31. Folkesson A, Jelsbak L, Yang L, Krogh Johansen H, Ciofu O, Høiby N, et al. Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective. Nat Rev Microbiol 2012;10:841-851.
32. Perez LRR, Costa MCN, Freitas ALP, Barth AL. Evaluation of biofilm production by Pseudomonas aeruginosa isolates recovered from Cystic fibrosis and non-cystic fibrosis patients. Braz J Microbiol 2011;42:476-479 .
33. Horrevortz AM, Borst J, Puyk RJT, de Ridder R, Dzoljic-Danilovic G, Degener JE, et al. Ecology of Pseudomonas aeruginosa in patients with cystic fibrosis. J Med Microbiol 1990;31:119-124.
34. Ortiz-Herrera M, Gerónimo-Gallegos A, Cuevas-Schacht F, Pérez-Fernández L, Coria-Jiménez R. [RAPD-PCR characterization of Pseudomonas aeruginosa strains obtained from cystic fibrosis patients].Salud Publica Mex 2004;46:149-157.
35. Cohen-Cymberknoh M, Gilead N, Gartner S, Rovira S, Blau H, Mussaffi H, et al. Eradication failure of newly acquired Pseudomonas aeruginosa isolates in cystic fibrosis. J Cyst Fibros 2016;15:776-782.
| Files | ||
| Issue | Vol 11 No 2 (2019) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v11i2.1068 | |
| Keywords | ||
| Pseudomonas aeruginosa Type 3 secretion system Cystic fibrosis Cytotoxicity Biofilm | ||
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How to Cite
1.
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(2):98-107.
