Case Report Open Access
Copyright ©2009 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Mar 28, 2009; 15(12): 1528-1529
Published online Mar 28, 2009. doi: 10.3748/wjg.15.1528
Successful isolation of Helicobacter pylori after prolonged incubation from a patient with failed eradication therapy
Yan Yin, Li-Hua He, Jian-Zhong Zhang, Department of Diagnosis, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Author contributions: Yin Y, Zhang JZ and He LH designed the research; Yin Y and He LH performed the research; Yin Y, He LH and Zhang JZ analyzed the data; Yin Y and Zhang JZ wrote the paper.
Correspondence to: Jian-Zhong Zhang, Professor, Department of Diagnosis, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing 102206, China. zhangjianzhong@icdc.cn
Telephone: +86-10-61739319
Fax: +86-10-61730233
Received: January 6, 2008
Revised: February 20, 2009
Accepted: February 27, 2009
Published online: March 28, 2009

Abstract

Helicobacter pylori (H pylori), a gastric pathogen, is a major cause of chronic gastritis and peptic ulcer disease, and is an important risk factor for the development of gastric malignancies. Culture of the bacterium from gastric biopsy is essential for the determination of drug resistance of H pylori. However, the isolation rates of H pylori from infected individuals vary from 23.5% to 97% due to a number of factors such as biopsy preparation, cultural environment, medium and the method adopted. In the present case, we found that a prolonged incubation period of up to 19 d allowed successful isolation of H pylori from a patient who received triple therapy that failed to eradicate the bacterium.

Key Words: Helicobacter pylori; Isolation; Eradication



INTRODUCTION

Helicobacter pylori (H pylori) is a gastric pathogen, which is present in approximately half of the world’s population. It is a major cause of chronic gastritis and peptic ulcer disease, and is an important risk factor for the development of gastric malignancies[14]. Although accurate non-invasive methods such as the urea breath test, the stool antigen test, and serology are available, biopsy-based invasive techniques, including the rapid urease test, histology and culture, are required to confirm the infection. Moreover, culture of the bacterium from gastric biopsy is essential for the determination of drug resistance of H pylori and thus for the subsequent treatment strategy after failed eradication therapy. However, the isolation rates of H pylori from infected individuals vary from 23.5% to 97%[56] due to a number of factors, such as biopsy preparation, cultural environment, medium and the method adopted. The duration of incubation for isolation of H pylori has been recommended to be 2 to 7 d. Here we reported our observation that a prolonged incubation period of up to 19 d allowed successful isolation of H pylori from a patient who received triple therapy that failed to eradicate the bacterium.

CASE REPORT

A patient (female, 59 years old) with an H pylori positive duodenal ulcer received two consecutive trials of 7-d triple regimens in a regional hospital. The regimen consisted of Metronidazole, Clarithromycin, and Cimetidine. Four weeks after the second trial, the patient was still positive for a 13C urea breath test. She then came to Beijing for a solution. To obtain the drug resistance profile of the H pylori strain, the patient underwent an upper gastrointestinal endoscopy, and four biopsy specimens were taken from gastric antrum. The biopsies were placed directly into transport medium at room temperature and processed for culture within 2 h. Biopsy samples were smeared on H pylori selective Dent Columbia agar plates (Oxoid Ltd., London, England) supplemented with 8% sheep blood (Hengzhaoxiang Science & Technology Co., Beijing), and incubated in a microaerophilic environment (5% O2, 10% CO2, and 85% N2) at 37°C. The plates were scheduled to be checked on days 3, 6, 8, and 10. However, there was no colony growing after 10 d. We decided to incubate the plates further, and check the plates every 3 d. On day 19, one suspected H pylori-like colony appeared on one of the plates. This colony was subcultured in non-selective 2 Columbia agar plates containing 8% sheep blood in the same environment mentioned above, for three days. The isolate was confirmed to be H pylori based on its typical colony morphology, negative Gram’s stain, and positive urease, catalase, and oxidase tests.

The antimicrobial susceptibilities of the isolate to metronidazole, clarithromycin, amoxicillin, ampicillin, levofloxacin, and rifampicin were determined by the E-test (AB Biodisk, Solna, Sweden). Briefly, a bacterial suspension (2 McFarland standard) was prepared with brain heart infusion (Oxoid Ltd., London, England) containing 10% heat-treated serum[7]. After the bacterial suspension was swabbed onto the entire Columbia plates, sterile E-test strips impregnated with the above antibiotics were placed on the agar surface of corresponding plates. Minimal inhibitory concentraction (MICs) were determined according to the manufacturer’s instructions after three to four days of incubation. The isolate exhibited high-level resistance to metronidazole (MIC > 256 &mgr;g/mL) and clarithromycin (MIC > 48 &mgr;g/mL), but was susceptible to amoxicillin, ampicillin, levofloxacin, and rifampicin (all MIC < 0.016 &mgr;g/mL), which explained the failure of the triple regimens containing metronidazole and clarithromycin.

DISCUSSION

Culture has been considered the “gold standard” in confirmation of the diagnosis of H pylori infection. Moreover, the isolation and identification of strains is important for the investigation of profiles of bacterial virulence and, particularly, drug resistance. Due to the gradually rising prevalence of H pylori resistance to many antibiotics commonly used in triple regimens, the determination of antibiotic susceptibility of individual isolates is of particular importance. However, primary isolation of H pylori from gastric biopsies is rather demanding, and is affected widely by the culture conditions in addition to the biopsy-related factors[8]. Our report also indicates that a prolonged incubation is necessary for some strains, especially those enduring hostile environment or a period of antibiotic force. It was reported that a longer incubation of 11 d is helpful for isolating H pylori strains from long-term-frozen specimens[9], but this is the first report of the bacteria recovered after 19 d incubation. Indeed, the isolate requiring 19 d recovery later exhibited normal growth characteristics of H pylori strains when compared to another strain, NCTC11637, indicating its unusually long incubation requirement was a temporary predicament.

It has been demonstrated in vitro that H pylori cells can transform from a cultivatable spiral-shaped form to a non- cultivatable coccoid form, in which the recovery of the bacterium is very difficult by routine culture methods[10]. We would propose that during the period of eradication therapy, some organisms transform into the so-called “uncultivatable form” with the propagation being stopped under the antimicrobial pressure in the local environment. However, these organisms, which may have been selectively resistant to the used antimicrobials, survive, possibly with some suppressed metabolic activities[11]. Once released from the medication at the end of the trial, these organisms gradually restore their normal growing features after prolonged incubation in an optimal environment and eventually become cultivatable. Therefore, these “uncultivatable form” organisms might contribute, at least partially, to treatment failures and the development of antimicrobial resistance. In the meantime, we suggest that a new H pylori culture after a first attempt to eradicate H pylori needs to be postponed, probably by four weeks or even longer. It is noticeable that the patient was positive for the 13C urea breath test four weeks after completion of treatment, indicating that there are a number of organisms that are able to produce urease activities after release from antimicrobial pressure after four weeks. The coccoid H pylori can produce urease, though at a decreased level[12], suggesting its potential pathogenicity.

Footnotes

Supported by The Grant “Research of the Helicobacter pylori gene engineering vaccine” from Hi-tech research and development (863) program of China, Grant No. 2001AA21516102 and National Science and Technology Infrastructure Program, Grant No. 2007BAI04B02

References
1.  Dunn BE, Cohen H, Blaser MJ. Helicobacter pylori. Clin Microbiol Rev. 1997;10:720-741.  [PubMed]  [DOI]  [Cited in This Article: ]
2.  Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, Sibley RK. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med. 1991;325:1127-1131.  [PubMed]  [DOI]  [Cited in This Article: ]
3.  Gastric cancer and Helicobacter pylori: a combined analysis of 12 case control studies nested within prospective cohorts. Gut. 2001;49:347-353.  [PubMed]  [DOI]  [Cited in This Article: ]
4.  Stolte M, Bayerdorffer E, Morgner A, Alpen B, Wundisch T, Thiede C, Neubauer A. Helicobacter and gastric MALT lymphoma. Gut. 2002;50 Suppl 3:III19-III24.  [PubMed]  [DOI]  [Cited in This Article: ]
5.  Fresnadillo Martinez MJ, Rodriguez Rincon M, Blazquez de Castro AM, Garcia Sanchez E, Garcia Sanchez JE, Trujillano Martin I, Cordero Sanchez M, Alvarez Alvarez P, Paz Bouza J, Garcia-Rodriguez JA. Comparative evaluation of selective and nonselective media for primary isolation of Helicobacter pylori from gastric biopsies. Helicobacter. 1997;2:36-39.  [PubMed]  [DOI]  [Cited in This Article: ]
6.  Heep M, Scheibl K, Degrell A, Lehn N. Transport and storage of fresh and frozen gastric biopsy specimens for optimal recovery of Helicobacter pylori. J Clin Microbiol. 1999;37:3764-3766.  [PubMed]  [DOI]  [Cited in This Article: ]
7.  Shibayama K, Nagasawa M, Ando T, Minami M, Wachino J, Suzuki S, Arakawa Y. Usefulness of adult bovine serum for Helicobacter pylori culture media. J Clin Microbiol. 2006;44:4255-4257.  [PubMed]  [DOI]  [Cited in This Article: ]
8.  van der Hulst RW, Verheul SB, Weel JF, Gerrits Y, ten Kate FJ, Dankert J, Tytgat GN. Effect of specimen collection techniques, transport media, and incubation of cultures on the detection rate of Helicobacter pylori. Eur J Clin Microbiol Infect Dis. 1996;15:211-215.  [PubMed]  [DOI]  [Cited in This Article: ]
9.  Boyanova L. Influence of transport conditions and media on Helicobacter pylori isolation. J Med Microbiol. 2003;52:1129-1130.  [PubMed]  [DOI]  [Cited in This Article: ]
10.  Shahamat M, Alavi M, Watts JE, Gonzalez JM, Sowers KR, Maeder DW, Robb FT. Development of two PCR-based techniques for detecting helical and coccoid forms of Helicobacter pylori. J Clin Microbiol. 2004;42:3613-3619.  [PubMed]  [DOI]  [Cited in This Article: ]
11.  Nilsson HO, Blom J, Abu-Al-Soud W, Ljungh A A, Andersen LP, Wadstrom T. Effect of cold starvation, acid stress, and nutrients on metabolic activity of Helicobacter pylori. Appl Environ Microbiol. 2002;68:11-19.  [PubMed]  [DOI]  [Cited in This Article: ]
12.  She FF, Su DH, Lin JY, Zhou LY. Virulence and potential pathogenicity of coccoid Helicobacter pylori induced by antibiotics. World J Gastroenterol. 2001;7:254-258.  [PubMed]  [DOI]  [Cited in This Article: ]