Eurasian Journal of Biosciences

Determination of genes responsible for some virulence factors of bacteria isolated from contaminated groundwater
  • Article Type: Research Article
  • Eurasian Journal of Biosciences, 2020 - Volume 14 Issue 2, pp. 4207-4215
  • Published Online: 21 Oct 2020
  • Open Access Full Text (PDF)


The current study is conducted to detect bacterial contamination of groundwater in Al-Dour district within Salah al-Din Governorate for a distance extending about 35 km by eight wells with depths ranging between (65-90) meters during the study period extending from spring 2018 to winter 2019. The study includes isolation and identification of the most important species of bacteria contaminating well water and investigating its virulence factors with molecular diagnosis. The Isolates were detected as being able to produce some virulence factors, which included hemolycin, lipase and protease, lecithinase, biofilm, urease, and beta-lactamases, is detected. The results show that Klebsiella pneumoniae produces biofilm, hemolysin, lipase and lecithinase by 100%, while beta-lactamase and both urease and protease enzymes are not produced. As for Enterobacter cloacae, it produced hemolysin, protease and lipase by 100%, while it produced the enzymes beta-lactamase and lecithinase by 50%, and did not form or produce the biofilm and the urease enzyme. Concerning Aeromonas veronii, it produced beta-lactamase, lipase, biofilm, protease and lecithinase by 100% and hemolysin by 50% and did not produce urease enzyme. Enterococcous cecorum produced beta-lactamase, urease, protease and biofilm by 100%, and produced hemolysin and lipase enzymes by 66.7% and lecithinase enzyme by 33.3%, while Enterococcous columbae produced beta- lactamases, urease and biofilm by 100% but unable to produce lipase, lecithinase, hemolysin and protease. Granulicatella elegans were shown to be producing beta-lactamase, lecithinase, biofilm and protease by 100%, but did not produce hemolysin, urease and lipase enzymes. As for Aeromonas hydrophila, they appeared to be producing lipase, urease and hemolysin by 100%, but produced protease, lecithinase and beta-lactamase by 50% but did not produce the biofilm. The results of Polymerase Chain Reaction (PCR) show the use of specialized primers of some genes of virulence factors in the isolated bacteria. The protease gene encoded ProA and beta-lactamases gene encoded MOXM are found in Enterobacter cloacae, the hemolysin gene encoded HemK and the biofilm gene encoded LuxS found in Klebsiella pneumonia, the urease gene encoded UreA and lipase gene encoded lip found in Aeromonas hydrophila. As for A.veronii, it has been detected holding the protease gene encoded ProA and the lipase gene encoded lip the. With respect to the genera Enterococcus sp., Tuf biofilm encoded is found in both Enterococcous columbae and Enterococcous cecorum. In Granulicatella elegans, the presence of the RpoB gene is detected.


  • Abreu R E, Magalhães T C, Souza R C, Oliveira S T, Ibelli A M, Demarqui F N, Gouveia G V (2018). Environmental factors on virulence of Aeromonas hydrophila. Aquaculture international, 26(2), 495-507.
  • Abu‐Elala N, Abdelsalam M, Marouf S, Setta A (2015). Comparative analysis of virulence genes, antibiotic resistance and gyrB‐based phylogeny of motile Aeromonas species isolates from Nile tilapia and domestic fowl. Letters in applied microbiology, 61(5), 429-436.
  • Alexander P (2008). Evaluation of groundwater quality of Mobi town in Admawa State, Nigeria. Africa Journal of Biotechnology. 7(11):1712-1715 p.
  • Al-Kulaby Z D A(2016). Safety of drinking water is essential for disease control in Al-Najaf Al-Ashraf Governorate. Journal of Al-Qadisiya for Engineering Sciences,.(9), No. 2.
  • Al-Rawi A M, and Al-Taey M F(2019). Contamination of domestic well water in Nineveh Governorate with some pathogenic bacteria and detecting its ability to form biofilms. Uloom Al-Rafidain Journal,28(3): 90-104
  • Al-Ukaili R M S Al-Ubaidy I MS and Nasir I H (2017). Isolation and diagnosis of Aeromonas hydrophila from the Tigris River and its ability to produce protease and hemolysin. Journal of Babil university for Pure and Applied Sciences, 1(25):89-96.
  • Bhowmik P, Bag PK, Hajra TK, et al. Pathogenic potential of Aeromonas hydrophila isolated from surface waters in Kolkata, India. Journal of Medical Microbiology. 2009 Dec;58(Pt 12):1549-1558. DOI: 10.1099/jmm.0.014316-0..
  • Christensen G D, Simpson W A, Bisno A L, Beachey E H (1982). Adherence of slime-producing strains of Staphylococcus epidermidis to smooth surfaces. Infection and immunity, 37(1), 318-326.‏
  • Collee J G, Fraser A, Marmion B P, Simmons A (1996). Mackie and Mac Carteny practical medical microbiology.14th ed. Churchill Livingstone Inc. New York.
  • Cotter J J, O’Gara J P, Mack D, Casey E (2009). Oxygen-mediated regulation of biofilm development is controlled by the alternative sigma factor σB in Staphylococcus epidermidis. Applied and environmental microbiology, 75(1), 261-264.
  • Dattelbaum J D, Lockatell C V, Johnson D E, and Mobley H L T(2003). Ure A, the transcriptional activator of the Proteus mirabilis urease gene cluster, is required for urease activity and virulence in expermintal urinary tract infection. Infect. Immune. 71(2): 1026-30.
  • Fady S (2011). Safety of drinking water is essential for disease control. Agricultural Journal, (1), (4), (7). Al-Ahram Institute.
  • Finlay J, Miller L, and Poupard J A (2003). Areview of the antimicrobial activity of clavulanate. J.Antimicrob. Chemother. 52 : 18-23.
  • Freeman D J, Falkiner F R and Keane C T (1989). New method for detecting slime production by coagulase negative staphylococci. J Clin Pathol. 42:872-4.
  • Gupta R, Gupta N, Rathi P, (2004). Bacterial lipases: an overview of production, purification and biochemical properties. Applied microbiology and biotechnology, 64(6), 763-781.‏
  • Hashemi T, Hemat J, (2016). Isolation of Klebsiella Species form Urinary Tract Infections and Study of Frequency of those LuxS Gene. scientific journal of ilam university of medical sciences, 23(6), 20-28.
  • Hayati H R, Hassan M D, Ong B L, Abdelhadi Y M, Hidayahanum H N, Sharifah R M, Alsaid M (2015). Virulence genes detection of Aeromonas hydrophila originated from diseased freshwater fishes. Advances in Environmental Biology, 9(22 S3), 22-27.‏
  • Husu J, Du J T, Shongleu H, and china Y (2003). Extended-epidemical of nosocomial. U.T.I caused by Serratia marcescens. J. Clin. Med., 41(10): 4726-4730.
  • Jung W K, Lim J Y, Kwon N H, Kim J M, Hong S K, Koo H C, Park Y H, (2007). Vancomycin-resistant enterococci from animal sources in Korea. International journal of food microbiology, 113(1), 102-107.
  • Koneman E W, Stephen D A, Dowell V R, William M J, and Herbert M S, (1988). color Atlas and text book of Diagnostic microbiology 3 rd ed., washing ton c. winn, Jn.
  • Leber A L, (2016).Clinical Microbiology Procedures Handbook, 4th ed, vol 2. Washington DC: ASM Press.
  • Lévy P Y, Fenollar F (2012). The role of molecular diagnostics in implant-associated bone and joint infection. Clinical microbiology and infection, 18(12), 1168-1175.
  • Liaw S J, Lai H, Cho S W, Luh K T, and Wang W B, (2003). Role of RsmA in the regulation of swarming motility and virulence factor Expression in proteus mirabilis. J. med. Microbial. 52:19-48.
  • Mansour A, Mahfouz N B, Husien MM, Omer A A, Moustafa E A (2019). Molecular characterisation and pathogenicity evaluation of Aeromonas hydrophila strains isolated from cultured tilapia Oreochromis niloticus in Egypt. Indian Journal Of Fisheries, 66(2), 93-100.‏
  • Nawaz M, Khan S A, Khan A A, Sung K, Tran Q, Kerdahi K, Steele R (2010). Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish. Food microbiology, 27(3), 327-331.
  • Omer S S, and Humadi H G, (2013).Qualitative and quantitative screening of alkaline protease production from some pathogenic bacteria. University of karbala.11..3:1-6.
  • Seo K W, Lee Y J, (2019). Detection of plasmid-mediated quinolone resistance genes in β-lactamase-producing Escherichia coli isolates from layer hens. Poultry science, 98(3), 1480-1487.
  • Sharaf E F, El-Sayed W S, Abosaif R M, (2014).Lecithinase-producing bacteria in commercial and home-made foods: Evaluation of toxic properties and identification of potent producers. Journal of Taibah University for science, 8(3), 207-215.
  • Stehr F, Kretschmar M, Kröger C, Hube B, Schäfer W (2003). Microbial lipases as virulence factors. Journal of Molecular Catalysis B: Enzymatic, 22(5-6), 347-355.‏
  • Sun J, Zhang X, Gao X, Jiang Q, Wen Y, Lin L (2016). Characterization of virulence properties of Aeromonas veronii isolated from diseased Gibel Carp (Carassius gibelio). International journal of molecular sciences, 17(4), 496.‏
  • Torzewska A, Staczek P, and Rozalski A (2003). Crystallization of urine mineral components may depend on chemical nature of Proteus endotoxin polysaccharide. J. Med. Microbiol. 52: 471-477.
  • ‏U.S.EPA.(2015) United State – Environmental Protection Agency Potential Threats to Our Groundwater. Washington, US.A.
  • UNESCO.(2012) United Nations Educational Scientific and Cultural Organization Groundwater Contamination Inventory: A Methodological Guide. United States Environmental Protiction Agency.
  • Vu B, Chen M, Crawford R, Ivanova E (2009). Bacterial extracellular polysaccharides involved in biofilm formation. Molecules, 14(7), 2535-2554.‏
  • Willley J M, Sherwood L, Woolverton C J,(2014). Prescott’s microbiology. McGraw-Hill Education,9th,793.


This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.