Novel chromogenic medium-based method for the rapid detection of Helicobacter pylori drug resistance

Abstract

BACKGROUND

Helicobacter pylori (H. pylori), a globally prevalent pathogen, is exhibiting increasing rates of antimicrobial resistance. However, clinical implementation of pre-treatment susceptibility testing remains limited due to the organism’s fastidious growth requirements and prolonged culture time.

AIM

To propose a novel detection method utilizing antibiotic-supplemented media to inhibit susceptible strains, while resistant isolates were identified through urease-mediated hydrolysis of urea, inducing a phenol red color change for visual confirmation.

METHODS

Colombia agar was supplemented with urea, phenol red, and nickel chloride, and the final pH was adjusted to 7.35. Antibiotic-selective media were prepared by incorporating amoxicillin (0.5 μg/mL), clarithromycin (2 μg/mL), metronidazole (8 μg/mL), or levofloxacin (2 μg/mL) into separate batches. Gastric antral biopsies were homogenized and inoculated at 1.0 × 10⁵ CFU onto the media, and then incubated under microaerobic conditions at 37 °C for 28-36 hours. Resistance was determined based on a color change from yellow to pink, and the results were validated via broth microdilution according to Clinical and Laboratory Standards Institute guidelines.

RESULTS

After 28-36 hours of incubation, the drug-resistant H. pylori isolates induced a light red color change in the medium. Conversely, susceptible strains (H. pylori 26695 and G27) produced no visible color change. Compared with the conventional 11-day protocol, the novel method significantly reduced detection time. Among 201 clinical isolates, 182 were successfully evaluated using the new method, resulting in a 90.5% detection rate. This was consistent with the 95.5% agreement rate observed when compared with microdilution-based susceptibility testing. The success rate of the novel approach was significantly higher than that of the comparative method (P < 0.01). The accuracy of the new method was comparable to that of the dilution method.

CONCLUSION

The novel detection method can rapidly detect H. pylori drug resistance within 28-36 hours. With its operational simplicity and high diagnostic performance, it holds strong potential for clinical application in the management of H. pylori antimicrobial resistance.

Key Words: Helicobacter pylori; Drug resistance; Antibiotic susceptibility testing; Chromogenic medium; Rapid detection method

Core Tip: Helicobacter pylori (H. pylori) drug resistance hinders clinical treatment, with slow, inaccurate detection prolonging therapy and reducing efficacy. This paper presents a novel rapid detection method for H. pylori resistance, identifying it in 28-36 hours - much faster than traditional approaches. It offers high accuracy, reliable resistance assessment, and easy operation, suitable for wide use in clinics and research. This method shows great potential to optimize clinical decisions, improve outcomes, and aid H. pylori infection management.

INTRODUCTION

Helicobacter pylori (H. pylori) is a microaerophilic, gram-negative bacterium[1], which has infected more than 50% of the world population[2]. Once an infection with H. pylori occurs, it can lead to gastritis, gastric cancer, and other diseases, posing a serious threat to human health[3]. Current treatment guidelines recommend quadruple therapy, which includes a proton pump inhibitor, bismuth, and two antibiotics[4]. However, with the widespread use of antibiotics, resistance in H. pylori has become increasingly problematic, particularly against metronidazole (MTZ) and clarithromycin (CLR), and resistance rates were reported as high as 71% and 55%, respectively[5,6]. In some areas, the resistance rate for MTZ has reached up to 81.7%[7], resulting in reduced eradication rates. Antimicrobial susceptibility testing is crucial for guiding effective therapies to address the growing challenges associated with drug resistance[8,9]. Currently, the primary methods for detecting H. pylori resistance involve classical culture-based methods. These methods require the collection of gastric mucosal biopsies, followed by isolation and culture to obtain pure bacterial strains. Resistance is therefore assessed using methods, such as the broth or agar dilution method and the Kirby-Bauer (disc diffusion) test[10]. However, such detection methods typically require 11-12 days to complete and are susceptible to procedural errors[11], which may compromise clinical decision-making An alternative strategy involves direct polymerase chain reaction (PCR) amplification of resistance genes from gastric mucosa or fecal samples An alternative strategy involves direct PCR amplification of resistance genes from gastric mucosa or fecal samples[12]. Although this approach is more rapid, the genotypic diversity of resistant H. pylori strains mainly does not correspond fully with phenotypic resistance[13], leading to false-negative results. For instance, CLR resistance in H. pylori is commonly associated with mutations at the A2142G and A2143G sites[14], while resistant strains without these mutations have also been identified[15]. Moreover, mutations in alternative genomic regions can lead to misinterpretation of resistance status. High-throughput genetic testing is conducted by examining stool samples to avoid invasive procedures, while the coincidence rate between genotypes from stool and biopsy samples is approximately 89%[16], indicating limited reliability. Therefore, resistance testing is recommended only for patients with treatment failure, and it remains underutilized. Some scholars have proposed the direct use of biopsy tissues for drug susceptibility testing[17], while the accuracy of this method needs further improvement. Consequently, the development of rapid, accurate, and clinically applicable methods for detecting H. pylori drug resistance essential. This study developed a colorimetric detection method using a specialized culture medium designed to identify H. pylori drug resistance. The approach determined the optimal antibiotic concentrations required to inhibit bacterial growth. In cases where drug-resistant strains proliferate, urease activity increases the medium’s alkalinity, resulting in a color change of phenol red. This change enables rapid identification of resistance. The method appeared fast, simple, accurate, and cost-effective, providing technical support for the clinical application of H. pylori drug resistance testing.

MATERIALS AND METHODS

Bacterial resuscitation, culture and passage

The H. pylori strains, including susceptible strains G27 and 26695, as well as resistant strains 159, 161, 162, 163, 286, 287, 289, and 290, were kindly provided by Professor Hong-Kai Bi from Nanjing Medical University. Furthermore, 201 clinical strains were collected from the Endoscopy Center of the Affiliated Hospital of Youjiang Medical University for Nationalities. All strains were retrieved from a -80 °C ultra-low temperature freezer. The bacterial pellets were resuspended in a Columbia medium (OXOID, Lot: 2179850) and incubated in a tri-gas incubator (85% N₂, 5% O₂, 10% CO₂) at 37 °C for resuscitation. Colonies in the logarithmic growth phase were selected for subculturing.

Determination of the minimum inhibitory concentration of H. pylori

Amoxicillin (AMX) (Lot: C15338727), CLR (Lot: C114967375), MTZ (Lot: C11.594013), and levofloxacin (LVX) (Lot: C10619762) were each prepared at a concentration of 1 mg/mL for subsequent use. In the first well of a 96-well microtiter plate, 173.6 μL of brain heart infusion (BHI) medium (OXOID, Lot: 7075659) was added, followed by the addition of 6.4 μL of the antimicrobial drug. Serial two-fold dilutions were performed from wells 1 to 11 to achieve final drug concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.3125 μg/mL. The 12^th well served as a drug-free control. A bacterial suspension was prepared by adjusting the OD₆₀₀ to 0.3 (approximately 1 × 10⁸ CFU/mL), followed by a 10-fold dilution. Ten microliters of the diluted suspension were added to wells 1-10 and 12, yielding a final concentration of 1 × 10⁶ CFUs/mL. Well 11, containing only sterile water and medium, served as the no-bacteria control. The plate was incubated for 72 hours under microaerophilic conditions. Negative controls (containing only sterile water, medium, and drug) and positive controls (containing only medium and bacteria) were included. The minimum inhibitory concentration (MIC) was defined as the lowest antibiotic concentration that completely inhibited visible bacterial growth. If a skipped well was observed, the MIC was determined based on the highest concentration showing complete inhibition.

Estimating the breakpoints for H. pylori resistance (1 × 10⁵ CFUs/mL)

Evaluation of drug resistance of H. pylori and calculation of the breakpoint range: To assess the breakpoint range, H. pylori suspensions were prepared at concentrations of 1 × 10⁶, 1 × 10⁵, and 1 × 10⁴ CFU/mL. Drug susceptibility to LVX, CLR, MTZ, and AMX was evaluated using the MIC determination method. The fold differences in MICs between bacterial concentrations were calculated to estimate breakpoint variability. According to the Clinical and Laboratory Standards Institute (CLSI) guidelines, the resistance breakpoints for H. pylori at 1 ×10⁶ CFU/mL were defined as 2 μg/mL for LVX, 2 μg/mL for CLR, 8 μg/mL for MTZ, and 0.5 μg/mL for AMX. Then, the range of resistance breakpoints was determined for a bacterial dose of 1 × 10⁵ CFUs.

Verification of the estimated breakpoint value: According to the estimated range of drug resistance breakpoints, Columbia agar was prepared with twofold serial concentrations of each antibiotic to create a solid medium gradient. H. pylori was suspended in BHI broth and adjusted to 1 × 10⁸ CFU/mL, followed by a 10-fold dilution. Ten microliters of the diluted suspension (final concentration 1 × 10⁷ CFU/mL) were inoculated onto each antibiotic-containing medium. Plates were incubated at 37 °C for 3-5 days in a tri-gas incubator (85% N₂, 5% O₂, 10% CO₂). Bacterial growth was monitored and recorded to verify the accuracy of the estimated resistance breakpoints.

Preparation of characteristic media

Preparation of solutions: The following stock solutions were prepared, sterilized, and stored at -20 °C for future use: 10 mL of 0.2% phenol red solution (Lot: SHBM7900), 50 mL of 20% urea solution (Lot: C15338727), 10 mL of 5% NaOH solution (Lot: 190826978E), and 10 mL of 100 μmol/L nickel chloride (NiCl₂) solution (Lot: C12077718).

Determining the urea concentration in the characteristic medium: Columbia medium (2.0 g) was dissolved in 45 mL of purified water, transferred to a conical flask, and sterilized. After cooling to approximately 55 °C, 7 mL of calf serum (Lot: PRXNXQ-500X) and 1% (v/v) of H. pylori selective supplement (Dent additive) were added. Solid media were subsequently prepared with urea concentrations of 0.6 mg/mL, 1.2 mg/mL, and 2.4 mg/mL (scheme 1). H. pylori suspensions (1 × 10⁵ CFU/mL) were inoculated onto the prepared media and incubated at 37 °C in a tri-gas incubator (85% N₂, 5% O₂, 10% CO₂) for 3-5 days. The results were recorded to evaluate the optimal urea concentration.

Determining the phenol red concentration in the characteristic medium: In scheme 1, the phenol red solution was added at final concentrations of 0.2 mg/100 mL, 0.4 mg/100 mL, 0.8 mg/100 mL, and 1.6 mg/100 mL, and the solutions were thoroughly mixed. Solid media (scheme 2) were subsequently prepared, and 1 × 10⁵ CFUs/mL of H. pylori were inoculated onto the media from scheme 2. After incubation for 3-5 days, bacterial growth and medium discoloration were evaluated to determine the optimal phenol red concentration.

Adjusting the pH value: As presented in scheme 2, the final pH of the media was adjusted to 7.15, 7.25, 7.35, or 7.45. Solid media (scheme 3) were prepared, inoculated with 1 × 10⁵ CFUs/mL of H. pylori, and incubated for 3-5 days. Observations of bacterial growth and time to color change were utilized to identify the optimal pH for the characteristic medium.

Determination of NiCl₂ concentration in the characteristic medium: In scheme 3, NiCl₂ solution was added at concentrations of 1 mmol/L, 10 mmol/L, 100 mmol/L, and 1000 mmol/L, which were thoroughly mixed to form a solid medium (scheme 4). Subsequently, 1 × 10⁵ CFUs/mL of H. pylori were inoculated onto the medium prepared in Scheme 4 and incubated for 3-5 days. During this period, a degree of medium discoloration was recorded to evaluate the effect of NiCl₂ concentration.

Preparation of a set of characteristic media: A H. pylori selective supplement (Dent) was added to Columbia agar medium to create the base formulation, which was designated as Medium A. Medium B was prepared according to the optimized conditions outlined in scheme 4, incorporating phenol red, urea, NaOH, and NiCl₂ at the determined optimal concentrations. Based on medium B, LVX (2 μg/mL), CLR (2 μg/mL), MTZ (8 μg/mL), and AMX (0.5 μg/mL) were added to generate medium C, D, E, and F, respectively. This complete set of characteristic media was assembled for the detection of H. pylori drug resistance (Figure 1). Each medium was inoculated with 10 μL of a bacterial suspension containing 1 × 10⁵ CFUs/mL of H. pylori and incubated at 37 °C for 3-5 days. Bacterial growth and color change of the medium were recorded to assess antimicrobial susceptibility.

Figure 1 Schematic illustration of the rapid detection of antibiotic resistance in Helicobacter pylori. For a given bacterial strain, its resistance to specific antibiotics is determined by the color change of the medium. The yellowish color of the medium indicates that no Helicobacter pylori growth has occurred, whereas the red color indicates the presence of Helicobacter pylori growth. In particular, the appearance of a red color in the medium was employed as an indicator of bacterial resistance. A: It represents Columbia medium; B: It stands for characteristic medium without antibiotics; C: It denotes characteristic medium with levofloxacin; D: It indicates characteristic medium with clarithromycin; E: It refers to characteristic medium with metronidazole; F: It symbolizes characteristic medium with amoxicillin. H. pylori: Helicobacter pylori.

Detection of laboratory strain resistance using characteristic media

Standard H. pylori strain G27 and resistant strains 159, 286, 287, and 290 were harvested and prepared as bacterial suspensions. For each strain, 10 μL of the working suspension (1 × 10⁵ CFUs/mL) was inoculated onto each type of characteristic medium (Figure 1). At designated time points (24 hours, 28 hours, 32 hours, 36 hours, 42 hours, 48 hours, and 72 hours), media plates were removed from the incubator and left at room temperature for 30 minutes to allow for optimal visualization. The presence or absence of bacterial growth and changes in medium coloration were recorded to evaluate resistance profiles.

Detection of drug resistance in clinical samples using characteristic media

Preparation of the transfer solution: The transfer solution was prepared by combining BHI, glycerol (30% of the total volume), and H. pylori selective supplement (1% of the total volume). In this experiment, 0.5 milliliter of this transfer solution was placed in a sterile EP tube, to which 6-8 sterile steel beads were, added for later use.

Selection of clinical samples: After obtaining approval from the Ethics Review Committee of the Affiliated Hospital of Youjiang University for Nationalities, patients were selected based on the following criteria: History of receiving antimicrobial drugs, availability of a positive 14c-urea breath test, had stopped taking bismuth or antibiotics for 4 weeks, had stopped taking proton pump inhibitor for 2 weeks, and signing the relevant informed consent form.

Specimen collection: After obtaining the patient’s consent (2024073001), during gastroscopy at the Digestive Endoscopy Center of the Affiliated Hospital of Youjiang Medical University for Nationalities, one tissue sample was collected from both the antrum and gastric body. In cases where lesions with erosion or ulcers were observed, two biopsy samples were obtained. These samples were aseptically transferred to sterile EP tubes and placed on ice, and the subsequent steps were completed as promptly as possible (within 2 hours), ensuring strict aseptic conditions throughout the experiment.

Preparation of the H. pylori suspension: The biopsy tissue in the EP tube was homogenized using a tissue disruptor (300 Hz for 5 minutes), then transferred to a sterile EP tube, and centrifuged at 12000 rpm for 2 minutes. The resulting pellet was resuspended in 200 μL of BHI to prepare a bacterial suspension. The concentration of H. pylori bacteria in the suspension was determined using H. pylori test strips (Supplementary Figure 1), and the concentration was adjusted to approximately 1 × 10⁷ CFUs/mL.

Inoculation and incubation: A total of 10 μL (containing approximately 1 × 10⁵ CFUs) of the H. pylori suspension was added dropwise to a set of characteristic media. These media were subsequently incubated for 28-36 hours, removed, and incubated at room temperature for 30 minutes before the results were interpreted.

Verification of strain resistance using classical culture-based methods

For the same clinical, an additional 100 μL of the bacterial suspension was simultaneously inoculated onto antibiotic-free Columbia agar and cultured using conventional methods. Identification tests, including urease, oxidase, and catalase assays, along with PCR analysis, confirmed the isolated bacteria as H. pylori. Following this confirmation, the MIC test was performed to determine whether H. pylori had developed drug resistance.

RESULTS

Estimating antibiotic resistance breakpoints

Various concentrations of H. pylori, including both susceptible and resistant strains, were tested to assess their sensitivity to LVX, CLR, MTZ, and AMX. As the bacterial load decreased from 1 × 10⁶ CFUs/mL to 1 × 10⁵ CFUs/mL, the MICs decreased by 1-4 times (Table 1). According to the CLSI guidelines, when the bacterial load was 1 × 10⁶ CFUs/mL, the resistance breakpoints of H. pylori against these four drugs were set at 2, 2 μg/mL, 8 μg/mL, and 0.5 μg/mL, respectively. Correspondingly, when the bacterial load was reduced to 1 × 10⁵ CFUs/mL, the resistance breakpoints for H. pylori are expected to fall within the following ranges: 0.5-2 μg/mL for LVX and CLR, 1-8 μg/mL for MTZ, and 0.06-0.5 μg/mL for AMX.

Table 1 Minimum inhibitory concentration of Helicobacter pylori determined via the microdilution method (μg/mL).

H. pylori	Counts (CFUs)	LVX	CLR	MTZ	AMX
G27	106	0.1250	0.0625	0.5	0.0313
	105	0.0625	0.0312	0.5	0.0156
	104	0.0312	0.0156	0.125	0.0078
26695	106	0.1250	0.1250	0.5	0.0625
	105	0.0625	0.0625	0.25	0.0325
	104	0.0312	0.0312	0.125	0.0153
159	106	2	2	4	0.1250
	105	1	2	2	0.0625
	104	0.5	1	0.5	0.0313
161	106	2	2	4	0.1250
	105	2	1	2	0.0625
	104	1	0.5	1	0.0313
162	106	8	8	2	0.1250
	105	4	4	1	0.0625
	104	2	1	0.5	0.03125
163	106	16	0.5	16	1
	105	4	0.25	4	0.5
	104	2	0.125	2	0.25
286	106	1	8	32	1
	105	0.5	4	16	0.5
	104	0.5	1	4	0.5
287	106	4	4	32	0.25
	105	2	2	16	0.1250
	104	1	8	8	0.0625
289	106	8	32	32	1
	105	4	8	16	0.5
	104	2	2	8	0.125
290	106	32	16	32	4
	105	32	4	16	2
	104	16	4	4	1

Helicobacter pylori: H. pylori; LVX: Levofloxacin; CLR: Clarithromycin; MTZ: Metronidazole; AMX: Amoxicillin.

Verification of antibiotic resistance breakpoints

LVX (0.5 μg/mL), CLR (0.5 μg/mL), MTZ (4 μg/mL), and AMX (0.06 μg/mL) were added to the solid medium. Under these conditions, the growth of sensitive H. pylori strains (1 × 10⁵ CFU) was effectively inhibited, whereas drug-resistant strains were not suppressed (Table 2).

Table 2 Breakpoints for verifying antibiotic concentrations (μg/mL).

H. pylori	LVX				CLR				MTZ				AMX
	0.25	0.5	1	2	0.25	0.5	1	2	1	2	4	8	0.03	0.06	0.13	0.25
G27	+	-	-	-	+	-	-	+	+	+	-	-	+	-	-	-
26695	+	-	-	-	+	-	-	+	+	+	-	-	+	-	-	-
159	+	+	-	-	+	+	-	+	+	+	+	-	+	+	-	-
161	+	+	-	-	+	+	-	+	+	+	+	-	+	+	-	-
162	+	+	-	-	+	+	-	+	+	+	-	-	+	-	-	-
163	+	+	-	-	-	-	-	+	+	+	+	-	+	+	-	-
286	+	+	-	-	+	+	-	+	+	+	+	-	+	+	-	-
287	+	+	-	-	+	+	-	+	+	+	+	-	+	+	+	-
289	+	+	+	-	+	+	+	+	+	+	+	-	+	+.	-	-
290	+	+	+	-	+	+	-	+	+	+	+	+	+	+	-	-

By adding antibiotics with certain concentrations to the Columbia medium, sensitive and resistant strains (working concentration of (1 × 10⁵ CFUs/mL) could be cultured. Adding 0.5 μg/mL of levofloxacin, 0.5 μg/mL of clarithromycin, 4 μg/mL of metronidazole, and 0.06 μg/mL of amoxicillin could inhibit the growth of sensitive bacteria (G27, 26695), while hardly affect the growth of resistant bacteria (159, 161, 162, 163, 286, 287, 289, 290). Helicobacter pylori: H. pylori; LVX: Levofloxacin; CLR: Clarithromycin; MTZ: Metronidazole; AMX: Amoxicillin.

Screening the optimal concentrations of urea and phenol red

The addition of 0.60 mg/mL urea did not impede the growth of H. pylori. However, at concentrations between 1.2 mg/mL and 2.4 mg/mL, H. pylori (1 × 10⁵ CFU/mL) exhibited poor growth. In the 0.60-1.2 mg/mL range, further evaluation of 0.8 mg/mL, 0.9 mg/mL, and 1.0 mg/mL urea revealed that 0.8 mg/mL was the optimal concentration (Table 3). When 0.8 mg/100 mL phenol red was added to the medium along with urea, it had no inhibitory or stimulatory effect on H. pylori growth. While it provided optimal colorimetric recognition, its sensitivity was limited. Similarly, 0.2 mg/100 mL phenol red had no influence on H. pylori growth, while both recognition and sensitivity were suboptimal. Finally, 0.4 mg/100 mL phenol red had no adverse effect on H. pylori growth and demonstrated notable recognition and sensitivity (Figure 2, Table 3).

Figure 2 The discoloration reaction observed in Helicobacter pylori cultures following the addition of urea and phenol red. 0.2-0.8 mg/100 mL phenol red had no influence on Helicobacter pylori growth. However, when 0.2 mg/100 mL phenol red was added to the medium, the cultured Helicobacter pylori (1 × 105 CFUs/mL) showed minimal color change. The medium turned light red with the addition of 40 mg, and dark red with 80 mg.

Table 3 Effects of different urea and phenol red concentrations on the growth of Helicobacter pylori.

H. pylori	Urea (mg/mL)						Phenol red (mg/100 mL)
	0.6	0.8	0.9	1	1.2	2.4	0.2	0.4	0.8	1.6
G27	++	++	+	+	+	-	2	3	1	1
26695	++	++	+	+	+	-	2	3	1	1
159	++	++	+	+	+	-	2	3	1	1
161	++	++	+	+	+	-	2	3	1	1
162	++	++	+	+	+	-	2	3	1	1
163	++	++	+	+	+	-	2	3	1	1
286	++	++	+	+	+	-	2	3	1	1
287	++	++	+	+	+	-	2	3	1	1
289	++	++	+	+	+	-	2	3	1	1
290	++	++	+	+	+	-	2	3	1	1

1: Indicates that bacteria can grow, with remarkable discoloration recognition, while exhibiting poor sensitivity; 2: Indicates bacterial growth with notable discoloration recognition, while with relatively poor sensitivity; 3: Represents bacterial growth with excellent discoloration and high sensitivity, for the discoloration and sensitivity assessments; +: Indicates that bacteria can grow, while their growth is affected to a certain extent; ++: Refers to the medium with urea added to the culture of Helicobacter pylori (1 × 10⁵ CFUs), where bacterial growth is not affected or has little impact; -: Signifies that bacteria are affected and do not grow. Helicobacter pylori: H. pylori.

Screening the optimal pH and NiCl₂ concentration

The sensitivity and visibility of the color change reaction were further enhanced by adjusting the pH of the medium to 7.15-7.35 while maintaining a urea concentration of 0.8 mg/mL and a phenol red concentration of 0.4 mg/100 mL. Various concentrations of NiCl₂ were subsequently introduced, resulting in a more notable discoloration reaction. In contrast, NiCl₂ did not alter the MIC value (Supplementary Table 1). The optimal outcome was achieved when the NiCl₂ concentration was set to 100 mmol/L (Figure 3, Table 4).

Figure 3 The effects of nickel chloride on the degree of discoloration of the medium. Nickel chloride can promote urease decomposition, phenol red color is more obvious, and it has no effect on bacterial growth and drug resistance. NiCl₂: Nickel chloride.

Table 4 Effects of pH value and nickel chloride concentration on the growth of Helicobacter pylori and the discoloration of media.

H. pylori	pH			NiCl2 (mmol/L)
	7.15	7.25	7.35	1	10	100
G27	1	1	2	+	+	++
26695	1	1	2	+	+	++
159	1	1	2	+	+	++
161	1	1	2	+	+	++
162	1	1	2	+	+	++
163	1	1	2	+	+	++
286	1	1	1	+	+	++
287	1	1	2	+	+	++
289	1	1	2	+	+	++
290	1	1	2	+	+	++

1: Indicates that the bacterial culture is viable and grows, while the medium shows no significant discoloration after 24 hours; 2: Indicates that the bacterial culture is viable and grows, and the medium exhibits obvious discoloration after 24 hours of incubation; +: Indicates slight discoloration; ++: Signifies obvious discoloration; NiCl₂: Nickel chloride; Helicobacter pylori: H. pylori.

Specialty media validation

The effects of different concentrations of antibiotics on the growth of H. pylori (sensitive and resistant strains) were consistent between the Columbia medium and the specialty medium. However, the specialty medium incorporated a colorimetric reaction not present in the Columbia medium. Additionally, compared with the antibiotic-free specialty medium, the presence of antibiotics extended the reaction time from approximately 28 hours to around 36 hours (Table 5). Orthogonal experimental design was employed to validate the optimal formulation of the specialty medium (Supplementary Table 2).

Table 5 Comparison of minimum inhibitory concentrations between Columbia medium and characteristic medium (μg/mL).

H. pylori	Columbia medium				Characteristic medium
	LVX	CLR	MTZ	AMX	LVX	CLR	MTZ	AMX
	0.5	0.5	4	0.0625	0.5	0.5	4	0.0625
G27	-	-	-	-	×	×	×	×
26695	-	-	-	-	×	×	×	×
159	+	+	+	+	√	√	√	√
161	+	+	+	+	√	√	√	√
162	+	+	+	+	√	√	√	√
163	+	+	+	+	√	√	√	√
286	+	+	+	+	√	√	√	√
287	+	+	+	+	√	√	√	√
289	+	+	+	+	√	√	√	√
290	+	+	+	+	√	√	√	√

+: Indicates bacterial growth; -: Signifies the absence of bacterial growth; ×: Denotes no bacterial growth and no color change in the medium after 72 hours of incubation. √: Indicates a discoloration observed in the medium after 28-36 hours of incubation. Notably, Helicobacter pylori growth was confirmed after 72 hours of culture. LVX: Levofloxacin; CLR: Clarithromycin; MTZ: Metronidazole; AMX: Amoxicillin; Helicobacter pylori: H. pylori.

Detection of H. pylori in characteristic media

When H. pylori strain G27 at a concentration of 1 × 10⁵ CFU/mL was incubated in the antibiotic-free characteristic medium containing phenol red for 28-36 hours, the medium turned red. However, no color change was found in the presence of antibiotics (LVX, CLR, MTZ, and AMX, Figure 4), indicating that G27 was sensitive to these antibiotics based on the characteristic medium assay. This result was consistent with findings from the microdilution method. Similarly, H. pylori strain 26695 showed concordant results (Supplementary Figure 2). Strain 159 exhibited a color change in media containing CLR, MTZ, and LVX, while showed no color change in the medium supplemented with AMX. These findings indicated that strain 159 appeared resistant to CLR, MTZ, and LVX, while remaining sensitive to AMX (Figure 5). Strains 287 and 290 turned red after 28-36 hours of incubation in both antibiotic-free and antibiotic-containing characteristic media. These findings suggested that strains were resistant to all four tested antibiotics (Supplementary Figures 3 and 4). The results obtained for all the strains tested on the characteristic media were in agreement with the conclusions drawn from the MIC values (Table 1).

Figure 4 Growth and discoloration of the standard susceptible strain G27 after 36 hours of culture in the characteristic medium. The strain G27 is a sensitive strain that showed a red color only in the medium without antibiotics (containing phenol red), with no visible color change in other conditions. LVX: Levofloxacin; CLR: Clarithromycin; MTZ: Metronidazole; AMX: Amoxicillin.

Figure 5 Growth and discoloration of the drug-resistant strain 159 after 36 hours culture in the characteristic medium. Note: The strain 159 is resistant to levofloxacin, clarithromycin, and metronidazole, showing a red color in media containing levofloxacin, clarithromycin, and metronidazole, and phenol red, while no visible color change in the medium with amoxicillin was found. LVX: Levofloxacin; CLR: Clarithromycin; MTZ: Metronidazole; AMX: Amoxicillin.

Separation and detection of clinical samples

In clinical practice, the drug sensitivity of H. pylori was evaluated using both the traditional culture method and the novel method. A total of 201 gastric mucosal samples were collected. Among them, 139 samples were subjected to traditional antimicrobial susceptibility culture, of which 6 samples were contaminated or failed to subculture successfully during the detection process, rendering them untestable. In contrast, the new method was employed to detect drug susceptibility, resulting in the successful culture of H. pylori colonies in 185 samples, and only 3 samples were contaminated by miscellaneous bacteria. Consequently, the success rates of traditional antimicrobial susceptibility testing and the new method were 66.3% and 90.5%, respectively (Table 6).

Table 6 Detection via two detection methods.

Detection method	The number of successful detections	Number of culture failures	Total	Detection success rate (%)
Dilution method	133	68	201	66.3
New approach	182	19	201	90.5

The dilution method is a classical approach for drug susceptibility testing, whereas the new method was developed in this study. The difference in the success rate of the two methods was statistically significant (P < 0.01).

Consistency between the two detection methods: Results from the two detection methods were considered consistent only if all four antibiotic susceptibility tests produced matching outcomes. Any discrepancy in any of the four tests between the methods classified the results as inconsistent. Among 133 samples that met the inclusion criteria, 127 exhibited concordant results, yielding a 95.5% agreement rate. Notably, the novel method demonstrated superior success rates and greater efficiency, providing results within 28-36 hours compared with 11-12 days required by the traditional method (P < 0.001). The difference in the success rates of the two methods was statistically significant (P < 0.01). These data highlight its utility in informing clinical treatment strategies.

DISCUSSION

Currently, the extensive use of antibiotics has led to severe drug resistance in H. pylori[18]. In 2017, the World Health Organization identified CLR-resistant H. pylori as one of the 12 bacterial pathogens urgently requiring the development of new antibiotics[19]. Hence, several countermeasures have been proposed by scholars to address this challenge. These countermeasures include improving patient adherence, for instance, by combining CLR and AMX into a single capsule to improve the medication experience[20], integrating traditional Chinese and Western medical approaches[21], and exploring emerging therapies, such as nanomaterials, probiotics, and other novel agents that have shown promising efficacy[22-24]. The ability to perform drug resistance testing and subsequently select an appropriate treatment strategy is critical[25,26]. This process is vital to achieve individualized and precise treatments for H. pylori infections. At present, CLSI recommends the use of the international microdilution method for drug resistance testing in H. pylori[27]. Based on the results, the antimicrobial susceptibility of H. pylori can be categorized as susceptible, resistant, or intermediate[28]. However, the microdilution method requires cultivation to obtain pure colonies of H. pylori, which can be challenging and time-consuming, taking up to 9-11 days. This limitation has hindered the widespread adoption of traditional antimicrobial susceptibility testing for H. pylori. Alternative drug resistance detection methods, such as those based on resistance genes, are not as accurate as the microdilution method and may have significant errors. Therefore, the development of faster, more accurate, and easier-to-perform drug resistance detection methods for H. pylori is urgently needed. Such advancements could significantly improve the management and treatment of H. pylori infections, leading to superior patient outcomes and reducing the spread of antibiotic resistance.

Calculations and verification of antibiotic interception were conducted based on resistance breakpoints established by the CLSI and various logarithmic concentrations of antibiotics (1 × 10⁵ CFUs/mL), aiming to enhance detection accuracy and timeliness. By bypassing multiple steps, such as isolation, cultivation, and bacterial enrichment, this method could directly inhibit the growth of susceptible H. pylori strains using targeted antibiotics, thereby improving both efficiency and precision. As H. pylori hydrolyzes urea to produce alkaline substances, and NiCl₂ enhances urease activity, a combination of urea, phenol red, and NiCl₂ was incorporated into the culture medium to enable a colorimetric response. This modified approach allows for resistance determination during 28-36 hours based on phenol red discoloration, in contrast to the prolonged timeline of traditional antimicrobial susceptibility testing. To validate this method, a comparative analysis of 133 clinical samples was performed, which were also assessed via the standard microdilution method. The results demonstrated a high concordance rate of 95.5%, supporting the reliability and feasibility of the method as a rapid alternative for antimicrobial susceptibility testing in H. pylori.

A key limitation of this study is its concentration on only four commonly used antibiotics (AMX, CLR, MTZ, and LVX). Future investigations should expand the antibiotic panel to include emerging therapeutic agents (e.g., tetracycline and rifabutin). Although the current sample size (n = 201) was regarded as representative, larger multicenter studies with broader geographic diversity are necessary to validate the generalizability of the findings. Another notable limitation is the invasive nature of the method, as it requires endoscopic sampling of gastric mucosa. While non-invasive tests, such as stool-based detection of H. pylori resistance genes, have gained clinical acceptance, these approaches are mainly limited by interference from commensal gut microbiota and a restricted range of detectable resistance markers. For patients with refractory H. pylori infection, invasive methods remain essential for obtaining accurate antimicrobial resistance profiles, with most patients prioritizing diagnostic precision over the inconvenience of invasiveness. Thus, by enabling targeted antimicrobial therapy, this approach holds significant translational potential for improving H. pylori treatment outcomes. Therefore, this study proposes a novel chromogenic medium-based method for the rapid detection of H. pylori drug resistance, demonstrating significant innovation and clinical application potential.

CONCLUSION

The novel detection method can rapidly identify H. pylori drug resistance within 28-36 hours. It is characterized by high accuracy, operational simplicity, and broad clinical applicability. This approach may provide a practical diagnostic tool for managing H. pylori infections and hold remarkable promise for advancing clinical strategies to eliminate antimicrobial resistance.

ACKNOWLEDGEMENTS

We would like to express our gratitude to Professor Bi Hongkai from Nanjing Medical University for providing the Helicobacter pylori strains. The authors wish to thank the participants of the study and the staff at the Affiliated Hospital of Youjiang University for Nationalities for their dedication to patient recruitment and data collection. We also thank Dr. Gguang-Zi Qi for statistical consultation.