Genotypic characterization and novel multilocus sequence types of exoU+ Pseudomonas aeruginosa from different clinical infections and environments

Hemin E.  Othman; Eric L.  Miller; Jaladet MS.  Jubrael; Ian S.  Roberts

doi:10.15649/2346075X.458

Authors

Hemin E. Othman Dr.Hemin E. Othman, PhD of Molecular Microbiology,Scientific Research Centre, College of Science, University of Duhok, Kurdistan Region-Iraq
Eric L. Miller Dr. Eric L. Miller, Faculty of Biology, Medicine, and Health, University of Manchester, Manchester, UnitedKingdom
Jaladet MS. Jubrael ProfessorJaladet MS. Jubrael, Professor of Molecular biology, Scientific Research Centre, College of Science, University of Duhok, Kurdistan Region-Iraq
Ian S. Roberts Professor Ian S. Roberts,Faculty of Biology, Medicine, and Health, University of Manchester, Manchester, UnitedKingdom

DOI:

https://doi.org/10.15649/2346075X.458

Keywords:

Pseudomonas aeruginosa; PCR; exoU; MLST; Phylogenetic diversity

Abstract

Introducción: The exoU gene, a marker for highly virulent strains of Pseudomonas aeruginosa, is the major contributor to a wide variety
of healthcare-associated infections. Methods: In this study, the antibiotic susceptibility profile, prevalence and genotyping of exoU+ P.
aeruginosa were demonstrated. A total of 101 isolates of P. aeruginosa were analysed from different clinical and environmental sources. Results: The antibiotic susceptibility profile classified these isolates as extensively drug resistant (35.6%), multidrug resistant (40.5%) and non-multidrug resistant (23.7%). The prevalence of exoU gene was screened by PCR and 23 exoU+ genotypes were detected which all were clinical isolates. Multilocus sequence typing (MLST) analysis of seven loci assigned these exoU+ genotypes to 21 sequence types (STs) from which 16 new STs were identified. The prevalent STs were ST-
308 and ST-235. Phylogenetic analysis using the concatenated nucleotide sequences of the seven housekeeping genes, exoU and the ITS region differentiated these exoU+ strains into five main groups. However, distinct evolutionary origins for some new sequence types were also indicated. Conclusions: The studied isolates showed the coexistence of exoU- and exoU+ genotypes of clinical P. aeruginosa in Kurdistan with a majority of MDR and XDR pattern. The prevalent STs found in other hospitals worldwide and at the international level.

Author Biographies

Hemin E. Othman, Dr.Hemin E. Othman, PhD of Molecular Microbiology,Scientific Research Centre, College of Science, University of Duhok, Kurdistan Region-Iraq

Dr.Hemin E. Othman, PhD of Molecular Microbiology,Scientific Research Centre, College of Science, University of Duhok, Kurdistan Region-Iraq

Eric L. Miller, Dr. Eric L. Miller, Faculty of Biology, Medicine, and Health, University of Manchester, Manchester, UnitedKingdom

Dr. Eric L. Miller, Faculty of Biology, Medicine, and Health, University of Manchester, Manchester, UnitedKingdom

Jaladet MS. Jubrael, ProfessorJaladet MS. Jubrael, Professor of Molecular biology, Scientific Research Centre, College of Science, University of Duhok, Kurdistan Region-Iraq

ProfessorJaladet MS. Jubrael, Professor of Molecular biology, Scientific Research Centre, College of Science, University of Duhok, Kurdistan Region-Iraq

Ian S. Roberts, Professor Ian S. Roberts,Faculty of Biology, Medicine, and Health, University of Manchester, Manchester, UnitedKingdom

Professor Ian S. Roberts,Faculty of Biology, Medicine, and Health, University of Manchester, Manchester, UnitedKingdom

References

Akanji B O, Ajele J O, Onasanya A and Oyelakin O. Genetic Fingerprinting of Pseudomonas aeruginosa Involved in Nosocomial Infection as Revealed by RAPD-PCR Markers. Biotechnology 2011; 10: 70-77. https://doi.org/10.3923/biotech.2011.70.77

Lister P D, Wolter D J and Hanson N D. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms.ClinMicrobiol Rev. 2009; 22(4):582-610. https://doi.org/10.1128/CMR.00040-09.

Fazeli H, Sadighian H, Esfahani B N and Pourmand M R. Genetic characterization of Pseudomonas aeruginosa-resistant isolates at the university teaching hospital in Iran. Adv Biomed Res 2015. https://doi.org/10.4103/2277-9175.161583

Streeter K and Katouli M. Pseudomonas aeruginosa: A review of their Pathogenesis and Prevalence in Clinical Settings and the Environment. Infect Epidemiol Med. 2016; 2(1): 25-32. https://doi.org/10.18869/modares.iem.2.1.25

Diaz M H and Hauser A R. Pseudomonas aeruginosa CytotoxinExoU Is Injected into Phagocytic Cells during Acute Pneumonia. Infect. Immun 2010;78 (No. 4): 1447-1456. https://doi.org/10.1128/IAI.01134-09

Miranda C C, de Filippis I, Pinto L H, Coelho-Souza T, Bianco K, Cacci L C, Picão R C and Clementino M M. Genotypic characteristics of multidrug-resistant Pseudomonas aeruginosa from hospital wastewater treatment plant in Rio de Janeiro, Brazil. J ApplMicrobiol. 2015; 118(6):1276-86. https://doi.org/10.1111/jam.12792.

Valot B, Guyeux C, Rolland J Y, Mazouzi K, Bertrand X and Hocquet D. What it takes to be a Pseudomonas aeruginosa? The core genome of the opportunistic pathogen updated. PLoS ONE. 2015; 10(5): e0126468. https://doi.org/10.1371/journal.pone.0126468.

Ansari A, Salman S M and Yaqoob S. Antibiotic Resistance Pattern in Pseudomonas aeruginosa Strains Isolated at Era s Lucknow Medical College and Hospital, Lucknow, India.Int.J.Curr.Microbiol.App.Sci.2015; Special Issue-1: 48-58.

Holban A, Chifiriuc M C, Cotar A I, Bleotu C, Grumezescu A M, Banu O and Lazar V. Virulence markers in Pseudomonas aeruginosa isolates from hospitalacquired infections occurred in patients with underlying cardiovascular disease. Romanian Biotechnological Letters 2013; 18 (No. 6): 8843-8854.

Sato H and Frank D W. Intoxication of Host Cells by the T3SS Phospholipase ExoU: PI (4, 5) P2-Associated, Cytoskeletal Collapse and Late Phase Membrane Blebbing. PLoS ONE 2014; 9(7): https://doi.org/10.1371/journal.pone.0103127

Howell H A, Logan L K and Hauser A R. Type III secretion of ExoU is critical during early Pseudomonas aeruginosa pneumonia. mBio 2013, https://doi.org/10.1128/mBio.00032-13.

Machado G, Oliveira A, Saliba A M, Lima C, Suassuna J H R and Plotkowski M. Pseudomonas aeruginosa toxin ExoU induces a PAF-dependent impairment of alveolar fibrin turnover secondary to enhanced activation of coagulation and increased expression of plasminogen activator inhibitor-1 in the course of mice pneumosepsis. Respiratory Research 2011; 12:104, https://doi.org/10.1186/1465-9921-12-104

Sawa T, Shimizu M, Moriyama K and Wiener-Kronish J P. Association between Pseudomonas aeruginosa type III secretion, antibiotic resistance, and clinical outcome: a review. Critical Care 2014, https://doi.org/10.1186/s13054-014-0668-9

Garey K W, Vo Q P, Larocco M T, Gentry L O and Tam V H. Prevalence of Type III Secretion Protein Exoenzymes and Antimicrobial Susceptibility Patterns from Bloodstream Isolates of Patients with Pseudomonas aeruginosa Bacteremia. Journal of Chemotherapy 2008; 20 (No. 6): 714-720.

https://doi.org/10.1179/joc.2008.20.6.714

Ahmed S S and Alp E. Genotyping methods for monitoring the epidemic evolution of A. baumannii strains. J Infect Dev Ctries2015; 9(4):347-354. https://doi.org/10.3855/jidc.6201

Maàtallah M, Bakhrouf A, Habeeb M A, Turlej-Rogacka A, Iversen A, et al. Four Genotyping Schemes for Phylogenetic Analysis of Pseudomonas aeruginosa: Comparison of Their Congruence with Multi-Locus Sequence Typing. PLoS ONE 2013, https://doi.org/10.1371/journal.pone.0082069

Gomila M, Gallegos M C, Fernández-Baca V, Pareja A, Pascual M, Díaz-Antolín P, et al. (2013). Genetic diversity of clinical Pseudomonas aeruginosa isolates in a public hospital in Spain. BMC Microbiology 2013; 13:138. https://doi.org/10.1186/1471-2180-13-138

Mano Y, Saga T, Ishii Y, Yoshizumi A, Bonomo R A, Yamaguchi K and Tateda K. Molecular analysis of the integrons of metallo-β-lactamase-producing Pseudomonas aeruginosa isolates collected by nationwide surveillance programs across Japan. BMC Microbiology 2015, https://doi.org/10.1186/s12866-015-0378-8

Maiden M C J. Multilocus sequence typing of bacteria. Annu Rev Microbiol. 2006; 60:561-588, https://doi.org/10.1146/annurev.micro.59.030804.121325.

Maatallah M, Cheriaa J, Backhrouf A, Iversen A, Grundmann H, Do T, Lanotte P, Mastouri M, Elghmati M S, Rojo F, Mejdi S and Giske C G. Population structure of Pseudomonas aeruginosa from five Mediterranean countries: evidence for frequent recombination and epidemic occurrence of CC235.PLoS One. 2011; 6(10):e25617. https://doi.org/10.1371/journal.pone.0025617

Chen S H, Chen R Y, Xu X L and Chen H T.Multilocus sequencing typing of Pseudomonas aeruginosa isolates and analysis of potential pathogenicity of typical genotype strains from occupational oxyhelium saturation divers.Undersea Hyperb Med. 2014; 41(2):135-41.

Curran B, Jonas D, Grundmann H, Pitt T, Dowson C G. Development of a multilocus sequence typing scheme for the opportunistic pathogen Pseudomonas aeruginosa. J ClinMicrobiol 2004; 42 (No. 3): 5644-5649. https://doi.org/10.1128/JCM.42.12.5644-5649.2004

Hassan K I, Rafik S A, and Mussum K. Molecular identification of Pseudomonas aeruginosa isolated from Hospitals in Kurdistan region. Journal of Advanced Medical Research 2012; 2 (No.3): 90-98.

Al-Dahhan H AA. Molecular Characterization of P. aeruginosa isolated from Patients with URTI. JCBPS; Section B 2015; 5 (No. 3): 2736-2747.

AbdulQader N K, Raoof W M, and Al-Mussawi A A. BiofimForming-Pseudomonas aeruginosa isolated from burn patients in Basra city, south of Iraq. Int. J. Curr.Res. 2015; 7(10):21750-21753.

Khan N H, Ahsan M, Yoshizawa S, Hosoya S, Yokota A and Kogure K. Multilocus Sequence Typing and Phylogenetic Analyses of Pseudomonas aeruginosa Isolates from the Ocean. Appl. Environ. Microbiol. 2008; 74 (No. 20): 6194-6205. https://doi.org/10.1128/AEM.02322-07

Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; twenty-fourth informational supplement.2014; CLSI document M100-S24. Wayne, PA. 34(1).

Magiorakos A P, Srinivasan A, Carey R B, Carmeli Y, Falagas M E, Giske C G, Harbarth S, Hindler J F, Kahlmeter G, Olsson-Liljequist B, Paterson D L, Rice L B, Stelling J, Struelens M J, Vatopoulos A, Weber J T and Monnet D L. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance.ClinMicrobiol Infect. 2012; 18(3):268-81. https://doi.org/10.1111/j.1469-0691.2011.03570.x

Spilker T, Coenye T, Vandamme P, LiPuma J J. PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. J. Clin. Microbiol. 2004, https://doi.org/10.1128/JCM.42.5.2074-2079.2004

Bradbury R. Pseudomonas aeruginosa in Tasmania. PhD thesis, The University of Tasmania, 2009.

Tyler S D, Strathdee C A, Rozee K R, Johnson W M. Oligonucleotide primers designed to differentiate pathogenic pseudomonads on the basis of the sequencing of genes coding for 16S-23S rRNA internal transcribed spacers. Clin. Diagn. Lab. Immunol. 1995; 2: 448-453.

Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P and Drummond A. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012; 28(12): 1647-1649. https://doi.org/10.1093/bioinformatics/bts199

Posada D. jModelTest: Phylogenetic Model Averaging. Mol. Biol. Evol. 2008, 25:1253-1256. https://doi.org/10.1093/molbev/msn083

Bazinet A L and Cummings M P. The lattice project: a grid research and production environment combining multiple grid computing models. Pages 2-13. In Weber, M. H. W. (Ed.) Distributed & Grid Computing - Science Made Transparent for Everyone. Principles, Applications and Supporting Communities. Rechenkraft.net, Marburg, 2008.

Bradbury R S, Roddam L F, Merritt A, Reid D W and Champion A C. Virulence gene distribution in clinical, nosocomial and environmental isolates of Pseudomonas aeruginosa. Journal of Medical Microbiology 2010, https://doi.org/10.1099/jmm.0.018283-0

Jabalameli F, Mirsalehian A, Khoramian B, Aligholi M, et al. Evaluation of biofilm production and characterization of genes encoding type III secretion system among Pseudomonas aeruginosa isolated from burn patients. Burns 2012, https://doi.org/10.1016/j.burns.2012.07.030

Yousefi-Avarvand A, Khashei R, SedighEbrahim-Saraie H, Emami A, Zomorodian K and Motamedifar M. The frequency of exotoxin A and exoenzymes S and U genes among clinical isolates of Pseudomonas aeruginosa in Shiraz, Iran. Int J Mol Cell Med. 2015; 4(3):167-73.

Azimi S, Kafil H S, Baghi H B, Shokrian S, Najaf K, et al. Presence of exoY, exoS, exoU and exoT genes, antibiotic resistance and biofilm production among Pseudomonas aeruginosa isolates in Northwest Iran.GMS Hygiene and Infection Control.2016; 11: ISSN 2196-5226.

Kulasekara B R, Kulasekara H D, Wolfgang M C, Stevens L, Frank D W and Lory S. Acquisition and evolution of the exoU locus in Pseudomonas aeruginosa.J Bacteriol. 2006; 188(11):4037-50. https://doi.org/10.1128/JB.02000-05

Streeter K, Neuman C, Thompson J, Hatje E and Katouli M. The characteristics of genetically related Pseudomonas aeruginosa from diverse sources and their interaction withhuman cell lines. Canadian Journal of Microbiology 2016; 62(3): 233-240,https://doi.org/10.1139/cjm-2015-0536

Oliver A, Mulet X, López-Causapé C and Juan C. The increasing threat of Pseudomonas aeruginosa high-risk clones. Elsevier Ltd. Drug Resistance Updates2015, https://doi.org/10.1016/j.drup.2015.08.002.

Willmann M, Bezdan D, Zapata L, Susak H, et al. Analysis of a long-term outbreak of XDR Pseudomonas aeruginosa: a molecular epidemiological study. J AntimicrobChemother 2015, https://doi.org/10.1093/jac/dku546.

Martinez E, Perez J E, Buelvas F, Tovar C, Vanegas N and Stokes H. W. Establishment and multi drug resistance evolution of ST235 Pseudomonas aeruginosa strains in the intensive care unit of a Colombian hospital. Research in Microbiology, 2014; 165: 852-856. https://doi.org/10.1016/j.resmic.2014.10.011

Wang M, Cao B, Yu Q, Liu L, Gao Q, Wang L and Feng L. Analysis of the 16S-23S rRNA Gene Internal Transcribed Spacer Region in KlebsiellaSpecies. J. Clin. Microbiol. 2008, https://doi.org/10.1128/JCM.00927-08

Jolley K A, Maiden M C J. BIGSdb; Scalable analysis of bacterial genome variation at the population level.BMC Bioinformatics 2010, 11:595. https://doi.org/10.1186/1471-2105-11-595