Identification of Pseudomonas aeruginosa strains isolated from Dorper sheep milk with subclinical-mastitis infection by uniplex PCR using 16S rRNA, lasI/R, gyrB and ecfX genes


  • Amirah Azemin Universiti Sultan Zainal Abidin
  • Nadiawati Alias Universiti Sultan Zainal Abidin
  • Asmad Kari Universiti Sultan Zainal Abidin



Identification, Pseudomonas aeruginosa, PCR, genes, sheep milk


Pseudomonas aeruginosa is an opportunistic and versatile pathogenic bacterium that can adapt in various environmental condition, which play a role in multiple virulence factor and resistance to antibiotics. Moreover, molecular identification techniques using single target gene is more susceptible to error and false positive. Thus, the detection of this strain with high specificity and sensitivity is crucial in order to control this pathogenic bacterium. The aim of this study was to evaluate six bacteria strains (13-1, 66-1, 00-1, 46-1, 10-R and 67-1) isolated from Dorper sheep milk and two P. aeruginosa ATCC strains (ATCC BAA-2108 and ATCC27853) for prompt identification of the strains based on uniplex polymerase chain reaction which targeting PA-SS, PA-GS, lasI/R, gyrB and ecfX genes. In the present study, the Dorper sheep milk’s samples (n = 32) were collected and tested with California mastitis test (CMT). Out of 32 milk’s samples, six of the samples were detected with strong mastitis, and thus were continued with inoculation process on selective media Pseudomonas Agar P (for pyocyanin) or F (fluorescein) and MacConkey agar for differentiation. After extraction of the bacteria chromosomal DNA, uniplex PCR amplification were carried out by using 16S rRNA (PA-SS and PA-GS) primers and specific P. aeruginosa genes (lasI/R, gyrB and ecfX) primers. The specificity of the primers was also examined by non-Pseudomonas species as a control for data comparison. Sequence analysis has revealed that six of the isolated samples were confirmed as P. aeruginosa strains with the respective genes sequence confirmed by BLAST and Clustal Omega. From this study, lasI/R, gyrB and ecfX were highly reliable primers with the percentage of identification of more than 95.0% as compared to PA-SS and PA-GS which were less than 90.0%. This study concludes that highly specific and sensitive assay has been developed using lasI/R, gyrB and ecfX targeted genes for the detection of P. aeruginosa strains isolated from fresh sheep milk samples.


M. A. Al-kafaween et al., “Effects of Trigona honey on the gene expression profile of Pseudomonas aeruginosa ATCC 10145 and Streptococcus pyogenes ATCC 19615,” Jordan J. Biol. Sci., vol. 13, no. 2, pp. 133–138, 2020.

F. V. Bambeke, J.-M. Pagès, and V. J. Lee, “Inhibitors of bacterial efflux pumps as adjuvants in antibacterial therapy and diagnostic tools for detection of resistance by efflux,” Front. Anti-Infection Drug Discov., vol. 1, pp. 138–175, 2010.

D. Dong et al., “Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Baijing, China,” Front. Microbiol., vol. 6, pp. 1–7, 2015,

H. Aghamollaei, M. M. Moghaddam, H. Kooshki, M. Heiat, R. Mirnejad, and N. S. Barzi, “Detection of Pseudomonas aeruginosa by a triplex polymerase chain reaction assay based on lasI/R and gyrB genes,” J. Infect. Public Health, vol. 8, pp. 314–322, 2015.

Z. J. A. Alshalah, W. A. Al-Daraghi, and A. I. Khaleel, “Rapid detection for lasI and lasR genes Of Pseudomonas aeruginosa at deference Iraqi Hospitals by polymerase chain reaction ( PCR ) technique .,” Int. J. Chem Tech Res., vol. 10, no. 1, pp. 409–414, 2017.

S. Alhogail et al., “Rapid colorimetric detection of Pseudomonas aeruginosa in clinical isolates using a magnetic nanoparticle biosensor,” ACS Omega, vol. 4, pp. 21684–21688, 2019.

Z. Zakaria et al., “Physicochemical composition, microbiological quality and consumers’ acceptability of raw and pasteurized locally produced goat milk,” Malaysian J. Fundam. Appl. Sci., vol. 16, no. 4, pp. 475–482, 2020.

Y. Tang et al., “Detection methods for Pseudomonas aeruginosa : history and future perspective,” R. Soc. Chem. Adv., vol. 7, pp. 51789–51800, 2017.

A. Sharma, V. K. Gupta, and R. Pathania, “Efflux pump inhibitors for bacterial pathogens : From bench to bedside,” Indian J. Med. Res., vol. 149, pp. 129–145, 2019.

K. I. Hassan and S. R. Abdullah, “Detection of Pseudomonas aeruginosa in clinical samples using PCR targeting ETA and gyrB genes,” Baghdad Sci. J., vol. 15, no. 4, pp. 401–405, 2018.

S. N. Anuj et al., “Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reaction assay targeting the ecfX and the gyrB genes,” Diagn. Microbiol. Infect. Dis., vol. 63, pp. 127–131, 2009.

X. Qin, 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 nonfermenting Gram-negative bacilli from patients with cystic fibrosis,” J. Clin. Microbiol., vol. 41, no. 9, pp. 4312–4317, 2003.

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. Mthodsthods, vol. 70, pp. 20–29, 2007.

C. S. Lee, K. Wetzel, T. Buckley, D. Wozniak, and J. Lee, “Rapid and sensitive detection of Pseudomonas aeruginosa in chlorinated water and aerosola targeting gyrB gene using real-time PCR,” J. Appl. Microbiol., vol. 111, no. 4, pp. 893–903, 2011.

M. Tam, T. Thi, D. Wibowo, and B. H. A. Rehm, “Pseudomonas aeruginosa biofilms,” Int. J. Mol. Sci., vol. 21, p. 8671, 2020, doi: doi:10.3390/ijms21228671.

M. E. Hillenbrand, P. P. Thompson, R. M. Q. Shanks, and R. P. Kowalski, “Validation of PCR for the detection of Pseudomonas aeruginosa from corneal samples,” Int. J. Opthalmology, vol. 4, no. 3, pp. 262–268, 2011.

C. Colinon et al., “Detection and enumeration of Pseudomonas aeruginosa in soil and manure assessed by an ecfX qPCR assay,” J. Appl. Microbiol., vol. 114, pp. 1734–1749, 2013.

M. Schuster, D. J. Sexton, S. P. Diggle, and E. P. Greenberg, “Acyl-homoserine lactone quorum sensing : From evolution to application,” Annu. Rev. Microbiol., vol. 67, pp. 43–63, 2013.

G. Girard and G. V. Bloemberg, “Central role of quorum sensing in regulating the production of pathogenicity factors in Pseudomonas aeruginosa,” Futur. Med., vol. 3, no. 1, pp. 97–106, 2008.

S. T. Rutherford and B. L. Bassler, “Bacterial quorum sensing : Its role in virulence and possibilities for its control,” Cold Spring Harb. Perspect. Med., vol. 2, pp. 1–25, 2012.

L. C. M. Antunes, R. B. R. Ferreira, M. M. C. Buckner, and B. B. Finlay, “Quorum sensing in bacterial virulence,” Microbiology, vol. 156, pp. 2271–2282, 2010.

J. L. da C. Lima, L. R. Alves, P. R. L. de A. Jacomé, J. P. B. Neto, M. A. V. Maciel, and M. M. C. de Morais, “Biofilm production by clinical isolates of Pseudomonas aeruginosa and structural changes in LasR protein of isolates non biofilm-producing,” Brazilian J. Infect. Dis., vol. 22, no. 2, pp. 129–136, 2018.

A. Wan-Azemin, A. Kari, and N. Alias, “Assessment of subclinical mastitis effects on live weight, body condition score (BCS) and external udder measurements of dorper sheep,” J. Teknol., vol. 83, no. 2, pp. 135–142, 2021.

A. M. Abdalhamed, G. S. G. Zeedan, and H. A. A. A. Zeina, “Isolation and identification of bacteria causing mastitis in small ruminants and their susceptibility to antibiotics, honey, essential oils, and plant extracts,” Vet. World, vol. 11, no. 3, pp. 355–362, 2018.

M. Rovai et al., “Identifying the major bacteria causing intramammary infections in individual milk samples of sheep and goats using traditional bacteria culturing and real-time polymerase chain reaction,” J. Dairy Sci., vol. 97, pp. 5393–5400, 2014.

M. Geetha, K. M. Palanivel, T. R. Gopalakrishnamurthy, S. Udayavel, and R. Ragavi, “An unsual occurrence of Pseudomonas mastitis in an ewe and its clinical management – a case report,” Int. J. Sci. Environ., vol. 5, no. 6, pp. 4618–4621, 2016.

E. J. Kelly and D. J. Wilson, “Pseudomonas aeruginosa mastitis in two goats associated with an essential oil – based teat dip,” J. Vet. Diagnostic Investig., vol. 3, no. 10, pp. 1–3, 2016.

G. Leitner and O. Krifucks, “Pseudomonas aeruginosa mastitis outbreaks in sheep and goat flocks : Antibody production and vaccination in a mouse model,” Vet. Immunol. Immunopathol., vol. 119, pp. 198–203, 2007.

V. S. Mavrogianni, P. I. Menzies, I. A. Fragkou, and G. C. Fthenakis, “Principles of mastitis treatment in sheep and goats,” Vet. Clin. Food Anim., vol. 27, pp. 115–120, 2011.

J. G. Al-ahmadi and Z. R. Roodsari, “Fast and specific detection of Pseudomonas aeruginosa from other Pseudomonas species by PCR,” Ann. Burns Fire Disasters, vol. 29, no. 4, pp. 264–267, 2016.

L. Scaccabarozzi et al., “Pseudomonas aeruginosa in dairy goats: Genotypic and phenotypic comparison of intramammary and environmental isolates,” PLoS One, vol. 10, no. 11, pp. 1–23, 2015.

E. A. Wright et al., “Divergence of a strain of Pseudomonas aeruginosa during an outbreak of ovine mastitis,” Vet. Microbiol., vol. 175, pp. 105–113, 2015.

V. Cattoir, A. Gilibert, J. -m. Le Glaunec, N. Launay, L. Bait-mérabet, and P. Legrand, “Rapid detection of Pseudomonas aeruginosa from positive blood cultures by quantitative PCR,” Ann. Clin. Microbiol. Antimicrob., vol. 9, no. 21, pp. 1–5, 2010.

A. K. Bej, M. H. Mahbubani, J. L. Dicesare, and R. M. Atlas, “Polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples,” Appl. Environ. Microbiol., vol. 57, no. 12, pp. 3529–3534, 1991.

R. K. Saiki et al., “Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase,” Science (80-. )., vol. 239, pp. 487–491, 1988, doi: DOI: 10.1126/science.239.4839.487 ARTICLE.

T. Spilker, T. Coenye, P. Vandamme, and J. J. Lipuma, “PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients,” J. Clin. Microbiol., vol. 42, no. 5, pp. 2074–2079, 2004.

H. O. M. Al-dahmoshi, N. S. Al-khafaji, A. A. Jeyad, H. K. Shareef, and R. F. Al-jebori, “Molecular detection of some virulence traits among Pseudomonas aeruginosa isolates , Hilla-Iraq,” Biomed. Pharmacol. J., vol. 11, no. 2, pp. 835–842, 2018,

A. E. LaBauve and M. J. Wargo, “Growth and laboratory maintenance of Pseudomonas aeruginosa,” Curr. Protoc. Microbiol., vol. 25, no. 1, pp. 1–11, 2012.

D. M. Whiley, S. B. Lambert, S. Bialasiewicz, and N. Goire, “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.

M. Salman, A. Ali, and A. Haque, “A novel multiplex PCR for detection of Pseudomonas aeruginosa: A major cause of wound infections,” Pakistan J. Med. Sci., vol. 29, no. 4, pp. 957–961, 2013.

M. Motoshima et al., “Rapid and accurate detection of Pseudomonas aeruginosa by real-time polymerase chain reaction with melting curve analysis targeting gyrB gene,” Diagn. Microbiol. Infect. Dis., vol. 58, pp. 53–58, 2007.

D. Subedi, A. K. Vijay, G. S. Kohli, S. A. Rice, and M. Willcox, “Comparative genomics of clinical strains of Pseudomonas aeruginosa strains isolated from different geographic sites,” Sci. Rep., vol. 8, pp. 1–14, 2018.

V. S. Nikbin, M. M. Aslani, Z. Sharafi, M. Hashemipour, F. Shahcheraghi, and G. H. Ebrahimipour, “Molecular identification and detection of virulence genes among Pseudomonas aeruginosa isolated from different infectious origins,” Iran. J. Microbiol., vol. 4, no. 3, pp. 118–123, 2012.

M. A. Khattab, M. S. Nour, and N. M. Elsheshtawy, “Genetic identification of Pseudomonas aeruginosa virulence genes among different isolates,” J. Microb. Biochem. Technol., vol. 7, no. 5, pp. 274–277, 2015.

B. C. Agaras and C. Valverde, “A novel oligonucleotide pair for genotyping members of the Pseudomonas genus by single-round PCR amplification of the gyrB gene,” Methods Protoc., vol. 1, no. 24, pp. 1–13, 2018.

S. G. Mulamattathil, C. Bezuidenhout, M. Mbewe, and C. N. A. Ateba, “Isolation of environmental bacteria from surface and drinking water in Mafikeng , South Africa , and characterization using their antibiotic resistance profiles,” Journals Pathog., vol. 2014, pp. 1–11, 2014.

M. S. E. M. Badawy, O. K. M. Riad, F. A. Taher, and S. A. Zaki, “Chitosan and chitosan-zinc oxide nanocomposite inhibit expression of LasI and RhlI genes and quorum sensing dependent virulence factors of Pseudomonas aeruginosa,” Int. J. Biol. Macromol., vol. 149, pp. 1109–1117, 2020.

P. Kiratisin, K. D. Tucker, and L. Passador, “LasR , a transcriptional activator of Pseudomonas aeruginosa virulence genes, functions as a multimer,” J. Bacterology, vol. 184, no. 17, pp. 4912–4919, 2002.

F. Longo et al., “A new transcriptional repressor of the Pseudomonas aeruginosa quorum sensing receptor gene lasR,” PLoS One, vol. 8, no. 7, pp. 1–9, 2013.

D. Bortolotti et al., “Conjugation of LasR quorum-sensing inhibitors with ciprofloxacin decreases the antibiotic tolerance of P . aeruginosa clinical strains,” J. Chem., vol. 2019, pp. 1–13, 2019.

N. Sabharwal, S. Dhall, S. Chhibber, and K. Harjai, “Molecular detection of virulence genes as markers in Pseudomonas aeruginosa isolated from urinary tract infections,” Int. J. Mol. Epidemiol. Genet., vol. 5, no. 3, pp. 125–134, 2014.

H. M. Aboushleib, H. M. Omar, R. Abozahra, A. Elsheredy, and K. Baraka, “Correlation of quorum sensing and virulence factors in Pseudomonas aeruginosa isolates in Egypt,” J. Infect. Dev. Ctries., vol. 9, no. 10, pp. 1091–1099, 2015.

G. Mangiaterra et al., “Detection of viable but non-culturable Pseudomonas aeruginosa in cystic fibrosis by qPCR : a validation study,” BMC Infect. Dis., vol. 18, no. 701, pp. 1–7, 2018.

P. Deschaght, S. daele Van, F. D. Baets, and M. Vaneechoutte, “PCR and the detection of Pseudomonas aeruginosa in respiratory samples of CF patients. A literature review,” J. Cyst. Fibros., vol. 10, pp. 293–297, 2011.

H. J. Choi et al., “Improved PCR for identification of Pseudomonas aeruginosa,” Appl. Microbiol. Biotechnol., vol. 97, pp. 3643–3651, 2013.