Evaluation of different culture media for detection and quantification of H. pylori in environmental and clinical samples

Abstract

The objective of the present study was to establish the most suitable culture medium for the isolation of H. pylori from environmental and clinical samples. Ten different culture media were compared and evaluated. Four of them had been previously described and were modified in this study. The rest of the media were designed de novo. Three different matrices, tap water, wastewater, and feces, were inoculated with serial dilutions of H. pylori NCTC 11637 strain at a final concentration of 104 and 103 CFU/ml and the recovery rates were calculated. From inoculated tap water and wastewater samples, H. pylori colonies were recovered from four out of the analyzed culture media. When fecal samples were analyzed, the isolation of the pathogen under study was only possible from two culture media. Different optimal media were observed for each type of sample, even for wastewater and stool samples. Nevertheless, our results indicated that the combination of Dent Agar with polymyxin B sulfate did not inhibit the growth of H. pylori and was highly selective for its recovery, regardless of the sample origin. Thus, we propose the use of this medium as a diagnostic tool for the isolation of H. pylori from environmental and clinical samples, as well as for epidemiological studies.

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References

  1. Adams BL, Bates TC, Oliver JD (2003) Survival of Helicobacter pylori in a natural freshwater environment. Appl Environ Microbiol 69:7461–7466. https://doi.org/10.1128/aem.69.12.7462-7466.2003

    Article  Google Scholar 

  2. Al-Sulami AA, Al-Edani TA, Al-Abdulaz AA (2012) Culture method and PCR for the detection of Helicobacter pylori in drinking water in Basrah governorate Iraq. Gastroenterol Res Pract 12:5–5. https://doi.org/10.1155/2012/245167

    Article  Google Scholar 

  3. Álvarez-Ruiz R, Cuñat A, Picó Y (2018) Pollution fingerprinting of sewage sludge and their related risk in Albufera's wetland, Valencia. 16th IWA International conference on wetland Systems for Water Pollution Control. http://hdl.handle.net/10261/184328

  4. Ansorg R, Von Recklinghausen G, Pomarius R, Schmid EN (1991) Evaluation of techniques for isolation, subcultivation and preservation of Helicobacter pylori. J Clin Microbiol 29:51–53

    CAS  Article  Google Scholar 

  5. Atapoor S, Safarpoor Dehkordi F, Rahini E (2014) Detection of Helicobacter pylori in various types of vegetables and salads. Jundishapur J Microbiol. 7(5):e10013. https://doi.org/10.5812/jjm.10013

    Article  PubMed  PubMed Central  Google Scholar 

  6. 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 40:490–493. https://doi.org/10.1128/AEM.70.1.490-493.2004

    CAS  Article  Google Scholar 

  7. Azevedo NF, Almedia L, Cerquerias S, Dias C, Keevil W, Vieira MJ (2007) Coccoid form of Helicobacter pylori as a morphological manifestation of cell adaptation to the environment. Appl Environ Microbiol 73(10):3423–3427. https://doi.org/10.1128/AEM.00047-07

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Bahrami AR, Rahimi E, Ghasemian Safaei H (2013) Detection of Helicobacter pylori in City water, dental units’ water, and bottled mineral water in Isfahan, Iran. Sci world J 5. https://doi.org/10.1128/AEM.00827-0810.1155/2013/280510

  9. Bai X, Xi C, Wu J (2016) Survival of Helicobacter pylori in the wastewater treatment process and the receiving river in Michigan, USA. J Water Health 14:692–698. https://doi.org/10.2166/wh.2016.259

    Article  PubMed  Google Scholar 

  10. Bode G, Mauch F, Malfertheiner P (1993) The coccoid forms of Helicobacter pylori. Criteria for their viability. Epidemiol Infect 111:483–490. https://doi.org/10.1017/S0950268800057216

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Boro S, Sarma M, Sarma P (2016). Helicobacter pylori and Steps for its Elimination: A Review Global J Med Res (F) 16: 31–36

  12. Carbone M, Maugeri TL, Gugliandolo C, La Camera E, Biondo C, Fera MT (2005) Occurrence of Helicobacter pylori DNA in coastal environment of southern Italy (straits of Messina). J Appl Microbiol 98:768–774. https://doi.org/10.1111/j.1365-2672.2004.02517.x

    CAS  Article  PubMed  Google Scholar 

  13. Cellini L (2014) Helicobacter pylori: a chameleon-like approach to life. World J Gastroenterol 20:5575–5582. https://doi.org/10.3748/wjg.v20.i19.5575

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Degnan AJ, Sonzogni WC, Standridge JH (2003) Development of a plating medium for selection of Helicobacter pylori from water samples. Appl Environ Microbiol 69:2914–2918. https://doi.org/10.1128/AEM.69.5.2914-2918.2003

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Dent JC, McNulty CA (1988) Evaluation of a new selective medium for Campylobacter pylori. Eur J Clin Microbiol Infect Dis 7:555–558. https://doi.org/10.1007/bf01962615

    CAS  Article  PubMed  Google Scholar 

  16. Duque-Jamaica R, Arévalo-Galvis A, Poutou-Piñales RA, Trespalacios AA (2010) Sequential statistical improvement of the liquid cultivation of Helicobacter pylori. Helicobacter 15:303–312. https://doi.org/10.1111/j.1523-5378.2010.00763.x

    Article  PubMed  Google Scholar 

  17. Fernández M, Contreras M, Suárez P, García-Amado MA (2007) Use of HP selective medium to detect Helicobacter pylori associated with other enteric bacteria in seawater and marine molluscs. Let Appl Microbiol 45:213–218. https://doi.org/10.1111/j.1472-765X.2007.02174.x

    CAS  Article  Google Scholar 

  18. Giao MS, Azevedo NF, Wilks SA, Vieira MJ, Keevil CW (2008) Persistence of Helicobacter pylori in heterotrophic drinking water biofilms. Appl Environ Microbiol 74:5898–5904. https://doi.org/10.1128/AEM.00827-08

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Graham DY (1991) Helicobacter pylori: its epidemiology and its role in duodenal ulcer disease. J Gastroenterol Hepatol 6:105–113. https://doi.org/10.1111/j.1440-1746.1991.tb01448.x

    CAS  Article  PubMed  Google Scholar 

  20. Hultén K, Enroth H, Nyström T, Engstrand L (1998) Presence of Helicobacter species DNA in Swedish water. J Appli Microbiol 85:282–286. https://doi.org/10.1046/j.1365-2672.1998.00500.x

    Article  Google Scholar 

  21. Iwamoto A, Tanahashi T, Okada R, Yoshida Y, Kikuchi K, Keida Y, Yoshida M (2014) Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes. Gut Pathog 6:27. https://doi.org/10.1186/1757-47496-27

    Article  PubMed  PubMed Central  Google Scholar 

  22. Jiang X, Doyle MP (2002) Optimizing enrichment culture conditions for detecting Helicobacter pylori in foods. J Food Protect 65:1949–1954. https://doi.org/10.4315/0362-028x-65.12.1949

    Article  Google Scholar 

  23. Lagier JC, Edouard S, Pagnier I, Mediannikov O, Drancourt M, Raoult D (2015) Current and past strategies for bacterial culture in clinical microbiology. Clin Microbiol Rev 28(1):208–236

    CAS  Article  Google Scholar 

  24. Loke MF, Ng CG, Vilashni Y, Lim J, Ho B (2016) Understanding the dimorphic lifestyles of human gastric pathogen Helicobacter pylori using the SWATH-based proteomics approach. Sci Rep 6(26784). https://doi.org/10.1038/srep26784

  25. Lopes AI, Vale FF, Oleastro M (2014) Helicobacter pylori infection-recent developments in diagnosis. World J Gastroenterol 20:9299–9313. https://doi.org/10.3748/wjg.v20.i28.9299

    Article  PubMed  PubMed Central  Google Scholar 

  26. Lu Y, Redlonger TE, Avitia R, Galindo A, Goodman G (2002) Isolation and genotyping of Helicobacter pylori from untreated municipal wastewater. Appl Environ Microbiol 68(3):1436–1439

    CAS  Article  Google Scholar 

  27. McGee D, Geore S, Trainor E, Hortonm K, Hildebrandt E, Testerman L (2011) Cholesterol enhances Helicobacter pylori resistance to antibiotics and LL-37. Antimicrob Agents Ch 55:2897–2904. https://doi.org/10.1128/AAC.00016-11

    CAS  Article  Google Scholar 

  28. Miendje Deyi VY, Van den Borre C, Fontaine V (2010) Comparative evaluation of 3 selective media for primary isolation of Helicobacter pylori from gastric biopsies under routine conditions. Diagn Micr Infec Dis 68:474–476. https://doi.org/10.1016/j.diagmicrobio.2010.08.009

    Article  Google Scholar 

  29. Moreno Y, Ferrús MA (2012) Specific detection of cultivable Helicobacter pylori cells from wastewater treatment plant. Helicobacter 17:327-32. https://doi.org/10.1128/AEM.00827-0810.1111/j.1523-5378.2012.00961. X

  30. Moreno Y, Pérez R, Ramirez MJ, Calvet X, Santiago P, Ferrús MA (2015) Rapid identification of viable H. pylori cells in feces by DVC-FISH. JSM. Gastroenterol Hepatol 3:1049

    Google Scholar 

  31. Nilsson HO, Blom J, Abu-Al-Soud W, Ljung AA, Andersen LP, Wadstrom T (2002) Effect of cold starvation, acid stress, and nutrients on metabolic activity of Helicobacter pylori. Appl Environ Microbiol 68:11–19. https://doi.org/10.1128/AEM.68.1.11-19.2002

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Percival S, Suleman L (2014) Biofilms and Helicobacter pylori: dissemination and persistence within the environment and host. World J Gastrointest Pathophysiol 5:122–132. https://doi.org/10.4291/wjgp.v5.i3.122

    Article  PubMed  PubMed Central  Google Scholar 

  33. Percival S, Thomas JG (2009) Transmission of Helicobacter pylori and the role of water and biofilms. J Water Health 7:469–477. https://doi.org/10.2166/wh.2009.070

    Article  PubMed  Google Scholar 

  34. Ranjbar R, Khamesipour F, Jonaidi-Jafari N, Rahimi E (2016) Helicobacter pylori isolated from Iranian drinking water: vacA, cagA, iceA, oipA and babA2 genotype status and antimicrobial resistance properties. FEBS Open Bio 6:433–441. https://doi.org/10.1002/2211-5463.12054

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Rizvi F, Hanna A (2000) Evaluation of different transport and enrichment media for the isolation of Helicobacter pylori. JAMC 12:31–33 http://ayubmed.edu.pk/JAMC/PAST/12-3/Farhat.pdf

  36. Santiago P, Moreno Y, Ferrús MA (2015) Identification of viable Helicobacter pylori in drinking water supplies by cultural and molecular techniques. Helicobacter 20:252–259. https://doi.org/10.1111/hel.12205

    CAS  Article  PubMed  Google Scholar 

  37. Shibayama K, Nagasawa M, Ando T, Minami M, Wachino J, Suzuki S, Arakawa Y (2006) Usefulness of adult bovine serum for Helicobacter pylori culture media. J Clin Microbiol 44(11):4255–4257

    Article  Google Scholar 

  38. Sjomina O, Pavlova J, Niv Y, Leja M (2018) Epidemiology of Helicobacter pylori infection Helicobacter e12514. https://doi.org/10.1111/hel.12514, Epidemiology ofHelicobacter pyloriinfection, 23

  39. Stevenson TH, Lucia LM, Acuff GR (2000) Development of a selective medium for isolation of Helicobacter pylori from cattle and beef samples. App Environ Microbiol 66:723–727

    CAS  Article  Google Scholar 

  40. Suzuki R, Shiota S, Yamoka Y (2012) Molecular epidemiology, population genetics, and pathogenic role of Helicobacter pylori. Infect Genet Evol 12:203–2013. https://doi.org/10.1016/j.meegid.2011.12.002

    Article  PubMed  Google Scholar 

  41. Testerman TL, Conn PB, Mobley HLT, McGee DJ (2006) Nutritional requirements and antibiotic resistance patterns of Helicobacter species in chemically defined media. J Clin Microbiol 44:1650–1658. https://doi.org/10.1128/JCM.44.5.1650-1658.2006

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Van Tongeren SP, Slaets JP, Harmsen HJM, Welling GW (2005) Fecal microbiota composition and frailty. Appl Environ Microbiol 71:6438–6442. https://doi.org/10.1128/AEM.71.10.6438-6442.2005

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Wang YK, Kuo FC, Liu CJ, Wu MC, Shih HY, Wang SS, Wu JY, Kuo CH, Huang YK, Wu DC (2015) Diagnosis of Helicobacter pylori infection: current options and developments. World J Gastroenterol 21:11221–11235. https://doi.org/10.3748/wjg.v21.i40.11221

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by the Consellería de Educación, Investigación, Cultura y Deporte, of the Community of Valencia, Spain, under project AICO/2018/273, and by the Spanish Ministry of Economy and Competitiveness AGL2014/53875-R Grant.

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Correspondence to Yolanda Moreno.

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Hortelano, I., Moreno, Y., Vesga, F.J. et al. Evaluation of different culture media for detection and quantification of H. pylori in environmental and clinical samples. Int Microbiol (2020). https://doi.org/10.1007/s10123-020-00135-z

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Keywords

  • H. pylori
  • Isolation
  • Identification
  • Culture media
  • Feces
  • Environmental samples