Open Access
Pseudomonas aeruginosa detection methods from fish samples
Belgin Sırıken1, Veli Öz2, Ceren Başkan3*
1Ondokuz Mayıs University, Samsun, Turkey
2Ondokuz Mayıs University, Samsun, Turkey
3Amasya University, Amasya, Turkey
* Corresponding author:

Presented at the 4th International Symposium on Innovative Approaches in Health and Sports Sciences (ISAS WINTER-2019 (HSS)), Samsun, Turkey, Nov 22, 2019

SETSCI Conference Proceedings, 2019, 12, Page (s): 141-145 ,

Published Date: 23 December 2019    | 767     19


A total of 70 fish samples were randomly purchased from different butchers and supermarkets brands in Samsun Province, Turkey in 2018, and analysed for present of Pseudomonas (P.) aeruginosa. For that purpose, P.  aeruginosa isolation was carried out in conventional culture technique; briefly, under aseptic condition 10 g fish samples were transferred into a sterile polyethylene bag and 90 ml of peptone water (PW-Oxoid CM 00099) broth was added. The mixture was homogenized and prepared decimal dilution up to 10-6. Following that, the broth was plated onto Pseudomonas CN Selective Agar [Oxoid SR 102E, suppl. Pseudomonas Agar base-(Oxoid CM 0559)] (EN ISO 13720) using spread plate technique and the plates was incubated aerobically for 24-48 h at 37 °C. After the incubation, up to five susceptible colonies grown on the Pseudomonas CN Selective Agar were subcultured onto Tryptone Soya Agar plates (TSA-Oxoid-CM0131-L21). The presumptive P. aeruginosa colonies were tested with the Gram staining, oxidase (Oxoid BR 64) and catalase test. In addition, the colonies were streaked onto Endo Agar Base (Oxoid, CM0479, suppl. BR0050), for confirmation of the isolates for being P. aeruginosa at molecular levels, two types of genes were detected in the isolates- oprL and PA-SS (16 S rDNA) genes using PCR assay.  In conclusion, we obtained 100 Pseudomonas spp. isolates. Of those, 65 isolates were identified as a P. aeruginosa using classing culture technique. However, 30 out of 65 isolates were confirmed at molecular levels. Between two genes regions, although only oprL gene detected in the 30 isolates, PA-SS (16 S rDNA) gene was not detected in any of the isolates. 

Keywords - Pseudomonas aeruginosa, fish, oprL gene, PA-SS (16 S rDNA) gene


[1] LPSN (List of procaryotik names with standing nomenclature. Bergey’s Manual of Systematcs of Archae and Bacteria. 2017. http://www. bacterio. net/-allnamesmr. Html (Erişim tarhi 9.06. 2017).
[2] K. Edit, S. Sándor, D. Gyula, R. Júlia, R. Balázs, and K. Balázs, Pathogenic and phylogenetic features of 2 multi-resistant Paeruginosa strains originated from remediated sites. International Journal of Occupational Medicine and Environmental Health, vol. 29, pp. 503-516, 2016.
[3] M. Mulet, A. Bennasara, J. Lalucata and B.E. Garcı´a-Valde. An rpoD-based PCR procedure for the identification of Pseudomonas species and for their detection in environ samples. Mol Cell Prob 2009;23:140–147.
[4] R. Abdullahi, S. Lihan, B. S. Carlos, M. L. Bilung, M. K. Mikal, and F. Collick, Detection of oprL gene and antibiotic resistance of Pseudomonas aeruginosa from aquaculture environment. European J Experimen Biol, vol. 3(6), pp. 148- 152, 2013.
[5] C. Paniagua, R. Octavio, A. Juan, and N. German, J. Clin. Microbiol, vol. 28 (2), pp.350–355, 1990.
[6] A. Domenech, J.F. Fernandez-Garayzabal, J.A. Garcia, M.T. Cutuli, M. Blanco,A. Gibello, M.A Moreno, L. Dominguez. Association of Pseudomonas anguilliseptica infection with 'winter disease' in sea bream, Sparus aurata L. J Fish Dis 1999;22(1):69–71.
[7] B. Austin, D. A. Austin, Bacterial Fish Pathogens. Diseases of Farmed and Wild Fish, Springer-Praxis Publishing Ltd., United Kingdom, 2007.
[8] S. A. Mastan, Pseudomonas septicaemia in Labeo rohita (Ham) and Cyprinus car-pio(Linn) in Andhra Pradesh—natural occurrence and artificial challenge, Int.J. Pharm. Pharm. Sci. vol. 5, pp. 564–568, 2013.
[9] J. Thomas, S.Thanigaivel, S.Vijayakumar, K.AcharyaK, D. Shinge, T.S. Jeba Seelan, A. Mukherjee, N. Chandrasekaran. Pathogenecity of Pseudomonas aeruginosa in Oreochromis mossambicus and treatment using lime oil nanoemulsion. Col Surf B: Bioint 2014;116:372-377.
[10] M. Rüger, M. Ackermann, U. Reichl, ¨ BMC Microbiol, vol. 14, pp. 1–15, 2014.
[11] Joyanes, P., M. del Carmen Conejo, L. Martinez-Martinez, and E. J. Perea. 2001. Evaluation of the VITEK 2 system for the identification and susceptibility testing of three species of nonfermenting gram-negative rods frequently isolated from clinical samples. J. Clin. Microbiol. 39:3247–3253.
[12] L. Saiman, J. L. Burns, D. Larone, Y. Chen, E. Garber and S. Whittier, J. Clin. Microbiol, vol. 41, pp. 492–494, 2003.
[13] M. D. Quesada, M. Gim´enez, S. Molinos, G. Fern´andez, M.D. S´anchez, R. Rivelo, A. Ram´ırez, G. Banqu´e and V. Ausina. Clin. Microbiol. Infect, vol. 16, pp.137–140, 2010.
[14] N. M. Kingsford and H. W. Raadsma, Vet. Microbiol, vol. 47, pp. 61–70, 1995.
[15] A. Hummel and G. Unger, Zentralbl. Hyg. Umweltmed, vol. 201, pp. 349–355, 1998.
[16] X. Quin, J. Emerson, J. Stapp, L. Stapp, P. Abe, and J. L. Burns, Use of real-time PCR with multiple targets to identify Pseudomonas aeruginosa and other non fermenting gram-negative bacilli from patients with cystic fibrosis. J Clin Microbiol, vol. 41, pp. 4312–4317, 2003.
[17] D. De Vos, A. Lim, J. P. Pirnay, M. Struelens, C. Vandenvelde, L. Duinslaeger, A. Vanderkelen, and P. Cornelis. Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL. J Clin Microbiol, vol. 35, pp. 1295–1299, 1997.
[18] S. N. Anuj, D. M. Whiley, T. J. Kidd, S. C. Bell, C. E.
Wainwright, M. D. Nissen and T. P. Sloots, Diagn. Microbiol.
Infect. Dis, vol. 63, pp. 127–131, 2009.
[19] P. Deschaght P., S. V. Daele, F. D. Baets and M. Vaneechoutte, J. Cystic Fibrosis, 2011, 10, 293–297.
[20] M.M. Moghaddam, S. Khodi, and A. Mirhosseini, Quorum Sensing in Bacteria and a Glance on Pseudomonas aeruginosa. Clin Microbiol, vol. 3, pp. 156. 2014.
[21] S. Boutina, M. Weitnauera, S. Hassela, S. Y. Graeberb, M. Stahlb, A.S. Dittrichc, M.A. Mallb, and A.H. Dalpkea, Onetime quantitative PCR detection of Pseudomonas aeruginosa to discriminate intermittent from chronic infection in cysticfibrosis. J Cyst Fibros, vol. 17, pp. 348-355, 2018.

[22] T. Spilker, T. Coenye, P.Vandamme, J.J. LiPuma. PCR-Based Assay for Differentiation of Pseudomonas aeruginosa from Other Pseudomonas Species Recovered from Cystic Fibrosis Patients. J Clinical Microbiol, 42(5):2074–2079, 2004.
[23] A. Deep, U. Chaudhary, and V. Gupta, Quorum sensing and bacterial pathogenicity: from molecules to disease. J Lab Physicians, vol. 3(1), pp. 4–11, 2011.
[24] Rodriguez-Herva JJ, Ramos-Gonzalez MI ve Ramos JL. The Pseudomonas putida peptidoglycan-associated outer membrane lipoprotein is involved in the maintenance of the integrity of the cell envelope. J Bacteriol, 178:1699–1706, 1996.
[25] D. Van der Waaij, J. Antimicrob. Chemother, vol. 10, pp. 263–270, 1982.
[26] Y. Tang, Z. Ali, J. Zou, G. Jin, J. Zhu, J. Yanga, and J. Dai. Detection methods for Pseudomonas aeruginosa: history and future perspective. RSC Adv, vol. 7, pp. 51789, 2017.
[27] R. Lavenir, D. Jocktane, F. Laurent, S. Nazaret, and B. Cournoyer, Improved reliability of Pseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene target. J Microbiol Methods, vol. 70, pp. 20–29, 2007.
[28] C. K. D. Benie, G. G. Nathalie, D. Adjéhi, A. Solange, K. Fernique, K. Desire, B. Bourahima, D.K., Marcellin, and D. Mireille, Prevalence and Antibiotic Resistance of Pseudomonas aeruginosa Isolated from Bovine Meat, Fresh Fish and Smoked Fish. Arc Clin Microbiol, vol. 8(3), pp. 40, 2017.
[29] D. M. Whiley, S.B. Lambert, S. Bialasiewicz, N. Goire, M. D. Nissen, and T. P. Sloots, False-negative results in nucleic acid amplification tests-do we need to routinely use two genetic targets in all assays to overcome problems caused by sequence variation. Crit Rev Microbiol, vol. 34, pp. 71–76, 2008.
[30] I. Mehri, Y. Turki, I. Daly, A.B. Rjab, A. Hassen, G. Maher, Molecular identification and assessment of genetic diversity of fluorescent Pseudomonads based on different polymerase chain reaction (PCR) methods. Afr J Microbiol Res, vol. 7, pp. 2103-2113. 2013.

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