Skip to main content
SpringerLink
Log in

Development of a real-time TaqMan assay to detect mendocina sublineage Pseudomonas species in contaminated metalworking fluids

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 101 colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 × 103 and 3.9 × 106 CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 101 to 1.4 × 105 CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Bakken LR, Olsen RA (1987) The relationship between cell size and viability of soil bacteria. Microbial Ecol 13:103–114

    Article  Google Scholar 

  2. Gudnason H, Dufva M, Bang DD, Wolff A (2007) Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res 35:e127

    Article  PubMed  Google Scholar 

  3. Guilbaud M, Coppet P, Bourion F, Rachman C, Prevost H, Dousset X (2005) Quantitative detection of Listeria monocytogenes in biofilms by Real-Time PCR. Appl Environ Microbiol 71:2190–2194. doi:10.1128/AEM.71.4.2190-2194.2005

    Article  CAS  PubMed  Google Scholar 

  4. John DL, Phillips ML (2002) UV disinfection of soluble oil metalworking fluids. AIHA J 63:178–183

    Article  Google Scholar 

  5. Khan UHI, Yadav JS (2004) Real-time PCR assays for the genus-specific detection and quantification of culturable and non-culturable mycobacteria and pseudomonads in metalworking fluids. Mol Cell Prob 18:67–73. doi:10.1016/j.mcp.2003.09.004

    Article  CAS  Google Scholar 

  6. Lee M, Chandler AC (1941) A study of the nature, growth and control of bacteria in cutting compounds. J Bacteriol 41:373–386

    CAS  PubMed  Google Scholar 

  7. Lin W, Reed BE, Rinkus KM, Jha A (1999) Investigation into the nature and extent of microbial contamination present in a commercial metalworking fluid. Res Env Biotech 2:185–196

    CAS  Google Scholar 

  8. Moore ERB, Mau M, Arnscheidt A, Bottger EC, Huston RA, Collins MD, de Peer V, de Wachter R, Timmins KN (1996) The determination and comparison of the 16s rRNA gene sequences of species of the genus Pseudomonas (sensu stricto) and estimation of the natural intrageneric relationships. Syst Appl Microbiol 19:478–492

    CAS  Google Scholar 

  9. Moore SJ, Christensen M, Wilson RW, Wallace RJ Jr, Zhang Y, Nash DR, Shelton B (2000) Mycobacterial contamination of metalworking fluids: involvement of a possible new taxon of rapidly growing mycobacteria. AIHA J 61:205–213

    Article  CAS  Google Scholar 

  10. Mumy LK, Findlay RH (2004) Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR. J Microbiol Methods 57:259–268. doi:10.1016/j.mimet.2004.01.013

    Article  CAS  PubMed  Google Scholar 

  11. Nadkarni MA, Elizabeth F, Jacques NA, Hunter N (2002) Determination of bacterial load by real-time PCR using a broad range (universal) probe and primer set. Microbiology 148:257–266

    CAS  PubMed  Google Scholar 

  12. Rhodes G, Fluri A, Gerber M, Henderson A, Pickup RW (2008) Detection of Mycobacterium immunogenum by realtime quantitative Taqman PCR. J Microbiol Meth 73:266–268. doi:10.1016/j.mimet.2008.03.006

    Article  CAS  Google Scholar 

  13. Rossmoore HW (1995) Microbiology of metalworking fluids: deterioration, disease and disposal. J Soc Tribiol Lub Eng 51:112–118

    Google Scholar 

  14. Saha R, Sproer C, Beck B, Bagley S (2009) Pseudomonas oleovorans subsp. lubricantis subsp. nov., reclassification of Pseudomonas pseudoalcaligenes ATCC17440T as later synonym of Pseudomonas oleovorans ATCC 8062T. Curr Microbiol 60:294–300. doi:10.1007/s00284-009-9540-6

    Article  PubMed  Google Scholar 

  15. Suzuki N, Nakano Y, Yoshida A, Yamashita Y, Kiyoura Y (2004) Real-time TaqMan PCR for quantifying oral bacteria during biofilm formation. J Clin Microbiol 42:3827–3830. doi:10.1128/JCM.42.8.3827-3830.2004

    Article  CAS  PubMed  Google Scholar 

  16. Thompson JD, Gibson TJ, Plewniak F et al (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882

    Article  Google Scholar 

  17. Tiedje JM, Stein JL (1999) Microbial biodiversity: strategies for its recovery. In: Demain AL, Davies JE (eds) Manual of industrial microbiology and biotechnology. American Society for Microbiology Press, Washington, DC, pp 682–692

    Google Scholar 

  18. van der Gast C, Knowles CJ, Wright MA, Thompson IP (2001) Identification and characterization of bacterial populations of an in-use metalworking fluid by phenotypic and genotypic methodology. Intl Biodet Biodeg 47:113–123

    Article  Google Scholar 

  19. van der Gast C, Whiteley AS, Lilley AK, Knowles CJ, Thompson IP (2003) Bacterial community structure and function in a metalworking fluid. Environ Microbiol 5:453–461

    Article  PubMed  Google Scholar 

  20. Veillette M, Pagé G, Thorne PS, Duchaine C (2005) Recovery and quantification of DNA from metalworking fluids using dual-labeled probes. J ASTM Int 2:1–9. doi:10.1520/JAI12840

    Article  Google Scholar 

  21. Virji MA, Woskie SR, Sama SR, Kriebel D, Eberiel D (2000) Identifying the determinants of viable microorganisms in the air and bulk metalworking fluids. AIHA J 61:788–797

    Article  CAS  Google Scholar 

  22. Wallace JR, Zhang Y, Wilson RW, Mann L, Rossmoore H (2002) Presence of a single genotype of the newly described species Mycobacterium immunogenum in industrial metalworking fluids associated with hypersensitivity pseumonitis. Appl Environ Microbiol 68:5580–5584

    Article  CAS  Google Scholar 

  23. Ward N, Rainey FA, Goebel B, Stackenbrandt E (1995) Identification and culturing the ‘unculturables’: a challenge for microbiologists. In: Allsopp D, Colwell RR, Hawksworth DL (eds) Microbial diversity an ecosystem function. CAB, Wallingford, pp 89–110

    Google Scholar 

  24. Yadav JS, Selvaraju SB, Khan IU (2006) Enhanced recovery and real-time PCR based quantification of mycobacteria from metalworking fluids. J ASTM Int 3:1–18. doi:10.1520/JAI12839

    Article  Google Scholar 

  25. Yamamoto S, Kasai H, Arnold DL, Jackson RW, Vivian A, Harayama S (2000) Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes. Microbiology 146:2385–2394

    CAS  PubMed  Google Scholar 

  26. Yoshida A, Suzuki N, Nakano Y, Kawada M, Oho T, Koga T (2003) Development of a 5′ nuclease-based real-time PCR assay for quantitative detection of carcinogenic dental pathogens Streptococcus mutans and Streptococcus sobrinus. J Clin Microbiol 41:4438–4441. doi:10.1128/JCM.41.2.863-866.2003

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors acknowledge the support of the Biological Sciences Department, Michigan Technological University, MI, USA, and NSF International, MI, USA, for facilities and funding.

Author information

Authors and Affiliations

  1. Biological Sciences Division, Michigan Technological University, Dow 740, 1400 Townsend Drive, Houghton, MI, 49931, USA

    Ratul Saha & Susan T. Bagley

  2. Microbiology Department, NSF International, 789 N Dixboro Road, Ann Arbor, MI, 48105, USA

    Ratul Saha & Robert S. Donofrio

Authors
  1. Ratul Saha
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert S. Donofrio
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Susan T. Bagley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ratul Saha.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Saha, R., Donofrio, R.S. & Bagley, S.T. Development of a real-time TaqMan assay to detect mendocina sublineage Pseudomonas species in contaminated metalworking fluids. J Ind Microbiol Biotechnol 37, 843–848 (2010). https://doi.org/10.1007/s10295-010-0731-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-010-0731-8

Keywords

Search

Navigation