Helicobacter pylori Isolation and Antibiotic Susceptibility Testing Using Rapidly Frozen Biopsy Samples
Article information
, Jung Mogg Kim2,*, Gwang Ho Baik3, Jun Chul Park4, Hye-Kyung Jung5, Han Seung Ryu6, Soo-Jeong Cho7, Cheol Min Shin8, Hwoon-Yong Jung,1, Korean College of Helicobacter and Upper Gastrointestinal ResearchAbstract
Objectives
To involve institutions without the ability to perform susceptibility testing, long-term storage of tissue sample is critical to isolate the bacteria in a central laboratory. The aim of the study was to investigate the feasibility of H. pylori isolation and antibiotic susceptibility testing using rapidly frozen biopsy specimens collected from various institutions.
Methods
Eight institutions located in various regions of Korea participated in the study. Patients requiring upper endoscopy and H. pylori testing were screened. Two biopsy samples were taken from the stomach. One was placed in a sterile Eppendorf tube and then immediately placed in a vacuum bottle containing dry ice, which was stored at -80°C. The other was used in a rapid urease test. Collected samples were delivered to a central laboratory. The bacteria were isolated from the frozen samples under microaerophilic conditions. The agar dilution method was used to determine the minimum inhibitory concentration (MIC) of amoxicillin, clarithromycin, metronidazole, tetracycline, ciprofloxacin, and levofloxacin for each H. pylori isolate.
Results
Patients with a positive rapid urease test result (n=113) were enrolled. The mean age was 56.6±12.3 years. The male:female ratio was 64:49. The overall culture success rate was 77.0% (87/113). MIC values were determined using isolated 87 H. pylori strains. Rates of resistance to amoxicillin, clarithromycin, metronidazole, tetracycline, levofloxacin, and ciprofloxacin were 23.0%, 25.3%, 28.7%, 1.1%, 33.3%, and 34.5%, respectively.
Conclusions
It is feasible to perform H. pylori isolation and antimicrobial susceptibility testing using rapidly frozen and transported biopsy specimens.
INTRODUCTION
Since 1998, when regimens for Helicobacter pylori eradication were first recommended in Korea, triple therapy—consisting of a proton pump inhibitor, clarithromycin, and amoxicillin—has been the primary treatment choice [1-3]. However, the eradication rate with standard triple therapy has proven suboptimal [4]. A meta-analysis of first-line triple therapy in Korea indicated a significant decline in eradication rates [5]. Several factors contribute to this reduced efficacy, with the most critical being the increased resistance of H. pylori to antibiotics, especially clarithromycin. Studies assessing H. pylori resistance have typically been conducted in a limited number of metropolitan centers in Korea [6,7], suggesting potential regional differences in antibiotic resistance and corresponding eradication rates [8,9]. Therefore, it is important to employ standardized susceptibility testing methods to gather nationwide data on the antibiotic resistance of H. pylori clinical isolates.
Because most centers lack a microbiology laboratory capable of performing H. pylori isolation and antimicrobial susceptibility tests, transporting gastric biopsy specimens to a central laboratory is essential. However, transport time and temperature significantly impact the viability of H. pylori [10]. Several freezing methods have been reported for the long-term storage of H. pylori [11-14]. A previous study showed that the recovery rate of bacteria from biopsy specimens stored at -70°C for 2 to 8 weeks was 97% [12]. Long-term preservation of H. pylori can be achieved through cryopreservation. In one study, H. pylori was successfully isolated from 72.4% of patients whose gastric biopsy specimens had been stored at -70°C for over 10 years [13]. However, standardized methods for freezing and transporting H. pylori for long-term storage have not been established. Additionally, most studies in this area have been conducted in a single laboratory.
This study aimed to assess the feasibility of H. pylori isolation and antibiotic susceptibility testing by applying a standardized protocol for the rapid freezing of gastric biopsy specimens and their transport from various institutions to a central laboratory.
METHODS
Patients
Eight institutions across various regions of Korea—including Seoul, Gyeonggi, Gangwon, Busan, and Jeolla—participated in the study. From January to August 2016, patients requiring upper endoscopy and H. pylori testing were screened. Informed consent was obtained from all participants, and the study received approval from the Institutional Review Boards of all participating institutions.
Isolation of H. pylori strains
Two biopsy samples were collected from the greater curvature of the stomach. One sample was placed in a sterile Eppendorf tube and immediately stored in a vacuum bottle with dry ice, while the other was used for the rapid urease test. The Eppendorf tubes, stored in the vacuum bottle, were then placed in a -80°C deep freezer. Only patients with positive rapid urease results were enrolled in the study. Frozen samples from these enrolled patients were delivered to the Asan Institute for Life Sciences in Seoul, Korea.
Frozen biopsy specimens were thawed at room temperature and inoculated onto H. pylori isolation medium (Brucella broth agar supplemented with 7% sheep blood, vancomycin [10 mg/L], trimethoprim [5 mg/L], amphotericin B [5 mg/L], and polymyxin B [2.5 IU]). H. pylori strains were cultured under microaerophilic conditions (5% O2, 10% CO2, 85% N2). After initial isolation, a single colony from each biopsy specimen was subcultured and identified as H. pylori based on colony morphology, Gram staining, and urease reaction. The strains were preserved at -70°C in Brucella broth with 15% glycerol, thawed, and subcultured before use in subsequent experiments [15,16].
Determination of the minimum inhibitory concentration
H. pylori isolates were examined using the serial 2-fold agar dilution method, as previously described [16]. Briefly, bacteria were subcultured for 48 h on Mueller–Hinton agar supplemented with 5% defibrinated sheep blood. The bacterial suspension was adjusted to 1×107 colony-forming units and inoculated directly onto antibiotic-containing agar dilution plates. After 72 hours, the minimum inhibitory concentration (MIC) of each antibiotic was determined. The standard strain, H. pylori ATCC 43504, served as a control. Resistance breakpoints for amoxicillin, clarithromycin, metronidazole, tetracycline, levofloxacin, and ciprofloxacin were set at ≥0.5, >1, ≥8, ≥4, ≥1, and ≥1 μg/mL, respectively [7,17].
Statistical analysis
Categorical variables, expressed as numbers and percentages, were compared using the chi-square test. All statistical analyses were conducted with SPSS software (version 21.0; IBM Corp., Armonk, NY, USA). A p-value of less than 0.05 was considered statistically significant.
RESULTS
Baseline characteristics
From January to August 2016, 137 patients from eight institutions were screened, and 113 patients with positive rapid urease test results were enrolled. The baseline characteristics of these patients are summarized in Table 1.
Baseline characteristics of the study patients
Culture success rate
The median duration of tissue storage at -80°C was 79 days (range, 8–186 days). The overall culture success rate was 77.0% (87/113). Fig. 1 shows the culture success rates by institution, with institution 5 showing the lowest success rate at 42.9%. The culture success rate for patients without a history of H. pylori eradication was 79.4% (77/97), compared to 69.2% (9/13) for those with a history of eradication.
Culture success rates according to institution. Institution 1, 2, and 3 from Seoul; 4 and 8 from Gyeonggi; 5 from Gangwon; 6 from Busan; and 7 from Jeolla.
Prevalence of antibiotic resistance
MIC values were determined for 87 isolated H. pylori strains. The distribution of antibiotic MIC values is shown in Table 2. Resistance breakpoints for amoxicillin, clarithromycin, metronidazole, tetracycline, levofloxacin, and ciprofloxacin were set at ≥0.5, >1, ≥8, ≥4, ≥1, and ≥1 μg/mL, respectively [7,17]. The rates of resistance to amoxicillin, clarithromycin, metronidazole, tetracycline, levofloxacin, and ciprofloxacin were 23.0%, 25.3%, 28.7%, 1.1%, 33.3%, and 34.5%, respectively.
Distributions of antibiotic MIC for H. pylori isolates
DISCUSSION
The present study demonstrated that the overall H. pylori recovery rate from rapidly frozen biopsy specimens across various institutions in the country was 77.0%. This result is promising, considering the relatively low sensitivity of the culture methods employed [18-20].
Previous studies have reported good H. pylori recovery from frozen biopsy specimens [11,12,14]. Han et al. [11] achieved H. pylori isolation in 14 patients (87.5%) after 5 to 6 years of storage at -70°C; however, the recovery rate dropped to 57% after 12 weeks at -20°C. Heep et al. [12] reported a recovery rate of 97% when samples were stored in glycerol broth at -70°C for 2 to 8 weeks. The present study differs from these previous studies in that no transport medium was used. Previous studies utilized Stuart’s transport medium, Portagerm pylori, or cysteine-albumin broth supplemented with 20% glycerol for rapid freezing [11,12]. In this study, biopsy specimens were immediately placed in sterile Eppendorf tubes without medium and then rapidly frozen. This approach resulted in a high recovery rate without the need for a transport medium.
Different institutions reported varying culture success rates. The recovery rate at Institution 1 was 100% (15/15), while at Institution 5, it was 42.9% (9/21). Therefore, we reviewed the sample processing procedures at Institution 5. Eppendorf tubes containing biopsy tissue were stored in a standard refrigerator (4°C) for several hours before being transferred to a vacuum bottle with dry ice. This deviation from the standard protocol raises particular concerns, as the biopsy samples were not immersed in a protective transport medium during the interim storage period. The lack of such a medium, combined with the relatively warm temperature of 4°C, likely resulted in cellular degradation and metabolic changes. Consequently, it can be assumed that improper initial handling contributed significantly to the low culture success rates. A study showed that when the transfer time exceeded 4 h, isolating H. pylori from biopsy specimens became difficult, even when the temperature was maintained at 4°C [21]. Recently, a large-scale multicenter study involving 66452 patients from 26 hospitals in China was conducted. Gastric mucosal biopsy specimens were transferred to a central laboratory at ambient temperatures, with transport times ranging from 5 to 72 h and temperatures varying between 4.7°C and 29.1°C. The specimens were categorized into three groups based on transport time: 5 h, 24 h, and 48 h. The culture rate for the 24-h transport group was 32.8%, and for the 48-h group, it was 26.3% (p<0.001) [22]. Thus, optimal recovery of H. pylori requires that biopsy specimens be cultured as soon as possible. However, transferring all biopsy specimens to a laboratory within 24 h can be challenging. To ensure optimal bacterial recovery, we believe that rapidly freezing biopsy specimens before transfer to the central laboratory is preferable.
After H. pylori eradication, the culture success rate in post-treatment biopsy samples tended to decrease in the present study. This reduction in culture positivity could be attributed to several factors, including the effect of antibiotics used in eradication therapy, which may suppress the bacterial load to undetectable levels. Further studies are needed to understand this mechanism better.
Understanding local patterns of antibiotic resistance is essential for selecting the most appropriate first-line treatment. Several centers in Korea, primarily single centers in Seoul and the Gyeonggi area, have documented antibiotic resistance rates [6,7,9,16,23,24]. However, resistance patterns may differ in other regions. For example, a previous study in Japan found that the prevalence of metronidazole resistance was significantly higher in Kyoto (23.8%) than in Sapporo (8.1%) [8]. Similarly, the prevalence of clarithromycin resistance in H. pylori strains isolated from Korean patients in the Gyeonggi area between 2009 and 2012 was 37% [6], whereas another study from Seoul reported lower resistance rates (8.5%), leading to higher eradication rates (84.9%) with clarithromycin-based triple therapy [9]. These results suggest the need for H. pylori resistance analysis at both nationwide and regional levels using standardized susceptibility testing. The agar dilution method is recommended for measuring the MIC of antibiotics against H. pylori. However, few centers have laboratories equipped to isolate and test H. pylori using the agar dilution method. Therefore, effective transport methods are essential to obtaining meaningful nationwide antibiotic susceptibility data.
This study had several limitations. First, we did not observe regional differences in antibiotic susceptibility, as only a small number of patients from each region were included. However, the purpose of this study was not to identify regional differences in antibiotic susceptibility but to confirm the feasibility of antibiotic susceptibility testing using rapidly frozen gastric biopsy specimens. A nationwide multicenter study with a larger patient cohort is currently underway to investigate regional differences in antibiotic susceptibility. Another possible limitation is the variation in culture success rates reported across institutions. Although this study was conducted prospectively with a standardized protocol, we identified protocol deviations that could explain the differing culture success rates (ranging from 42.9% to 100%). Despite these limitations, this was the first prospective study to evaluate the feasibility of H. pylori isolation and antibiotic susceptibility testing using rapidly frozen gastric biopsy specimens collected from multiple institutions.
In conclusion, the isolation and antimicrobial susceptibility testing of H. pylori using frozen and transported biopsy specimens is both feasible and effective. This method can facilitate the collection of nationwide antibiotic resistance data, which can then inform the development of empirical regimens for H. pylori eradication.
Notes
Availability of Data and Material
The datasets generated or analyzed during the study are available from the corresponding author on reasonable request.
Conflicts of Interest
Soo-Jeong Cho, the Editor-in-Chief of the Korean Journal of Helicobacter and Upper Gastrointestinal Research, was not involved in the editorial evaluation or decision to publish this article. All remaining authors have declared no conflicts of interest.
Funding Statement
This study was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (grant number: HI15C1234).
Authors’ Contribution
Conceptualization: Kee Don Choi, Jung Mogg Kim, Hwoon-Yong Jung. Data curation: Kee Don Choi, Gwang Ho Baik, Jun Chul Park, Hye-Kyung Jung, Han Seung Ryu, Soo-Jeong Cho, Cheol Min Shin, Hwoon-Yong Jung. Formal analysis: Kee Don Choi. Funding acquisition: Jung Mogg Kim, Hwoon-Yong Jung. Investigation: Kee Don Choi, Jung Mogg Kim. Methodology: Jung Mogg Kim. Project administration: Hwoon-Yong Jung. Resources: Jung Mogg Kim, Hwoon-Yong Jung. Software: Kee Don Choi. Supervision: Jung Mogg Kim, Hwoon-Yong Jung. Validation: Jung Mogg Kim, Hwoon-Yong Jung. Visualization: Kee Don Choi. Writing—original draft: Kee Don Choi. Writing—review & editing: Jung Mogg Kim, Hwoon-Yong Jung. Approval of final manuscript: all authors.
Acknowledgements
None