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

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.

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.

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.

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.

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.

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).