Clarithromycin resistance in Seattle

From the Salama Lab, Human Biology Division

Almost four decades ago, Warren and Marshall reported the first successful cultivation of the human pathogen Helicobacter pylori from gastric biopsy samples. Dr. Barry Marshall obtained incontrovertible proof of H. pylori pathogenicity through studies that would be impossible to replicate today. Through self-ingestion experiments, Marshall showed that this bacterium truly caused gastroduodenal disorders, including gastric ulcers. For their discovery, Warren and Marshall were awarded the Nobel Prize for Physiology or Medicine in 2005.  Their work revolutionized the treatment of uncurable gastroduodenal disorders, such as peptic ulcer disease, from an uncertain, chronic ailment into a curable infectious disease. Today, the H. pylori bacterium is known to cause acute and chronic gastritis, and also contributes to gastric carcinoma and gastric lymphoma. Furthermore, this bacterium can be found in the stomachs of about half of the world's population, accentuating the need for effective treatment strategies.

Antibiotics can be used to kill H. pylori, however many of the antibiotics that the bacterium is sensitive to in vitro are not used in human patients.  Patients are typically given clarithromycin (a ribosomal protein synthesis inhibitor) along with amoxicillin (a bacterial cell-wall biosynthesis inhibitor) and a proton-pump inhibitor (PPI), as first-line treatment.  The high frequency of antimicrobial therapy, together with the limited choice of antibiotics, has resulted in the development of widespread antibiotic resistance in H. pylori, which substantially impairs the treatment of patients infected with the bacterium.  Despite the rising prevalence of antibiotic resistance, patients are generally not tested for antibiotic resistance before prescription of antibiotics, largely due to the difficulty of obtaining separate gastric biopsies for bacterial culture. Although current guidelines advise against the use clarithromycin, amoxicillin and PPI when at least 20% of the local population has clarithromycin resistance, local patterns of antibiotic resistance are often unknown, leaving clinicians with no choice but to prescribe antibiotics empirically.

To overcome these pressing limitations to effective treatment, Dr. Nina Salama’s Lab in the Human Biology Division, along with their colleagues at the University of Washington, developed a culture-free method to detect antibiotic resistance, and used this to retrospectively measure local resistance patterns in a patient population.  Using digital droplet polymerase chain reaction (ddPCR), a precise, sensitive and highly quantitative assay that partitions the PCR reaction into thousands of smaller reactions within separate droplets, the authors showed that mutations that confer resistance to clarithromycin can be accurately detected from DNA extracted from previously archived, formalin-fixed paraffin-embedded (FFPE) gastric tissue samples.  These point mutations (A2142G, A2142C and A2143G) occur in the 23S ribosomal RNA gene and account for >90% of resistance to clarithromycin by altering its ability to bind to the ribosome and inhibit bacterial protein synthesis. Using multiple fluorescent probes in the ddPCR assay, the authors were able to distinguish and quantify the wild-type from the mutant 23S rDNA alleles, allowing for population resistance rates to be determined retrospectively and bypassing the need for H. pylori bacterial culture.

The authors conducted the clarithromycin resistance ddPCR assay on archived FFPE gastric tissue samples from 110 H. pylori-positive patients at the University of Washington Medical Center.  The results of this study were recently published in Helicobacter, and showed a 44% clarithromycin resistance rate among the 102 patients in which the H.pylori 23S rDNA was detected.  The authors further ascertained that the amount of gastric tissue used had no effect on the detection level of 23S rDNA. Of the 45 patients that had clarithromycin resistant mutations, 33 had a mix of both wild-type and mutant alleles.  The researchers then looked closely at different aspects of the patient population. While the age and sex of patients were not considerably different in patients with wild-type versus resistance alleles and resistance rates were definitely above 20% across all racial groups, clarithromycin resistance was found to be highest among Asians, at 67% compared to 38% in non-Asian patients.  The authors speculate that this observation may stem from a high representation of foreign-born patients their study population, who come from countries with a higher prevalence of clarithromycin resistance.


Seattle patient population graph
Clarithromycin resistance in 102 patients within the Seattle area with detectable 23S rRNA. Prevalance of antibiotic resistance was highest among Asians. Figure by Yiting Lim

Since culture-based sensitivity was not performed for these patients, the authors were unable to compare their ddPCR assay to the traditional culture-based method.  Nevertheless, the Salama lab and their colleagues established a sensitive assay using ddPCR that can detect resistance mutations even if present in small proportions, and revealed that alternative treatment regimes should be used in the Seattle patient population as first-line treatment for H.pylori infection due to the high prevalence of clarithromycin resistance.

Antibiotic resistance is a very pressing problem, and this exciting platform can help facilitate solutions. Dr. Nina Salama revealed: “We are developing similar assays to measure resistance mutations for other antibiotics. We also want to do studies where we have pre-treatment samples to test for presence of resistance alleles coupled with treatment outcome data.”

Talarico S, Korson AS, Leverich CK, Park S, Jalikis FG, Upton MP, Broussard E, Salama NR. 2018. High prevalence of Helicobacter pylori clarithromycin resistance mutations among Seattle patients measured by droplet digital PCR. Helicobacter. Apr;23(2):e12472

Funding was provided by the National Institutes of Health.

This study was a Cancer Consortium collaboration between Nina Salama (Fred Hutch) and Elizabeth Broussard (UW).