Clarithromycin Resistance Mutations in Helicobacter pylori in Association with Virulence Factors and Antibiotic Susceptibility of the Strains

Abstract

Antibiotic resistance is the major cause for Helicobacter pylori eradication failure. H. pylori clarithromycin resistance mutations were evaluated in 84 (82 phenotypically clarithromycin resistant and 2 intermediately susceptible) strains by allele-specific PCR and 3′-mismatched PCR. Many (57.1%) of these strains were metronidazole resistant. Prevalence of cagA⁺, cagE⁺, vacA s1a, m1, i1, and i2 strains was 76.2%, 58.0%, 82.1%, 35.7%, 50.0%, and 50.0%, respectively. A2143G, A2142G, A2142C, and A2143G+A2142G mutation rates were 64.3%, 23.8%, 1.2%, and 10.7%, respectively. Strains harboring the A2142G mutation showed 5.3-fold higher clarithromycin MIC₅₀ than those harboring the A2143G mutation. The A2143G mutation alone was 1.7-fold more common in vacA i2 strains compared with vacA i1 strains, while the A2142G mutation alone was 3-fold more frequent in vacA i1 strains than vacA i2 strains and 3.1-fold more common in metronidazole-susceptible compared with metronidazole-resistant strains. Briefly, clarithromycin resistance mutations were significantly linked to vacA i allele and metronidazole susceptibility. This is the first report about associations between the A2143G mutation and less virulent vacA i2 strains, and between the A2142G mutation and more virulent vacA i1 strains. As the 2143G mutation often predicts eradication failure by clarithromycin-based regimens, the results may be linked to the better eradication of more virulent strains compared with the less virulent strains.

Introduction

H elicobacter pylori virulence factors are linked to the clinical outcome of the gastroduodenal diseases.⁷ Two of the most important virulence factors of H. pylori are cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA) gene proteins.⁴ CagA protein alters cell-signaling mechanisms in gastric cells and induces morphological changes, cell proliferation and apoptosis, interleukin-8 secretion, chromosomal instability, and activation of proto-oncogenes.^4,7,19 H. pylori cag pathogenicity island (cagPAI) containing cagA, cytotoxin-associated gene E (cagE), and many other genes is important for CagA translocation into host cells and for induction of proinflammatory cytokine (interleukin-8) release.^7,25,29

About 40–50% of the strains form the most active VacA protein due to sequence variation in the genes causing mosaic combinations in the signal (s), middle (m), and intermediate (i) regions.⁴ VacA protein causes cellular vacuolation, pore formation, disturbance of the endosome/lysosomal activity, and gastric cell apoptosis, as well as development of a proinflammatory response.²⁹

Severe outcomes of H. pylori infection such as gastric cancer and peptic ulcers have been strongly associated with cagA, cagPAI, and vacA status, for example, vacA s1/i1/m1 type of strains in Western countries.⁷

The cagE gene has been associated with severe diseases as well. In Turkey as a neighboring country, cagA, cagE, and vacA s1a strains were linked to duodenal ulcer and gastric cancer, cagE and vacA s1a types were predictors of duodenal ulcer, and cagE strains were associated with gastric cancer.¹⁴

Eradication of the infection is necessary for the interruption of its natural course. However, the main cause for the therapeutic failure is the antibiotic resistance of H. pylori.²² Clarithromycin resistance in H pylori reduces the success of the standard clarithromycin-based triple therapy by 70%.²² Important factors associated with H. pylori resistance to antibiotics are the emergence of the resistance most often from point mutations and the long-term character of H. pylori infection if not eradicated.²²

Clarithromycin is a key antibiotic for H. pylori eradication. The main reason of H. pylori resistance to macrolides is the lack of binding of the agents to the 23S ribosomal subunit of the bacterial ribosome because of modification of the target site through methylation or point mutations in the domain V (peptidyl transferase loop) of the 23S rRNA.¹ The most common mutation for H. pylori resistance to clarithromycin has been A2143G (also known as A2147G in about 70% of the resistant strains, with a range from 53% to 95%), followed by A2142G (also called A2146G, 11.7%) and A2142C (also known as A2146C, 2.6%).^6,12,23

Primary resistance rates to clarithromycin have been high (≥20%) mostly in some developed countries, while primary resistance rates to metronidazole have been high (≥76%) in some developing countries.⁶ The difference can be explained by the more common use of newer macrolides for respiratory infections or otitis media in the developed countries compared with the developing countries and the frequent use of nitroimidazoles for parasitic infections in many developing countries.^1,6

Double resistance to both clarithromycin and metronidazole has usually been <10% of H. pylori strains but has been higher (>10–20%) in some countries like Brazil, France, Spain, and Poland.^6,13 The main reasons for H. pylori antibiotic resistance are prior treatment for H. pylori or other infections and outpatient antibiotic consumption in the country.⁶ In some studies, the resistance to both clarithromycin and metronidazole has been linked to ethnicity, female sex, older age, and nonulcer diseases.⁶

The aim of the present study was to detect the prevalence of the mutations causing H. pylori resistance to clarithromycin and the associations between the mutations and the characteristics of the patients and the strains.

Materials and Methods

In total, 84 strains, including 82 phenotypically clarithromycin-resistant and two intermediately susceptible strains, were included in the study. The strains were isolated from 9 children and 75 adults, involving 41 men/boys and 43 women/girls. According to the endoscopic diagnosis, 11 patients had peptic ulcers (7 patients with duodenal ulcer and 4 subjects with gastric ulcer) and 73 patients had nonulcer diseases (44 cases of chronic gastritis, 2 cases of acute gastritis, 1 case of chronic gastritis+gastroesophageal reflux disease [GERD], 25 cases of GERD, and 1 case with pancreatic cyst). Data about the place of living and previous therapy for H. pylori infection were available for 66 and 83 patients, respectively. Baseline characteristics of the patients and the strains are given in Table 1.

Table 1.

Clarithromycin Resistance Mutations in Association with the Characteristics of the Patients and Strains

			No. of strains with mutations (%)
Characteristics of patients and strains	Groups of patients and strains	No. of strains evaluated	A2143G	A2142G	A2142C	Mixed ^a
Age	Children	9	5 (55.6)	3 (33.3)	0 (0)	1 (11.1)
	Adults	75	49 (65.3)	17 (22.7)	1 (1.3)	8 (10.7)
Sex	Men	41	25 (61.0)	10 (24.4)	0 (0)	6 (14.6)
	Women	43	29 (67.4)	10 (23.3)	1 (2.3)	3 (7.0)
Disease	Ulcer	11	8 (72.7)	3 (27.3)	0 (0)	0 (0)
	Nonulcer	73	46 (63.0)	17 (23.3)	1 (1.4)	9 (12.3)
Treatment	Untreated	69	42 (60.9)	17 (24.6)	1 (1.4)	9 (13.0)
	Treated	14	11 (78.6)	3 (21.4)	0 (0)	0 (0)
Place of living	The capital	48	30 (62.5)	11 (22.9)	1 (2.1)	6 (12.5)
	Elsewhere	18	11 (61.1)	5 (27.8)	0 (0)	2 (11.1)
Metronidazole	Susceptible	36	19 (52.8)	14^b (38.9)	0 (0)	3 (8.3)
	Resistant	48	35 (72.9)	6^b (12.5)	1 (2.1)	6 (12.5)
cagA	Positive	64	39 (60.9)	18 (28.1)	0 (0)	7 (10.9)
	Negative	20	15 (75.0)	2 (10.0)	1 (5.0)	2 (10.0)
cagE	Positive	47	26 (55.3)	15 (31.9)	1 (2.1)	5 (10.6)
	Negative	34	25 (73.5)	5 (14.7)	0 (0)	4 (11.8)
vacA s alleles	vacA s1a	69^c	43 (62.3)	17 (24.6)	1 (1.4)	8 (11.6)
	vacA s1b	1	1 (NA)	0 (NA)	0 (NA)	0 (NA)
	vacA s2	17^c	13 (76.5)	3 (17.6)	0 (0)	1 (5.9)
vacA m alleles	vacA m1	30^c	18 (60.0)	8 (26.7)	0 (0)	4 (13.3)
	vacA m2	57^c	38 (66.7)	13 (22.8)	1 (1.8)	5 (8.8)
vacA i alleles	vacA i1	39	18^b (46.2)	15^b (38.5)	1 (2.6)	5 (12.8)
	vacA i2	39	31^b (79.5)	5^b (12.8)	0 (0)	3 (7.7)
Clarithromycin MIC (mg/L)	0.5 to 12	20	18^b (90.0)	2^b (10.0)	0 (0)	0 (0)
	16 to >256	26	12^b (46.2)	11^b (42.3)	0 (0)	3 (11.5)
Total		84	54 (64.3)	20 (23.8)	1 (1.2)	9 (10.7)

Mixed, both A2143G and A2142G mutations.

Statistical difference (p < 0.05).

Two strains had vacA s1a/s2 and m1/m2 alleles, one strain had s1a/s2 alleles and one strain harbored m1/m2 alleles.

MIC, minimal inhibitory concentration; NA, not appropriate.

Phenotypic clarithromycin and metronidazole resistance rates of the strains included in the study were assessed by a breakpoint susceptibility testing (BST) method using the twofold dilution technique as previously described.⁸ BST was chosen as a simplified agar dilution method that can be used to test the susceptibility of single isolates.⁸ Briefly, ∼30–60 μl of H. pylori suspensions in the Mueller-Hinton broth (density of 2 McFarland standard; Oxoid, Hampshire, United Kingdom) were inoculated onto blood Mueller-Hinton agar plates (Oxoid) containing 0.25, 0.5, 1, and 2 mg/L clarithromycin or 4, 8, 16, and 32 mg/L metronidazole. The antibiotics were purchased from Sigma-Aldrich (St. Louis, MO). The plates were incubated microaerophilically (CampyGen; Oxoid) at 37°C for 2–3 days. H. pylori growth on the agar containing the lowest antibiotic concentration indicated resistance. Nonselective Mueller-Hinton blood agar plates were used for a control of strain viability.

Clarithromycin susceptibility of 46 strains was also assesses by E tests (Liofilchem s.r.l., Roseto degli Abruzzi, Italy). H. pylori suspensions (0.5 ml, density of 2–3 McFarland standards) in Mueller-Hinton broth were inoculated onto Mueller-Hinton agar pates (Oxoid) containing 5% sheep blood. E test strips were placed on the dried plates (1 strip per 90-mm-diameter plate) and the plates were incubated in microaerophilic atmosphere (as above) at 37°C for 48–72 hr. The results were read according to the supplier's recommendations. H. pylori resistance found using the E tests was compared with the results of the BST method.

Resistance to clarithromycin and metronidazole was determined as minimal inhibitory concentrations (MICs) of >0.5 and >8 mg/L, respectively (The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 5.0, 2015. www.eucast.org.). The breakpoint for intermediately susceptible strains to clarithromycin was >0.25–0.5 mg/L.

Metronidazole resistance rate was compared with that in total H. pylori strains from 75 untreated children, 607 untreated adults, and 91 treated adults in our previous study.⁸

Gene ureA was used to confirm the species identity of the strains.³⁰ The ureA and virulence genes (cagA and vacA sla, slb, s2, m1, m2, i1, and i2 alleles) were evaluated by the primers described in our previous studies.^9,32 The prevalence of cagA⁺ and vacA i1 strains in the clarithromycin-resistant/intermediately susceptible strains in the present study was compared to that in a total of 216 H. pylori strains evaluated in our previous study.³²

To detect A2143G and A2142G mutations, allele-specific PCR (AS-PCR) was carried out by the method of Furuta et al.¹⁶ Phusion^® High-Fidelity DNA Polymerase (New England Labs, Ipswich, MA) was used for PCRs. The total volume of PCR was 20 μl, including 0.4 unit Fusion Taq polymerase per reaction, 4 mmol dNTP, 1× final concentration 5× Phusion HF, and 5 pmol primers FP-1 (5′-TCGAAGGTTAAGAGGATGCGTCAGTC-3′), RP-1 (5′-GACTCCATAAGAGCCAAAGCCCTTAC-3′), and 10 pmol RP2142G (5′-AGTAAAGGTCCACGGGGTATTCC-3′) and FP2143G (5′-CCGCGGCAAGACAGAGA-3′). An AS-PCR was performed as follows: denaturation at 98°C for 30 sec, followed by 35 cycles of denaturation at 98°C for 10 sec, annealing at 61.5°C for 20 sec, and extension at 72°C for 20 sec, with a final extension at 72°C for 5 min. The 320 bp fragment indicates the presence of the wild-type H. pylori 23S rRNA gene, while 238 and 118 bp fragments indicate the presence of mutations A2142G and A2143G, respectively, which cause clarithromycin resistance. All PCR products were subjected to electrophoresis on a 2% agarose gel along with a 100 bp DNA marker.

To detect A2142C point mutation, a 3′-mismatched PCR was performed using published primers according to Alarcón et al.² The primers used were CLA 18 (AGTCGGGACCTAAGGCGAG) and CLA 3 (AGGTCCACGGGGTCTTG).²

The chi-square with Fisher's exact test was used to compare the groups, and p-values of ≤0.05 were considered statistically significant.

Results

Within the clarithromycin-resistant/intermediately susceptible strains evaluated, the cagA⁺, cagE⁺, vacA s1a, m1, i1, and i2 strains were 76.2% (64/84 strains), 58.0% (47/81), 82.1% (69/84), 35.7% (30/84), 50.0% (39/78), and 50.0% (39/78), respectively. Many (57.1%, 48/84) strains were metronidazole resistant.

The most virulent cagA⁺ vacA s1a/m1/i1 genotype was found in 21 (26.9%) of the 78 strains tested for all the aforementioned genes. The prevalence of cagA⁺ strains (76.2%) in the present study was similar to that (84.7%, p = 0.082) in a total of 216 H. pylori strains evaluated in our previous study.³² The prevalence of vacA i1 (50.0%) in the present study was lower compared with that (63.4%, p = 0.038) in our previous study.³²

E test results for clarithromycin were compared with the results of the BST. The metronidazole resistance rate (57.1%) in the clarithromycin-resistant/intermediately susceptible strains in the present study was much higher compared with that (25.4%, p = 0.0001) in the total H. pylori strains in our previous study.⁸

The A2143G, A2142G, and A2142C point mutations were detected in all 84 (100%) of the clarithromycin-resistant/intermediately susceptible strains evaluated.

Heteroresistance was found in patients (10.7%, 9/84), which strains harbored several resistant alleles (both A2143G and A2142G mutations) and in patients (6.5%, 3/46), which specimens yielded both clarithromycin-susceptible and clarithromycin-resistant strains/variants by E test. In case of both susceptible and resistant subpopulations, only the phenotypically resistant ones were considered.

The overall prevalence of A2143G (including strains with intermediate susceptibility to clarithromycin, MIC, 0.5 mg/L), A2142G, A2142C, and A2143G+A2142G mutations was 64.3% (54/84 strains), 23.8% (20/84), 1.2% (1/84), and 10.7% (9/84), respectively (Table 1).

The A2142G point mutation was more frequent (38.9%, 14/36 strains) in metronidazole-susceptible strains compared with metronidazole-resistant strains (12.5%, 6/48, p = 0.009).

The A2143G mutation alone was found in 79.5% (31/39) of the vacA i2 strains versus 46.2% (18/39, p = 0.002) in vacA i1-positive strains. The prevalence of both single A2143G and double A2143G+A2142G mutations was 87.2% (34/39) in vacA i2 strains versus 59.0% (23/39, p = 0.01) in vacA i1 strains.

The vacA i1 strains had more often A2142G point mutation alone (38.5%, 15/39) compared with vacA i2 strains (12.8%, 5/39, p = 0.018), and more frequently A2142G and mixed A2143G+A2142G mutations (51.3%, 20/39) compared with vacA i2 strains (20.5%, 8/39, p = 0.005).

There was no interpretive category disagreement between the BST- and E test for clarithromycin in the 46 strains, in which clarithromycin susceptibility was assessed by both methods.

Strains with lower (0.5–12 mg/L) clarithromycin MICs had more frequently A2143G point mutation (90.0%, 18/20 strains) compared with those with MICs 16 to >256 mg/L (46.2%, 12/26, p = 0.002). Clarithromycin MIC₅₀ was 12 mg/L in the strains with A2143G point mutation alone and 64 mg/L in the strains with A2142G point mutation alone (Table 2).

Table 2.

MICs of Clarithromycin in Strains According to the Mutation

Mutation	No. of strains	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	Range (mg/L)
2142G	13	64	>256	1 to >256
2143G	30	12	256	0.5 to 256
2143G+2142G	3	NA	NA	256 to >256
Total	46	16	256	0.5 to >256

There was no statistical difference (p > 0.05) between the mutations for clarithromycin resistance and groups according to the age (children or adults), sex, disease (ulcer or nonulcer), treatment, place of living (in the capital or elsewhere), cagA, cagE, and vacA s and m status.

Discussion

In the present study, we observed a full category agreement between BST- and E test results for clarithromycin. Similarly, in our previous study,⁸ the interpretive category agreement between the BST- and E test results for clarithromycin was 95%.

Metronidazole susceptibility testing was performed by BST because using the E test can lead to false resistance and because in our previous study, the category agreement between the BST and agar dilution method results for metronidazole was good (>93%).⁸

The metronidazole resistance rate in the clarithromycin-resistant/intermediately susceptible strains in the present study was high (57.1%). However, although H. pylori harbors different nitroreductase enzymes to reduce the drug and the treatment with high doses of metronidazole can overcome the resistance, the double resistance can hinder the success of many eradication regimens.^6,23

The prevalence of cagA⁺ strains (76.2%) was similar to those (77.4%) in total strains in Greece as a neighboring country and in Italy (72.0%), while the prevalence of vacA s1 strains in the present study was similar to those in Germany (83.5%) and Ireland (85.4%).^3,5,7

The types of clarithromycin resistance mutations are of high importance for the treatment success by triple clarithromycin-based regimens for H. pylori eradication. The A2143G mutation has been associated with a lower eradication success (50%) compared with the other mutations (89%) in Italian children.¹⁵ Other authors like Hwang et al.¹⁸ have reported similar results in Korea. However, the sequential therapy has achieved a higher eradication rate than the triple therapy in patients with A2143G mutant strains.¹⁵

In addition to the A2143G, A2142G, and A2142C point mutations, some authors have detected other mutations, for example, A2115G, A2223G, G2141A, T2182C, T2245C, and C2131T in clarithromycin-resistant isolates.^1,6 However, in the present study, all clarithromycin-resistant/intermediately susceptible strains harbored A2143G, A2142G, and A2142C point mutations alone or in combinations. Similar results were found by Alarcón et al.² in a Spanish study,² by Raymond et al.²⁷ in a French study, as well as by Caliskan et al.¹¹ in Turkey as a neighboring country, although no A2142C point mutation was found in 36 clarithromycin-resistant strains.

The A2143G mutation, mixed with other clarithromycin resistance mutations, has been uncommon (2.1–21.1%) in France, Japan, and Brazil but frequent in Taiwan (44.4%).^6,12,16 The proportion of strains with mixed clarithromycin resistance mutations (10.7%) in the present study was similar to those (8.8–11.4%) found in clarithromycin-resistant clinical strains in French and Spanish studies.^1,12

The A2143G mutation alone or mixed was less frequent (75.0%) compared with that in Spain (85.3%) according to Agudo et al.¹ and in Chinese children (84.6%) according to Liu et al.,²¹ but was most common compared with that (62.5%) in Poland¹³ and was similar to that in France.¹²

In some studies, A2142G point mutation has been associated with one to eight times (often two to eight times) higher clarithromycin MICs compared with A2143G mutation,^6,16,20 while no such difference has been found by other authors.^1,24,27

In the present study, the strains with the 2142G mutation had 5.3-fold higher MIC₅₀ of clarithromycin compared with those with the 2143G mutation. Moreover, two strains exhibiting intermediate susceptibility to clarithromycin had clarithromycin MICs of 0.5 mg/L and harbored A2143G mutation.

The allele of the intermediate vacA region, vacA i1, was associated with higher virulence of the strains and greater risk for gastric cancer compared with i2 allele.^28,29 The vacA i1 allele has been linked to H. pylori toxicity and has been determined as the best marker of VacA-linked vacuolating activity.²⁸ In Italy, vacA s1 and i1 alleles were associated with gastric cancer and vacA i1 allele was linked to peptic ulcers.⁵

Importantly, in our previous study, vacA i status was a superior predictor for the strain virulence compared with the other vacA alleles.³² Similarly, in Greece, vacA i1 allele was a risk factor for augmented activity and severity of gastritis.²⁶

To our knowledge, this is the first report about the association (p = 0.002) between the A2143G mutation alone and vacA i2 strains. In these strains, A2143G mutation was 1.7-fold more frequent compared with that in vacA i1-positive strains. Significant association was found between the prevalence of both A2143G and A2143G+A2142G mutations and vacA i2 strains. Conversely, the A2142G mutation alone was threefold more frequent in vacA i1-positive strains compared with vacA i2 strains.

Conversely, in Iraq, an association was observed between the A2143G (A2147G) clarithromycin resistance mutation and the functional duodenal ulcer promoting gene A (dupA1) genotype.¹⁷ However, in this study, the dupA1 genotype was not associated with the secretion of interleukin-8 from the gastric mucosa.¹⁷ More studies are required to assess the association between different H. pylori virulence factors and clarithromycin resistance mutations.

According to Broutet et al.¹⁰ and Suzuki et al.,³¹ less virulent (cagA negative) strains have been associated with a twofold higher risk for treatment failure by clarithromycin-based triple regimen compared with more virulent (cagA positive) strains.

In the present study, the less virulent (vacA i2) strains have been associated with A2143G mutations as a good predictive marker for unsuccessful eradication of H. pylori.^15,18 As the 2143G mutation has been linked to lower eradication success compared with other clarithromycin resistance mutations, these results of the present study may contribute to the understanding about the better eradication of more virulent H. pylori strains compared with the less virulent strains. The higher inflammation in the gastric mucosa may increase the chance of eradication success.¹⁰

The association between A2142G mutation and metronidazole-susceptible strains is interesting and should be more evaluated in further studies.

There was no difference between the clarithromycin resistance mutations and groups according to the age, sex, disease, treatment, place of living, cagA, cagE, and vacA s and m status. The results may be explained, at least partially, by the relatively low (≤14 patients) number of children, ulcer patients, and treated subjects, as well as by the high (83.3%) prevalence of vacA s1 strains.

In conclusion, in the clarithromycin-resistant/intermediately susceptible strains, the metronidazole resistance was much higher and the prevalence of vacA i1 strains was lower compared with those in the total strains evaluated recently. The prevalence of cagA⁺ and vacA s1 strains in the present study was in the range of those found in other European countries. The mutations responsible for clarithromycin resistance in H. pylori were associated with vacA i alleles and metronidazole susceptibility. The A2143G point mutation was common in less virulent vacA i2 strains, while the A2142G point mutation was prevalent in more virulent vacA i1 strains. As the 2143G mutation most often predicts eradication failure by triple clarithromycin-based regimens, the present results may be linked to the better eradication of more virulent H. pylori strains compared with the less virulent strains.

Footnotes

Acknowledgment

This research was funded by the Grant/Contract B02/17 (12.12.2014) from the National Science Fund at the Ministry of Education and Science of Bulgaria, Project Ref. B02/2 (14.07.2014) entitled “Complex study of Helicobacter pylori virulence and resistance factors and epidemiology of the infection.”

Disclosure Statement

No competing financial interests exist.

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