Liquid culture medium for the rapid cultivation of Helicobacter pylori from biopsy specimens

  • N. Sainsus
  • V. Cattori
  • C. Lepadatu
  • R. Hofmann-Lehmann
Article

Abstract

The goal of this study was to develop a liquid culture medium for the rapid isolation, cultivation, identification and subsequent antibiotics susceptibility testing of Helicobacter pylori directly from biopsy specimens. Five liquid media were tested: Ham’s F-12, Brucella broth, tryptic soybroth, brain heart infusion broth and Mueller-Hinton broth. After optimisation of the medium, it was applied in order to investigate biopsy samples from 150 patients with gastro-duodenal disorders and compared with traditional culture methods, microscopy and an H. pylori-specific TaqMan real-time polymerase chain reaction (PCR). The most reliable and rapid growth of H. pylori, even at a small inoculum size, was obtained in Ham’s F-12 medium with 5% horse serum. The developed system allowed the primary isolation of H. pylori in clinical samples and provided 87% sensitivity and 100% specificity.

References

  1. 1.
    NIH Consensus Conference (1994) Helicobacter pylori in peptic ulcer disease. JAMA 272:65–69CrossRefGoogle Scholar
  2. 2.
    Lee A (1995) Helicobacter infections in laboratory animals: a model for gastric neoplasias. Ann Med 27:575–582. doi:10.3109/07853899509002472 PubMedCrossRefGoogle Scholar
  3. 3.
    Talley NJ, Hunt RH (1997) What role does Helicobacter pylori play in dyspepsia and nonulcer dyspepsia? Arguments for and against H. pylori being associated with dyspeptic symptoms. Gastroenterology 113(Suppl 6):S67–S77PubMedGoogle Scholar
  4. 4.
    Gilman RH (1990) Helicobacter pylori and gastritis in Peruvian patients: relationship to socioeconomic level, age, and sex. Am J Gastroenterol 85:819–823Google Scholar
  5. 5.
    Hegarty JP, Dowd MT, Baker KT (1999) Occurrence of Helicobacter pylori in surface water in the United States. J Appl Microbiol 87:697–701. doi:10.1046/j.1365-2672.1999.00912.x PubMedCrossRefGoogle Scholar
  6. 6.
    Yingzhi L, Redlinger TE, Avitia R, Galindo A, Goodman K (2002) Isolation and genotyping of Helicobacter pylori from untreated municipal wastewater. Appl Environ Microbiol 68:1436–1439. doi:10.1128/AEM.68.3.1436-1439.2002 CrossRefGoogle Scholar
  7. 7.
    Fendrick AM, Chernew ME, Hirth RA, Bloom BS, Bandekar RR, Scheiman JM (1999) Clinical and economic effects of population-based Helicobacter pylori screening to prevent gastric cancer. Arch Intern Med 159(2):142–148. doi:10.1001/archinte.159.2.142 PubMedCrossRefGoogle Scholar
  8. 8.
    Aguemon B, Struelens M, Deviere J, Denis O, Golstein P, Salmon I, Nagy N (2005) Primary antibiotic resistance and effectiveness of Helicobacter pylori triple therapy in ulcero-inflammatory pathologies of the upper digestive tract. Acta Gastroenterol Belg 68(3):287–293PubMedGoogle Scholar
  9. 9.
    Buzás GM, Lotz G, Kiss A (2007) The Hungarian epidemiology of clarithromycin resistance in Helicobacter pylori infection. Orv Hetil 148(31):1461–1467. doi:10.1556/OH.2007.28013 PubMedCrossRefGoogle Scholar
  10. 10.
    Fischbach L, Evans EL (2007) Meta-analysis: the effect of antibiotic resistance status on the efficacy of triple and quadruple first-line therapies for Helicobacter pylori. Aliment Pharmacol Ther 26(3):343–357PubMedCrossRefGoogle Scholar
  11. 11.
    Vakil N, Megraud F (2007) Eradication therapy for Helicobacter pylori. Gastroenterology 133(3):985–1001. doi:10.1053/j.gastro.2007.07.008 PubMedCrossRefGoogle Scholar
  12. 12.
    Han SW, Flamm R, Hachem C, Kim HY, Clarridge JE, Evans DG, Beyer J, Drnec J, Graham DY (1995) Transport and storage of Helicobacter pylori from gastric mucosal biopsies and clinical isolates. Eur J Clin Microbiol Infect Dis 14:349–352. doi:10.1007/BF02116531 PubMedCrossRefGoogle Scholar
  13. 13.
    Siu LK, Leung WK, Cheng AF, Sung JY, Ling TK, Ling JM, Ng EK, Lau JY, Chung SC (1998) Evaluation of a selective transport medium for gastric biopsy specimens to be cultured for Helicobacter pylori. J Clin Microbiol 36(10):3048–3050PubMedGoogle Scholar
  14. 14.
    Soltesz V, Zeeberg B, Wadström T (1992) Optimal survival of Helicobacter pylori under various transport conditions. J Clin Microbiol 30:1453–1456PubMedGoogle Scholar
  15. 15.
    Shahamat M, Mai U, Paszko-Kolva C, Yamamoto H, Colwell RR (1991) Evaluation of liquid media for growth of Helicobacter pylori. J Clin Microbiol 29:2835–2837PubMedGoogle Scholar
  16. 16.
    Jiang X, Doyle MP (2000) Growth supplements for Helicobacter pylori. J Clin Microbiol 38(5):1984–1987PubMedGoogle Scholar
  17. 17.
    Deshpande M, Calenoff E, Daniels L (1995) Rapid large-scale growth of Helicobacter pylori in flasks and fermentors. Appl Environ Microbiol 61(6):2431–2435PubMedGoogle Scholar
  18. 18.
    Walsh EJ, Moran AP (1997) Influence of medium composition on the growth and antigen expression of Helicobacter pylori. J Appl Microbiol 83(1):67–75. doi:10.1046/j.1365-2672.1997.00164.x PubMedCrossRefGoogle Scholar
  19. 19.
    Stevenson TH, Castillo A, Lucia LM, Acuff GR (2000) Growth of Helicobacter pylori in various liquid and plating media. Lett Appl Microbiol 30(3):192–196. doi:10.1046/j.1472-765x.2000.00699.x PubMedCrossRefGoogle Scholar
  20. 20.
    Nedenskov P (1994) Nutritional requirements for growth of Helicobacter pylori. Appl Environ Microbiol 60(9):3450–3453PubMedGoogle Scholar
  21. 21.
    Reynolds DJ, Penn CW (1994) Characteristics of Helicobacter pylori growth in a defined medium and determination of its amino acid requirements. Microbiology 140:2649–2656PubMedGoogle Scholar
  22. 22.
    Albertson N, Wenngren I, Sjöström JE (1998) Growth and survival of Helicobacter pylori in defined medium and susceptibility to Brij 78. J Clin Microbiol 36(5):1232–1235PubMedGoogle Scholar
  23. 23.
    Morshed MG, Karita M, Konishi H, Okita K, Nakazawa T (1994) Growth medium containing cyclodextrin and low concentration of horse serum for cultivation of Helicobacter pylori. Microbiol Immunol 38(11):897–900PubMedGoogle Scholar
  24. 24.
    Kitsos CM, Stadtländer CT (1998) Helicobacter pylori in liquid culture: evaluation of growth rates and ultrastructure. Curr Microbiol 37(2):88–93. doi:10.1007/s002849900344 PubMedCrossRefGoogle Scholar
  25. 25.
    Vega AE, Cortiñas TI, Mattana CM, Silva HJ, Puig De Centorbi O (2003) Growth of Helicobacter pylori in medium supplemented with cyanobacterial extract. J Clin Microbiol 41(12):5384–5388. doi:10.1128/JCM.41.12.5384-5388.2003 PubMedCrossRefGoogle Scholar
  26. 26.
    Azevedo NF, Pacheco AP, Keevil CW, Vieira MJ (2004) Nutrient shock and incubation atmosphere influence recovery of culturable Helicobacter pylori from water. Appl Environ Microbiol 70(1):490–493. doi:10.1128/AEM.70.1.490-493.2004 PubMedCrossRefGoogle Scholar
  27. 27.
    Bury-Moné S, Kaakoush NO, Asencio C, Mégraud F, Thibonnier M, De Reuse H, Mendz GL (2006) Is Helicobacter pylori a true microaerophile? Helicobacter 11(4):296–303. doi:10.1111/j.1523-5378.2006.00413.x PubMedCrossRefGoogle Scholar
  28. 28.
    Testerman TL, McGee DJ, Mobley H (2001) Helicobacter pylori growth and urease detection in the chemically defined medium Ham’s F-12 nutrient mixture. J Clin Microbiol 39(11):3842–3850. doi:10.1128/JCM.39.11.3842-3850.2001 PubMedCrossRefGoogle Scholar
  29. 29.
    Testerman TL, Conn PB, Mobley HL, McGee DJ (2006) Nutritional requirements and antibiotic resistance patterns of Helicobacter species in chemically defined media. J Clin Microbiol 44(5):1650–1658. doi:10.1128/JCM.44.5.1650-1658.2006 PubMedCrossRefGoogle Scholar
  30. 30.
    Greig MA, Neithercut WD, Hossack M, McColl KE (1991) Harnessing of urease activity of Helicobacter pylori to induce self-destruction of the bacterium. J Clin Pathol 44:157–159. doi:10.1136/jcp.44.2.157 PubMedCrossRefGoogle Scholar
  31. 31.
    Clyne M, Labigne A, Drumm B (1995) Helicobacter pylori requires an acidic environment to survive in the presence of urea. Infect Immun 63(5):1669–1673PubMedGoogle Scholar
  32. 32.
    Sjöström JE, Larsson H (1996) Factors affecting growth and antibiotic susceptibility of Helicobacter pylori: effect of pH and urea on the survival of a wild-type strain and a urease-deficient mutant. J Med Microbiol 44(6):425–433PubMedCrossRefGoogle Scholar
  33. 33.
    Xia HX, English L, Keane CT, O’Morain CA (1993) Enhanced cultivation of Helicobacter pylori in liquid media. J Clin Pathol 46:750–753. doi:10.1136/jcp.46.8.750 PubMedCrossRefGoogle Scholar
  34. 34.
    Morgan DR, Freedman R, Depew CE, Kraft WG (1987) Growth of Campylobacter pylori in liquid media. J Clin Microbiol 25:2123–2125PubMedGoogle Scholar
  35. 35.
    Henriksen TH, Lia A, Schøyen R, Thoresen T, Berstad A (2000) Assessment of optimal atmospheric conditions for growth of Helicobacter pylori. Eur J Clin Microbiol Infect Dis 19(9):718–720. doi:10.1007/s100960000340 PubMedCrossRefGoogle Scholar
  36. 36.
    Kobayashi D, Eishi Y, Ohkusa T, Ishige I, Suzuki T, Minami J, Yamada T, Takizawa T, Koike M (2002) Gastric mucosal density of Helicobacter pylori estimated by real-time PCR compared with results of urea breath test and histological grading. J Med Microbiol 51:305–311PubMedGoogle Scholar
  37. 37.
    Cattori V, Hofmann-Lehmann R (2008) Absolute quantitation of feline leukemia virus (FeLV) proviral DNA and viral RNA loads by TaqMan real-time PCR and RT-PCR. Methods Mol Biol 429:73–87PubMedCrossRefGoogle Scholar
  38. 38.
    King A (2001) Recommendations for susceptibility tests on fastidious organisms and those requiring special handling. J Antimicrob Chemother 48(Suppl 1):77–80PubMedGoogle Scholar
  39. 39.
    Ellerbrok H, Nattermann H, Ozel M, Beutin L, Appel B, Pauli G (2002) Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR. FEMS Microbiol Lett 214(1):51–59PubMedCrossRefGoogle Scholar
  40. 40.
    Boddicker JD, Rota PA, Kreman T, Wangeman A, Lowe L, Hummel KB, Thompson R, Bellini WJ, Pentella M, Desjardin LE (2007) Real-time reverse transcription-PCR assay for detection of mumps virus RNA in clinical specimens. J Clin Microbiol 45(9):2902–2908. doi:10.1128/JCM.00614-07 PubMedCrossRefGoogle Scholar
  41. 41.
    Lindh M, Görander S, Andersson E, Horal P, Mattsby-Balzer I, Ryd W (2007) Real-time Taqman PCR targeting 14 human papilloma virus types. J Clin Virol 40(4):321–324. doi:10.1016/j.jcv.2007.09.009 PubMedCrossRefGoogle Scholar
  42. 42.
    Deltenre M, Glupczynski Y, De Prez C, Nyst JF, Burette A, Labbé M, Jonas C, DeKoster E (1989) The reliability of urease tests, histology and culture in the diagnosis of Campylobacter pylori infection. Scand J Gastroenterol Suppl 160:19–24. doi:10.3109/00365528909091730 PubMedCrossRefGoogle Scholar
  43. 43.
    Shadowen RD, Sciortino CV (1989) Improved growth of Campylobacter pylori in a biphasic system. J Clin Microbiol 27(8):1744–1747PubMedGoogle Scholar
  44. 44.
    Frenck RW Jr, Clemens J (2003) Helicobacter in the developing world. Microbes Infect 5(8):705–713. doi:10.1016/S1286-4579(03)00112-6 PubMedCrossRefGoogle Scholar
  45. 45.
    National Committee for Clinical Laboratory Standards (NCCLS) (1999) Performance standards for antimicrobial susceptibility testing: 9th informational supplement: document M100-S9. NCCLS, Villanova, PAGoogle Scholar
  46. 46.
    McNulty C, Owen R, Tompkins D, Hawtin P, McColl K, Price A, Smith G, Teare L; PHLS Helicobacter Working Group (2002) Helicobacter pylori susceptibility testing by disc diffusion. J Antimicrob Chemother 49(4):601–609. doi:10.1093/jac/49.4.601 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • N. Sainsus
    • 1
  • V. Cattori
    • 2
  • C. Lepadatu
    • 3
  • R. Hofmann-Lehmann
    • 2
  1. 1.Department of MicrobiologyState University of Medicine and Pharmacy “N. Testemiţanu”ChishinauMoldova
  2. 2.Clinical Laboratory, Vetsuisse FacultyUniversity of ZurichZurichSwitzerland
  3. 3.Department of Surgery, Faculty of CME, Clinical Republican HospitalState University of Medicine and Pharmacy “N. Testemiţanu”ChishinauMoldova

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