Nội dung

Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 194-199 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 2 (2020) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2020.902.024 PCR Based Detection of Pseudomonas aeruginosa in Mastitic Cow Milk T. Lurthu Reetha*, R. Manickam and B. Puvarajan Department of Veterinary Microbiology, Veterinary College and Research Institute, Orathanadu, Thanjavur, India *Corresponding author ABSTRACT Keywords Cattle, Bovine Mastitis, Pseudomonas aeruginosa, PCR Article Info Accepted: 05 January 2020 Available Online: 10 February 2020 Mastitis is the most economically significant disease of dairy animals. This condition is associated with a significant reduction in milk yield, increased costs of production and deteriorated milk quality. In the present study a total number of 40 bovine mastitis milk samples were collected to isolate and identify the Pseudomonas aeruginosa. All the samples were inoculated into nutrient broth which was further cultured on agar plates. Colonies showing typical Pseudomonas morphological characteristics were processed for biochemical analysis. Based on the morphological, cultural and biochemical characterization indicated that 6 isolates were identified as Pseudomonas aeruginosa. Investigations of the Pseudomonas aeruginosa proteolysis activity is the basal objective of this study. To achieve this objective 16S rRNA primer was used in the amplification of DNA extracted from bacterial isolates. In the present study six bacterial isolates were subjected to molecular identification. The 16SrRNA based PCR results, revealed 956bp amplicon sized product. Introduction animal and less occasionally (Radostits et al., 2007). Bovine mastitis is an inflammatory condition of udder (Bradley, 2002) of cattle and buffaloes which results in altered quality and decreased milk production (Sharma et al., 2012). In severe cases, mastitis may results in partial or complete damage to udder tissues leading to decrease productive lifespan of the Mastitis is caused by many bacteria, which include the coliform group (Specifically Escherichia coli, Enterobacter, Klebsiella species), Streptococci, Staphylococci, Corynebacteria, Pasteurella, Mycoplasma, Leptospira, Yersinia, Mycobacteria, 194 fatalities Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 194-199 Psuedomonas, Serratia and other organisms like fungi, yeasts and virus (Burns et al., 1996). (TVCC) with apparent clinical signs of mastitis and were referred for antimicrobial sensitivity test. All the samples were subjected to routine laboratory procedures for the bacterial growth in nutrient broth medium (Cruickshank et al., 1975). Pseudomonas aeruginosa is a motile gramnegative rod from the family of Pseudomonadaceae. It is a frequently isolated from clinical specimens and identified as an accidental cause of bovine mastitis (Radostits, 2009). Most of the P. aeruginosa strains that have been isolated are strong biofilm producers that can reduce the efficacy of antibiotics and lead to chronic mastitis (Melchior et al., 2006). The pathogen uses a wide range of virulence factors, including proteins capable of inducing toxicity, which effectively damage tissues (Lyczak et al., 2000). Isolation of Pseudomonas aeruginosa Inoculation into broth A total of 40 milk samples were collected from the mastits affected animals. The collected milk samples were brought to laboratory and transferred to the nutrient broth. After inoculation, the test tube was incubated at 37°C for overnight (Cruickshank et al., 1975). Each of the mastitic milk samples were streaked on to nutrient agar, tryptic soy agar (TSA, Himedia - India), violet red bile glucose agar (VRBG, Liofilchem- Italy), MacConkey agar, EMB agar, blood agar and Cetrimide agar. The inoculated plates were incubated for 24-48 hours at 37°C. Suspected colonies were confirmed primarily as Gram-negative rods (Cruickshank et al., 1975). P. aeruginosa infections are generally detected by standard microbiological techniques such as phenotypic and biochemical profiles (Qin et al., 2003), however these commercial tests tend to be lengthy and unreliable (Procop, 2007). Molecular techniques, such as polymerase chain reaction (PCR) are rapid and reliable for the identification of microbial pathogens (Procop, 2007); many PCR based diagnostic methods have been developed for P. aeruginosa (Motoshima et al., 2007). Identification of Pseudomonas aeruginosa The pure colonies subjected for biochemical test using Indole test, Methyl red test Sugar fermentation reaction, Citrate utilization test and Voges-Proskauer test (Kavitha and Devasena, 2013). 16S rRNA gene based identification method was also developed to identify P. aeruginosa (Spilker et al., 2004). The aim of this study was PCR based molecular characterization of Pseudomonas aeruginosa from bovine affected with mastitis. DNA Extraction and PCR Total DNA was extracted from bacterial isolates by using commercially genomic DNA mini kit (Qiagen- Germany) following the mini spin protocol according to the manufacturer’s instructions. To confirm the presence of P. aeruginosa DNA, a standard diagnostic PCR was carried using one 16SrRNA; Primer DG10-03F: 5’ Materials and Methods Samples A total of forty milk samples from the cases of clinical mastitis in cattle were reported to Teaching Veterinary Clinical Complex 195 Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 194-199 GGGGGATCTTCGGACCTCA 3’ and the primer DG10-04R: 5’ TCCTTAGAGTGCCC ACCCG 3’ as a primer sequence (5’-3’). Eosin Methylene Blue agar (EMB) Eosin Methylene Blue (EMB) Agar is used for identification of microorganism. Pseudomonas aeruginosa produced light purple colonies with spreading edges in Eosin Methylene Blue (EMB) Agar. In the present study 6 isolates which produced typical colonies indicating the Pseudomonas aeruginosa. The PCR amplification mixture (20μl) includes (10.0 μl) of master mix which contains bacterially derived Taq DNA polymerase, dNTPs, MgCl2 and reaction buffer at optimal concentration for efficient amplification of DNA templates by PCR (5 μl) of template DNA, (1 μl) of each forward and reverse primers and (3.0 μl) of nuclease free water to complete the amplification mixture to (20 μl). The PCR tubes containing amplification mixture were transferred to preheated thermocycler and started the program as follow; predenaturtion at 95°C for 3 min, then 35 cycles of denaturation at 95°C for 60 sec, annealing at 63°C for 30 Sec, extension at 72°C for 60 sec with one final extension of 10 min at 72°C and a subsequent hold temperature of 4°C using a thermocycler (Techne-UK). MacConkey agar MacConkey agar is used to isolate Pseudomonas aeruginosa. In this media colourless colony with black tinge, typical fruity odour and metallic sheen was identified as Pseudomonas aeruginosa. Nutrient agar The samples were streaked on nutrient agar plates and the plates were incubated at 37 C° for 24 hours as described by (Pawel et al., 2008). Pseudomonas aeruginosa isolated from milk samples were produces green tinged, circular, mucoid smooth colonies with emits sweat grape odour with metallic sheen on nutrient agar. The results of PCR were detected after the amplification process. (8μl) from amplification sample was directly loaded in a 1.2% agarose gel containing (3.0μl/30ml) ethidium bromide and DNA size marker as standard in electrophoresis and run at 50 V. The DNA was observed and photographed by using gel documentation system. Blood agar Blood agar is used to isolate Pseudomonas aeruginosa along with other media. In this media pseudomonas species produced gray coloured colony, β haemolytic with metallic sheen was identified as typical Pseudomonas species. Results and Discussion Identification of Pseudomonas aeruginosa Forty milk samples were collected from mastitis affected cattle. The samples were inoculated in nutrient broth. After the overnight period of overnight incubation the samples were streaked on to different media namely MacConkey agar, Eosine Methylene blue agar, Cetrimide agar, Nutrient agar and Blood agar. Cetrimide agar Cetrimide agar is used as a selective media to isolate Pseudomonas aeruginosa. In this media pseudomonas species produced green tinged colony with metallic sheen was identified Pseudomonas aeruginosa. 196 Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 194-199 aeruginosa on the basis of cultural and morphological properties were subjected to biochemical tests. All the 6 isolates were confirmed to be Pseudomonas aeruginosa after the biochemical results. Gram staining Gram staining is used for identification of Gram positive and Gram negative organism. Pseudomonas is a gram negative rod Shaped bacterium. In the present study all the 6 isolates were subjected to gram staining and all the isolates were found to be gram negative typically thin rods. The isolates were sub-cultured on the same medium until pure cultures were achieved. The isolated bacteria were grown in nutrient broth (Oxoid - UK) and were then stored in 15% (v/v) glycerol at−20 °C and used as stock cultures in subsequent analysis (Dikbas, 2010). Biochemical characterization The phenotypic characteristics of P. aeruginosa were determined using tests including Simmon's citrate utilization (Oxoid - UK), oxidase (1.0% tetramethyl), Urease test ( Himedia - India) Gelatine liquefaction test, Oxidase test, Catalase test, Indole test, Methyl red test, Voges-Proskauer test, Sugar fermentation reaction, Phenyl alanine deaminase test, TSI and Carbohydrate fermentation tests (Smibert and Krieg, 1984; MacFadden, 2000). PCR Analysis To determine the presence of proteolytic P. aeruginosa in mastitis affected milk, 16S rRNA primer was used. Genomic DNA from a Pseudomonas positive isolate was used as the source of template DNA. The positive results for the prescence of 16S rRNA (956 bp) gene amplified products were displayed (Fig. 1). All suspected colonies of Pseudomonas Fig.1 PCR for Pseudomonas aeruginosa M: 1500bp DNA marker; Lane 1-6: Positive sample (956bp) 197 Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 194-199 Pseudomonas aeruginosa was isolated by using nutrient agar which was promoted primarily based on characteristics colony morphology in nutrient agar, blood agar and MacConkey agar media and Gram’s staining technique, Many strains of P. aeruginosa produce various species of pyocins and this pyocin producing strain of P. aeruginosa afford pigment on agar media (Cheesbrough, 1985). Pseudomonas aeruginosa produces circular mucoid smooth colonies with emits sweat grape odour on nutrient agar, these characteristics colonies were similar with finding of Haleem et al., 2011. References Bradley AJ. 2002. Bovine mastitis: an evolving disease. Vet. J. 164:116– 128.doi:10.1053/tvjl.2002.0724. Burns, A.L. Ball, H.J. and Finlay, D.A, 1996. CNF producing Escherichia coli isolated from cattle in Northern Ireland. Vet Microbiol. 49: 235 – 241 Cheesbrough M, 1985. Medical Laboratory manual for tropical countries. Vol. II. Microbiol., P. 248-264. Cruickshank R., Duguid J.P., Marmion B.P. and Swain R.H.A, 1975. Medical Microbiology Vol. II, 12th edn, Crurchill Living stone, New York, Pp. 31-57 and 96-218. Dikbas, N. 2010. Determination of antibiotic susceptibility and fatty acid methyl ester profiles of Bacillus cereus strains isolated from different food sources in Turkey. African Journal of Microbiology Research., 9(11): 16411647. Haleem H, Kadhim J, Ilham T, Banyan A, 2011. Isolation of Pseudomonas aeruginosa from Clinical Cases and Environmental Samples, and Analysis of its Antibiotic Resistant Spectrum at Hilla Teaching Hospital. Med. J. Babylon. 8: 618-624. Kavitha J R and Devasena T, 2013. Molecular and Bacteriological examination of cow milk in coliform mastitis. J. Pharmay and Biological Science., 6(2): 23197676. Lyczak, J. B., Cannon, C. L. and Pier, G.B, 2000: Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect. 2, 1051–1060. MacFadden JF, 2000. Biochemical tests for Identification of Medical Bacteria 3rd Ed. The Williams & Wilkins Co., USA: 689-691. Melchior, M., Vaarkamp, H. and Fink- In cultural isolation, colonies are selected based on phenotypic characteristics like size, colour, shape etc. Similarly, possible reasons for no growth in milk samples can include low concentration of bacteria in the milk samples, pathogens not growing in standard culture media, or presence of substances in the milk decreasing the viability of bacteria in culture (Taponen, 2009). All 6 isolates which had been biochemically identified as P. aeruginosa were subjected to genotyping using species specific primers reported earlier (Spilkeret al., 2004). In conclusion, PCR assay can be used for rapid, sensitive, specific and reliable identification of the major mastitis pathogens from milk. As several bacterial species are involved in mastitis infection, inadequate pathogen detection or identification techniques often delay timely interventions in disease treatment and control. Acknowledgement We are thankful to all the Staff, Department of Veterinary Clinical Complex, Veterinary College and Research Institute, Orathanadu, who alerted us of and assisted us in the collection of milk samples. 198 Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 194-199 Gremmels, J, 2006. Biofilms: A role in recurrent mastitis infections? Vet. J. 171, 398–407. Motoshima M, Yanagihara K, Fukushima K, Matsuda J, Sugahara K, Hirakata Y, et al., 2007. Rapid and accurate detection of Pseudomonas aeruginosa by realtime polymerase chain reaction with melting curve analysis targeting gyrB gene. Diagn Microbiol Infect Dis. 58:53-58. Pawel S, Piotr W, Tomasz H, Marcin Z, Dorota O, Elzbieta T, 2008. Metallo –βlactamases of Psudomonas aeruginosaa noval mechanism risistance to βlactam antibiotics. Folia histochemica and Cytobiologica vol.46, No..2, PP.137-142. Procop GW, 2007. Molecular diagnostics for the detection and characterization of microbial pathogens. Clin Infect Dis.1:99-111. Qin X, Emerson J, Stapp J, Stapp L, Abe P, Burns JL, 2003. 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. 41:4312-4317. Radostits OM, Gay CC, Hinchcliff KW, Constable PD, 2009. Bovine Leukemia. In: Veterinary Medicine. 10th ed. pp: 1210-1221 Radostits, OM, Blood DC, Gay CC, et al., 2007. Veterinary Medicine: A textbook of the diseases of cattle, horses, sheep, pigs and goats, 10th ed. Saunders Elsevier Limited, Philadelphia, USA. Sharma N, Rho GJ, Hong YH, et al., 2012. Bovine mastitis: an Asian perspective. Asian Journal of Animal and Veterinary Advances 7(6): 454–476. doi:10.3923/ajava.2012.454.476. Smibert, R.M., Krieg, N.R., 1984. General characterization. In: Manual of Methods for General Bacteriology, (Gerhardt, P., Murray, R.G.E., Costilow, R.N., Nester, E.W., Wood, W.A., Krieg, N.R., Phillips, G.B.(Eds.), American Society for Microbiology, Washington, DC, pp. 411–420. Spilker T, Coenye T, Vandamme P, LiPuma JJ, 2004. PCR-Based Assay for Differentiation of P. aeruginosa from other Pseudomonas Species Recovered from Cystic Fibrosis Patients. Journal of Clinical Microbiology 42(5): 20742079. Taponen S, Salmikivi L, Simojoki H, Koskinen MT, Pyorala S, 2009. Real Time Polymerase chain reaction based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing. Journal of Dairy Science 92(6): 2610-2617. How to cite this article: Lurthu Reetha, T., R. Manickam and Puvarajan, B. 2020. PCR Based Detection of Pseudomonas aeruginosa in Mastitic Cow Milk. Int.J.Curr.Microbiol.App.Sci. 9(02): 194-199. doi: https://doi.org/10.20546/ijcmas.2020.902.024 199