Molecular Identification and Genotyping of Pseudomonas aeruginosa Isolates Using Double-Locus Sequence Typing (DLST) Analysis

Main Article Content

Hemin Esmael Othman


Pseudomonas aeruginosa, antibiotic sensitivity, PCR, DLST, Phylogenetic analysis.


     The opportunistic pathogen Pseudomonas aeruginosa is responsible for many life-threatening nosocomial and environmental acquired infections. In this study, the antibiotic susceptibility, molecular identification and genotyping of P. aeruginosa were performed. A total of 100 isolates of P. aeruginosa out of 523 specimens (19.12%) from clinical and environmental sources were analyzed. The results of antibiotic sensitivity profile grouped these isolates into non-multidrug resistant (non-MDR), multidrug resistant (MDR), and extensively drug resistant (XDR) by the ratios of 23.7%, 40.5% and 35.6%, respectively. The double-locus sequence typing (DLST) scheme was employed for genotyping a collection of 36 isolates of P. aeruginosa recovered from different clinical and environmental sources. Isolates were successfully typed into 19 different DLST genotypes with high discriminatory power (0.9206). In addition, three new alleles were recognized for the locus ms172 namely; ms172-128, ms172-129 and ms172-130. Thus, three novel DLST genotypes of this pathogen have been identified, which were previously not reported, with the combinations of DLST128-60, DLST129-79 and DLST130-17, respectively. All new genotypes, which were exclusively belonged to the clinical sources, were exhibited XDR pattern. The phylogenetic analysis differentiated these genotypes into seven different genetic clusters supported by strong bootstrap values. However, there were indications of distinct evolutionary origins for some of the un-clustered genotypes (5/8). The DLST type 32-39 was the predominant cluster in this region with a majority of XDR pattern. Hereby, it can be concluded that DLST was capable of discriminating the phenotypically and genetically related isolates of P. aeruginosa and offered a reliable phylogenetic analysis.

Abstract 188 | PDF Downloads 193


1. AbdulQader N K, Raoof W M, and Al-Mussawi A A. (2015). BiofimForming-Pseudomonas aeruginosa isolated from burn patients in Basra city, south of Iraq. International Journal of Current Research; 7(10):21750-21753.
2. Al-Dahhan H A A. (2015). Molecular Characterization of P. aeruginosa isolated from Patients with URTI. JCBPS; Section B; 5 (No. 3): 2736-2747.
3. Al-Zaidi J R. (2016). Antibiotic susceptibility patterns of Pseudomonas aeruginosa isolated from clinical and hospital environmental samples in Nasiriyah, Iraq. Afr J Microbiol Res., 10(23):844-849. doi: 10.5897/AJMR2016.8042.
4. Basset P and Blanc D S. (2014). Fast and simple epidemiological typing of Pseudomonas aeruginosa using the double-locus sequence typing (DLST) method. Eur J Clin Microbiol Infect Dis, 33:927–932. doi: 10.1007/s10096-013-2028-0.
5. Burstein D, Satanower S, Simovitch M, et al., (2015). Novel type III effectors in Pseudomonas aeruginosa. mBio., 6(2):e00161-15. doi:10.1128/mBio.00161-15.
6. Chen Y, Frazzitta A E, Litvintseva A P, Fang C, Mitchell T G, Springer D J, Ding Y, Yuan G and Perfect J R. (2015). Next generation multilocus sequence typing (NGMLST) and the analytical software program MLSTEZ enable efficient, cost-effective, high-throughput, multilocus sequencing typing. Fungal Genetics and Biology, 75:64-71. doi: 10.1016/j.fgb.2015.01.005.
7. Cholley P, Stojanov M, Hocquet D, et al., (2015). Comparison of double-locus sequence typing (DLST) and multilocus sequence typing (MLST) for the investigation of Pseudomonas aeruginosa populations. Diagnostic Microbiology and Infectious Disease 82, 274–277.
8. Clinical and Laboratory Standards Institute (CLSI). (2014). Performance standards for antimicrobial susceptibility testing; twenty-fourth informational supplement. CLSI document M100-S24. Wayne, PA. 34(1).
9. Firouzi-Dalvand L, Pooladi M, Nowroozi J, et al., (2016). Presence of exoU and exoS genes in Pseudomonas aeruginosa isolated from urinary tract infections. Infect Epidemiol Med., 2(2):8-11. doi: 10.7508/iem.2016.02.003.
10. Gba K M K, Guessennd N K, Makaya N P N D, et al., (2018). Detection of Metallo-beta-lactamase Producing Pseudomonas aeruginosa in an Abidjan Hospital, Côte d’Ivoire. JAMB, 8(1): 1-8. doi: 10.9734/JAMB/2018/38792.
11. Hare N J, Solis N, Harmer C, et al., (2012). Proteomic profiling of Pseudomonas aeruginosa AES-1R, PAO1 and PA14 reveals potential virulence determinants associated with a transmissible cystic fibrosis-associated strain. BMC Microbiology, 12:16.
12. Hassan K I, Rafik S A and Mussum K. (2012). Molecular identification of Pseudomonas aeruginosa isolated from Hospitals in Kurdistan region. J Adv Med Res., 2(3): 90-98.
13. Janam R, Gulati A K and Nath G. (2011). Antibiogram and genotyping of pseudomonas aeruginosa isolated from human, animal, plant, water and soil sources in north India. Southeast Asian J Trop Med Public Health, Vol. 42 No. 6, p.1477-1488.
14. Kearse M, Moir R, Wilson A, et al., (2012). Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics ; 28(12): 1647-1649.
15. Khan N H, Ahsan M, Yoshizawa S, et al., (2008). Multilocus sequence typing and phylogenetic analyses of Pseudomonas aeruginosa isolated from the ocean. Appl Environ Microbiol., 74:6194-6205. doi:10.1128/AEM.02322-07.
16. Khosravi A D, Hoveizavi H, Mohammadian A, et al., (2016). Genotyping of multidrug-resistant strains of Pseudomonas aeruginosa isolated from burn and wound infections by ERIC-PCR. Acta Cirúrgica Brasileira, Vol. 31 (3).p.206-2011. doi:
17. Li W, Raoult D and Fournier P E. (2009). Bacterial strain typing in the genomic era. FEMS Microbiol Rev., 33(5):892-916. doi: 10.1111/j.1574-6976.2009.00182.x.
18. Maatallah M, Cheriaa J, Backhrouf A, et al., (2011). Population structure of Pseudomonas aeruginosa from five mediterranean countries: Evidence for frequent recombination and epidemic occurrence of CC235. PLoS ONE, 6(10): e25617. doi:10.1371/journal.pone.0025617.
19. Magiorakos A P, Srinivasan A, Carey R B, et al., (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect., 18(3):268-81. doi: 10.1111/j.1469-0691.2011.03570.x.
20. Miranda C C, de Filippis I, Pinto L H, et al., (2015). Genotypic characteristics of multidrug-resistant Pseudomonas aeruginosa from hospital wastewater treatment plant in Rio de Janeiro, Brazil. J Appl Microbiol., 118(6):1276-86. doi: 10.1111/jam.12792.
21. Pappa O, Beloukas A, Vantarakis A, et al., (2017). Molecular Characterization and Phylogenetic Analysis of Pseudomonas aeruginosa Isolates Recovered from Greek Aquatic Habitats Implementing the Double-Locus Sequence Typing Scheme. Microb Ecol, 74:78–88. doi: 10.1007/s00248-016-0920-8.
22. Park J-W, Shin I-S, Ha U-H, et al., (2015). Pathophysiological changes induced by Pseudomonas aeruginosa infection are involved in MMP-12 and MMP-13 upregulation in human carcinoma epithelial cells and a pneumonia mouse model. Infect Immun, 83:4791–4799. doi:10.1128/IAI.00619-15.
23. Spilker T, Coenye T, Vandamme P and LiPuma J J. (2004). PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. J Clin Microbiol., 42: 2074-2079. doi: 10.1128/JCM.42.5.2074-2079.2004.
24. Stover C K, Pham X Q, Erwin A L, Mizoguchi S D, et al., (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959-964.
25. Tang Y W and Stratton C W. (2013). Advanced techniques in diagnostic microbiology, Second Edition. Springer Science, Springer New York Heidelberg Dordrecht London. doi: 10.1007/978-1-4614-3970-7.
26. Valot B, Guyeux C, Rolland J Y, et al., (2015). What it takes to be a Pseudomonas aeruginosa? The core genome of the opportunistic pathogen updated. PLoS ONE, 10(5): e0126468. doi:10.1371/journal.pone.0126468.