Evaluation of Antibiotic Resistance of Helicobacter pylori Strains Isolated in Bari,Southern Italy,in 2017–2018 by Phenotypic and Genotyping Methods

Abstract

Objective

: Helicobacter pylori antibiotic resistance is a constantly evolving process and local surveillance is warranted to guide clinicians in the choice of therapy.

Materials and Methods:

Antibiotic susceptibility testing was performed by E-test on 92 H. pylori strains, and resistance to clarithromycin and levofloxacin was also evaluated using a commercially available genotyping method.

Results:

In naïve patients the resistance to clarithromycin, levofloxacin, and metronidazole was 37.7%, 26.2%, and 16.4%, respectively, significantly lower than the percentage found in treated patients. Concomitant resistance to ≥2 antibiotics was also observed in naïve patients. The A2143G mutation of the 23S-rRNA gene was the most frequently detected, also in naïve patients. The highest minimum inhibitory concentration (MIC)₅₀ value (256 mg/L) was associated with A2142 mutations in all the patients carrying them. For levofloxacin resistance a mutation in codon 87 was detected in 63.9% and in codon 91 in 36.1% of the H. pylori strains, without significant differences in the patients groups. A mutation in codon 87 was associated with the highest MIC₅₀ value (32 mg/L).

Conclusions:

In our area, a high prevalence of H. pylori primary resistance was detected; these rates were higher in patients who had experienced failure of several courses of therapy. A better knowledge of the local epidemiology of resistance, and the genotypes responsible, will improve the H. pylori eradication rates.

Introduction

Helicobacter pylori is a Gram negative spiral bacterium that colonizes the human stomach. Infected subjects develop gastritis, and if not treated, the infection may evolve to peptic ulcer and gastric mucosa-associated lymphoid tissue lymphoma. Furthermore, H. pylori is the main risk factor for the development of gastric carcinoma. The infecting H. pylori strains, host response, and environmental factors may all play a role in the different outcomes of the infection.¹ First line triple therapy (clarithromycin, amoxicillin, or metronidazole) plus a proton pump inhibitor (PPI) has been recommended in several international guidelines.² The decline in the H. pylori cure rates with this empiric therapy is due to the emergence of H. pylori strains resistant to these antibiotics, which has been observed worldwide. Second line and third line “rescue” therapies have been used and various combination of antibiotics have been proposed, including the new quadruple regimen (PPI, bismuth, tetracycline, and metronidazole).^3–5 Biopsy cultures are carried out mainly in research centers, and in the updated Maastricht Consensus Report it was recommended that culture and antimicrobial susceptibility testing should be routinely performed only after two treatments failures with different antibiotics.² The prevalence of H. pylori antimicrobial resistance varies in different geographic areas and has been correlated with the consumption of antibiotics in the general population, their frequent or inadequate use, and poor patients compliance to treatment.⁶ In Europe, the overall prevalence of primary antibiotic resistance was recently reported to be 18% for clarithromycin, 32% for metronidazole, 11% for levofloxacin, and 0% for both amoxicillin and tetracycline.⁷ In the last years an increase in resistance has occurred in Italy, the highest prevalence being in South Italy.⁸

Resistance to clarithromycin is due to the acquisition of point mutations in domain V of the rrl gene encoding the 23S rRNA. Several point mutations in this site have been described worldwide. In Italy, the presence of six of them has been reported and new mutations such as the T2717C have also been detected.^9,10 Mutations in the gyrase A gene are associated with fluoroquinolones resistance; only three of these are responsible for most cases of resistance in developed countries.¹¹ To avoid the spread of H. pylori resistant strains a knowledge of the local H. pylori antibiotic resistance is crucial to planning tailored eradication regimens.

The aim of this work was to evaluate the level of primary and secondary resistance of H. pylori to antibiotics used to treat this infection. In addition, point mutations responsible for antibiotic resistance to clarithromycin and levofloxacin were determined.

Materials and Methods

Patients and samples

This observational study was conducted at the Microbiology and Virology Laboratory of I.R.C.C.S. Saverio de Bellis, Castellana Grotte, Bari, Italy between October 2017 and November 2018. A total of 224 patients referred to the Endoscopic Unit of Gastroenterology of this hospital were included.

In 92 of these patients, H. pylori infection was detected by rapid urease test (44 patients), urea breath test (32 patients), or stool antigen test of H. pylori (16 patients). Gastric biopsies were performed in all 92 patients: 2 from the antrum, 2 from the body-bottom, and 1 from the angulus. Two of these specimens (antrum and body) were used for H. pylori culture and the remaining one for histologic examination. Another sample was taken for the rapid urease test when indicated. Gastric biopsies for culture of H. pylori were placed in a saline solution containing 20% glucose and processed within 2 hours. Informed consent was obtained from all patients. Demographic data including name, gender, age, place of birth and residence, symptoms, and treatment history were collected.

Isolation of H. pylori

H. pylori was isolated on Pylori agar (bioMérieux, Florence, Italy). The plates were incubated in a microaerobic environment (6% O₂, 10% CO₂, and 84% N₂) using the Gas Pack incubation method (bioMérieux) at 37°C up to 10 days. Isolates were identified as H. pylori on colony morphology, Gram staining, and positive reactions for urease, catalase, and oxidase tests.¹²

Antibiotic susceptibility testing

Minimum inhibitory concentrations (MICs) were determined using the E-test methodology. The following antimicrobial agents were tested: amoxicillin, clarithromycin, levofloxacin, metronidazole, tetracycline, and rifampicin. A bacterial suspension equivalent to McFarland turbidity standard no. 3 (∼1.0 × 10⁹ CFU/mL) was used. Aliquots of this suspension were spread by sterile, disposable L-Shaped Cell Spreaders on Mueller Hinton Fastidious II agar plates (Liofilchem, Roseto degli Abruzzi, Italy) and antibiotic E-test strips (bioMérieux) were placed. The plates were incubated in a microaerobic atmosphere at 37°C. After 72 hours of incubation the inhibition zones were measured. All the tests were performed in duplicate. The MIC interpretative breakpoints were those recommended by the European Committee on Antimicrobial Susceptibility Testing: clarithromycin: R > 0.5 mg/L, amoxicillin R > 0.12 mg/L, metronidazole R > 8 mg/L, levofloxacin, tetracycline R > 1 mg/L, and rifampicin R > 1 mg/L. H. pylori ATCC 43504 was used as control strain.¹³

DNA extraction

For genotyping resistance and sequencing genomic of H. pylori isolates, DNA was extracted using the QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. DNA purity and concentrations were estimated using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA).

To detect the point mutations conferring resistance to clarithromycin and fluoroquinolones, a DNA strip methodology (GenotypeHelicoDR assay; Hain Life Science, Nehren, Germany) was used. Briefly, it consists of multiplex PCR followed by DNA strip hybridization. The probes hybridize with the sequence of wild-type alleles (WT probes) or mutated alleles (MUT probes). This test has 94% sensitivity and 99% specificity for the detection of clarithromycin resistance, and 87% and 98.5%, respectively, for the detection of fluoroquinolone resistance.¹⁴

Library preparation, WGS, and in silico analysis of H. pylori strains

Purified genomic DNA of H. pylori strains were subjected to whole genome sequencing using the Nextera XT library preparation workflow (Illumina, San Diego, CA), according to the manufacturer's instructions. Sequencing was performed on an Illumina MiSeq platform generating tagged paired-end reads (2 × 250 bp). The paired-end raw reads (fastq) were filtered and trimmed using Trimmomatic. De novo genome assembly and scaffolding were performed with SPAdes v. 3.11.

Antibiotic resistance and plasmids prediction was performed with the ABRicate pipeline using ARG-ANNOT, NCBI, CARD, ResFinder, and PladmidFinder as reference databases. In addition, assembled genomes were submitted to the BLASTn Basic Local Alignment Search Tool for nucleotide sequences of CARD.¹⁵

To confirm mutation T87I, the genes sequences were extracted and aligned with Bionumerics v7.6 (Applied Maths NV, Sint-Martens-Latem, Belgium) to search for all specific single nucleotide polymorphisms and amino-acid mutations in codon 87 that confer resistance. Sequencing of each isolate was performed at the laboratories of the Istituto Zooprofilattico Sperimentale di Puglia and Basilicata.

Statistical analysis

We assumed the prevalence of clarithromycin and fluoroquinolone resistance to be 20–30% and 10–15%, respectively, based on the following reference.⁷ Therefore more than 200 patients were included to obtain a representative sample. The results of the study were statistically analyzed by Pearson's χ² test when possible and Fisher exact test was used when appropriate. A p-value less than 0.1 was considered statistically significant.

Results

A total of 224 patients who underwent endoscopy for a variety of gastrointestinal symptoms was examined: 91 men (aged 19–65 years; mean age 48.7 years) and 133 women (aged 22–65 years; mean age 48.5). Of these patients, 92 (41.1%) resulted H. pylori positive. Data including age, gender, symptoms, endoscopic findings, and previous treatment for H. pylori infection are shown in Table 1. Sixty-one (66.3%) of the patients had never been treated for H. pylori eradication (naïve patients), whereas 33.7% had failed to respond to several therapy lines. H. pylori resistance to clarithromycin, levofloxacin, and metronidazole was significantly associated with prior therapy (p-value <0.01). Resistance to ≥2 antibiotics was found in 39.1% of strains and was higher in those isolated from treated patients. Concomitant resistance to clarithromycin and levofloxacin was most frequently detected, in particular in H. pylori isolated from naïve patients. Concomitant resistance to clarithromycin, levofloxacin, and metronidazole was found in 14.1% of strains and resistance to four antibiotics (clarithromycin, levofloxacin, metronidazole, and amoxicillin) in 2.2% of strains. Multiple resistance was significantly (p-value <0.01) associated with previous treatment (Table 2).

Table 1.

Demographic and Clinical Characteristics of Helicobacter pylori Positive Naïve and Treated Patients

Characteristic	Total, n (%)	Naïve, n (%)	Treated, n (%)
Age (years)
≤29	6 (6.6)	4 (6.6)	2 (6.5)
30–41	13 (14.1)	8 (13.1)	5 (16.1)
42–53	30 (32.6)	22 (36.1)	8 (25.8)
≥54	43 (46.7)	27 (44.2)	16 (51.6)
Gender
Male	38 (41.3)	26 (42.6)	12 (38.7)
Female	54 (58.7)	35 (57.4)	19 (61.3)
Symptoms
Epigastralgia	67 (72.8)	41 (67.2)	26 (83.9)
Gastroesophageal reflux	17 (18.5)	14 (22.9)	3 (9.7)
Dysphagia	11 (12.0)	9 (18.0)	2 (35.5)
Anemia and/or intestinal hemorrhage	6 (6.5)	6 (9.8)	0 (0)
Endoscopic findings
Gastritis	87 (94.6)	57 (93.4)	30 (96.8)
Hiatal hernia and/or cardial incontinence	29 (31.5)	24 (39.3)	5 (16.1)
Duodenitis and/or duodenal ulcer	20 (21.7)	17 (27.9)	3 (9.7)
Esophagitis	15 (16.3)	12 (19.7)	3 (9.7)
No. of patients	92	61	31

More than two symptoms and endoscopic findings could be present in the same patient.

n (%), number and percentage of patients.

Table 2.

Helicobacter pylori Resistance Rates in Naïve and Treated Patients

Antibiotics	Resistant strains
Antibiotics	Total, n (%)	Naïve, n (%)	Treated, n (%)
^*
Clarithromycin	49 (53.3)	23 (37.7)	26 (83.9)
Levofloxacin	36 (39.1)	16 (26.2)	20 (64.5)
Metronidazole	30 (32.6)	10 (16.4)	20 (64.5)
Amoxicillin	3 (3.3)	1 (1.6)	2 (6.5)
Rifampicin	1 (1.1)	1 (1.6)	0 (0.0)
Tetracycline	0 (0.0)	0 (0.0)	0 (0.0)
Clarithromycin + levofloxacin	10 (10.9)	7 (11.5)	3 (9.7)
Levofloxacin + metronidazole	2 (2.2)	0 (0.0)	2 (6.5)
Clarithromycin + metronidazole	7 (7.6)	3 (4.9)	4 (12.9)
Metronidazole + amoxicillin	1 (1.1)	1 (1.6)	0 (0.0)
Levofloxacin + rifampicin	1 (1.1)	1 (1.6)	0 (0.0)
Clarithromycin + levofloxacin + metronidazole	13 (14.1)	1 (1.6)	12 (38.7)
Clarithromycin + levofloxacin + metronidazole + amoxicillin	2 (2.2)	0 (0.0)	2 (6.5)
^**
S to all antibiotics	26 (28.3)	24 (39.3)	2 (6.5)
R to one antibiotic	30 (32.6)	24 (39.3)	6 (19.4)
R to ≥2 antibiotics	36 (39.1)	13 (21.3)	23 (74.2)

The plus sign after antibiotic indicates the resistance found to 2, 3, or 4 antibiotics.

p-Values <0.01, resistance (naïve vs. treated patients);

p-Values <0.01, multiple resistance (naïve vs. treated patients). p Values were calculated by χ² tests.

n (%), number and percentage of resistant strains; R, resistant; S, susceptible.

The MIC₅₀ of clarithromycin, metronidazole, and levofloxacin were higher in H. pylori strains isolated from treated patients in comparison to MIC₅₀ found in naïve patients. MIC₉₀ was similar for H. pylori isolated from both naïve and treated patients (Table 3). A significantly higher rate of resistance to levofloxacin was detected in strains isolated from the population over 41 years (p-value = 0.02), whereas no significant differences were found for metronidazole and clarithromycin (Fig. 1A). No statistically significant differences were found between antibiotic resistance and gender, except for resistance to clarithromycin, found mainly in H. pylori isolated from treated female patients (p-value = 0.06) (Fig. 1B).

FIG. 1.

Resistance rate of Helicobacter pylori by age (A) and gender (B). *p-Value = 0.02, levofloxacin resistance in two different class age. p-Value was calculated by χ² test. **p-Value = 0.06, clarithromycin resistance in treated patients male versus female. p-Value was calculated by Fisher exact test. CH, clarithromycin; LE, levofloxacin; MZ, metronidazole.

Table 3.

MIC₅₀ and MIC₉₀ to Antibiotics in Helicobacter pylori Strains Isolated from Naïve and Treated Patients

Antibiotic	Total		Naïve		Treated
Antibiotic	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)
Clarithromycin	2	256	0.032	256	8	256
Levofloxacin	0.25	32	0.19	32	8	32
Metronidazole	0.75	256	0.5	192	256	256
Amoxicillin	0.016	0.094	0.016	0.064	0.016	0.125
Rifampicin	0.38	1.0	0.38	1.0	0.38	1.0
Tetracycline	0.032	0.094	0.032	0.094	0.032	0.25

MIC, minimum inhibitory concentration.

Mutations in the 23S-rRNA gene (rrl gene) (A2142G, A2143G, and A2142C) were investigated and 53.3% of resistant strains showed mutations in this gene. A mixture of resistant and susceptible H. pylori genotypes was found in 15.2%. The single A2143G, A2142G, and A2142C mutations were found in 75.5%, 12.2%, and 2% of H. pylori resistant strains, respectively. A double point mutation was detected in 10.2% of strains, A2142G plus A2142C being the type most frequently present (Table 4). A significant association between a mutation in position 2143 and previous treatment was detected (p-value = 0.06). No association with gender or age was found (data not shown). The mutation in 2143 was significantly associated (p-value = 0.06) with a lower MIC range (≤16 mg/L). The highest MIC₅₀ and MIC₉₀ values (over 16 mg/L) were associated with a mutation in 2142, while the mutation in 2143 was associated with lower MIC₅₀ values (Table 5).

Table 4.

Distribution of Point Mutations in Helicobacter pylori Isolated from Naïve and Treated Patients

Genotype	Total, n (%)	Naïve, n (%)	Treated, n (%)
Clarithromycin
S
No mutation (WT)	43 (46.7)	38 (62.3)	5 (16.1)
R
WT + mutation	7 (7.6)	7 (11.5)	0 (0.0%)
Mutation	42 (45.6)	16 (26.2)	26 (83.9)
Mutation
Single mutation
A2143G	37 (75.5)	14 (61.0)	23 (88.5)
A2142G	6 (12.2)	4 (17.4)	2 (7.7)
A2142C	1 (2.0)	1 (4.3)	0 (0.0)
Double mutation
A2142G + A2142C	2 (4.1)	2 (8.7)	0 (0.0)
A2142G + A2143G	3 (6.1)	2 (8.7)	1 (3.8)
^*
Mutation in 2143	37 (40.2)	14 (23.0)	23 (74.2)
Mutation in 2142	9 (9.8)	7 (11.5)	2 (6.5)

Levofloxacin
S
87 WT1 + 91WT	32 (57.1)	25 (55.6)	7 (63.6)
87WT2 + 91 WT	24 (42.9)	20 (44.4)	4 (36.4)
R
87 MUT +91 WT
N87K	21 (91.3)	8 (88.9)	13 (92.9)
T87I	2 (8.7)	1 (11.1)	1 (7.1)
87 WT +91 MUT
D91N	6 (46.1)	2 (28.6)	4 (66.5)
D91Y	4 (30.8)	3 (42.8)	1 (16.7)
D91G	3 (23.1)	2 (28.6)	1 (16.7)

p-Value = 0.06, mutation in 2143 and in 2142 (naïve vs. treated patients). p-Value was calculated by Fisher exact test.

MUT, mutated codon; n (%), number and percentage of strains; R, resistant; S, susceptible; WT, wild type; WT1, wild-type profile of codon 87; WT2, wild-type profile of codon 87.

Table 5.

Association Between Minimum Inhibitory Concentration Values and Helicobacter pylori Genotype

Clarithromycin
MIC range (mg/L)	Mutation in 2143	Mutation in 2142
^*
0.75–16	18	1
>16	19	8
MIC₅₀	16 mg/L	256 mg/L
MIC₉₀	256 mg/L	256 mg/L

Levofloxacin
MIC range (mg/L)	Mutation in codon 87	Mutation in codon 91
^**
1.5–6	1	6
>6	22	7
MIC₅₀	32 mg/L	12 mg/L
MIC₉₀	32 mg/L	32 mg/L

p-Value = 0.06, MIC range mutation in 2143 versus mutation in 2142.

p-Value = 0.005, MIC range mutation in codon 87 versus mutation in codon 91. p Values were calculated by Fisher exact test.

The resistance of H. pylori to quinolones is due to point mutations in the gyrA gene coding for the DNA gyrase subunit. Susceptible strains have both an 87 and a 91 wild-type codon and this type was found in 60.9% of our strains. In the literature, an Asn(N)/Thr(T) susceptible polymorphism at codon 87 is described. The genotypes indicated as WT1 and WT2 had N87, while genotypes WT3 and WT4 had T87. It is reported that only 15% of the strains had T87.¹⁴ In our study, none of the 92 strains showed the presence of the T87 polymorphism. The genotype WT1 was found in 57.1% and the genotype WT2 in 42.9% of susceptible strains. A resistant phenotype shows a mutation in codon 87 or in 91. A mutation in gyrA gene was found in 39.1% of H. pylori strains. Mutations in codon 87 were found in 63.9% of the levofloxacin-resistant strains, the N87K mutation being detected in 91.3% and the T87I in the remaining 8.7%. The presence of the T87I mutation was confirmed by Next Generation Sequencing. Mutations in codon 91 were found in 36.1% of resistant strains. In particular, 46.1% of strains had the D91N mutation, 30.8% the D91Y mutation, and 23.1% the D91G mutation (Table 4). No association between a specific mutation and previous treatment, age, or gender was found (data not shown). The mutation in codon 87 is significantly associated with a higher MIC range (>6 mg/L, p-value = 0.005) (Table 5). No association was found between the type of mutation and previous treatment, age, or gender (data not shown). There was a concordance between phenotypic and genotyping results in detecting H. pylori resistance to clarithromycin and levofloxacin.

Discussion

H. pylori antibiotic resistance is a globally emerging problem and is the main factor contributing to treatment failure of H. pylori infection. In 2017, the World Health Organization (WHO) ranked clarithromycin-resistant-H. pylori as a high priority microbe that needs more attention, and designated it as high priority for new antibiotic development. Primary and secondary resistance rates to clarithromycin, metronidazole, and levofloxacin were ≥15% in almost all WHO regions and an increasing resistance is being observed.^7,16

Therefore, it is important to know the regional data regarding primary resistance to antibiotics used for the eradication of this infection. In addition, recent data have shown that the main genetic mutations involved in clarithromycin resistance may contribute to the success of therapy. It has been demonstrated that the A2143G mutation is associated to failure of therapy.^17,18 Therefore, it is important to know the type of mutation not only for epidemiological purposes but also for planning personalized tailored-therapy based on the specific resistance pattern. The rate of resistance varies from region to region depending on several factors, including the widespread use of macrolides, fluoroquinolones, and metronidazole to treat bacterial infections other than H. pylori infection.¹⁹

In the present study the overall clarithromycin, levofloxacin, and metronidazole resistance of H. pylori isolated from naïve patients was significantly lower than the resistance found in the treated patients. Levofloxacin resistance was detected mainly in older patients. This is probably due to the common use, in our area, of fluoroquinolones to treat genitourinary and respiratory tract infections. We hypothesize that the higher rate of clarithromycin resistance detected in treated women, compared to treated men, may be due to the use of clarithromycin to treat respiratory and sexually transmitted diseases or to poor compliance.

The rates of resistance found in our study are slightly higher than those reported in a European multicenter study that included our region, Bari, Italy, conducted in the period 2008–2009. In naïve patients the resistance to clarithromycin, levofloxacin, and metronidazole in Southern Europe was 21.5%, 13.1%, and 29.7%, respectively.⁸ The resistance rate found in the present study was quite similar to the one previously reported in the same area by our group in the period 2006–2012, except for resistance to clarithromycin, which was 50% in strains isolated from naïve patients.²⁰

A study conducted in Foggia, Southern Italy, showed a twofold increase (from 10% to 21.34%) in H. pylori primary clarithromycin resistance in the two periods considered; A2143G was the most prevalent mutation detected.²¹ A further study conducted in 2010 on patients in three geographic areas of Italy (Northern, Central, and Southern), using genotypical methods performed on paraffin-embedded gastric biopsies, reported an overall primary clarithromycin resistance of 9.9%, without significant differences among these areas, except for Tuscany and Apulia, which were the areas with higher percentages of resistance (19.1%) even if the number of patients examined for each geographic area was low. The most common point mutation detected was A2143G and a high prevalence of clarithromycin resistance was found in female patients.²²

Different data were reported in another study conducted in a Central region of Italy (Abruzzo) in the period 2010–2014, showing high rates of resistance (72.44% to clarithromycin, 34.7% to metronidazole, and 42% to levofloxacin).²³ It has been also reported that the rates of clarithromycin phenotypic resistance were lower in comparison to those detected by genotypic methods (18.4% vs. 37.6%) and the A2143G mutation was associated to a low eradication rate.¹⁷

Therefore, several studies have demonstrated a patchy distribution of resistance patterns even in the same country. This may be due to different methods used to assess clarithromycin resistance in H. pylori (genotypic methods often performed on paraffin-embedded samples or phenotypic detection using E-test or agar dilution methods), to a different local consumption of antibiotics or to the different mutations responsible for clarithromycin resistance.

The percentages of resistance found in our study are also different from those reported for metronidazole in an Italian study conducted in Bologna of 1,006 naïve patients in the period 2010–2015: 34.0% of strains were resistant to clarithromycin and 34.8% were resistant to metronidazole.²⁴ These differences may be due to the higher number of patients examined, to the different period evaluated, and finally, to a different consumption of these antibiotics.

These data were also confirmed by a study of 1,325 naïve patients observed in the same period in the same area. Resistance to clarithromycin was 36.1%, to metronidazole 38.6%, and 28.7% to levofloxacin. In addition, a significant increase in primary resistance over time was observed for all these antibiotics.²⁵ Another study collecting data from 1,424 naïve patients showed a significant increasing trend from 2010 to 2013 for all the antibiotics, whereas no increased resistance was observed in the period 2013–2016, suggesting that the prevalence of H. pylori primary resistance probably reached a plateau in the last years.²⁶

Several point mutations in the 23S rRNA gene have been described worldwide but only three (A2142G, A2143G, and A2142C) are responsible for most cases of clarithromycin resistance in many countries.¹⁰ Interestingly, resistance to clarithromycin and the mutations associated with it shows a different distribution among geographic areas even within the same regions, so further investigation of the regional differences in point mutation patterns in the 23S rRNA of H. pylori are needed. In Europe, several studies have dealt with the mutations associated with H. pylori clarithromycin resistance, the A2143G being most commonly found, followed by the mutation 2142.^27–29 As found in our study, the 2142 mutation was associated with a higher MIC value (>256 mg/L).³⁰ In Italy, the presence of six point mutations has been observed. A2143G was the most prevalent (52–59%), followed by A2142G (34.6–44%) and A2142C (52–59%).^17,22,31 Three novel point mutations associated to H. pylori clarithromycin resistance, never previously described in Western countries (A2144T, A2115G, and G2141A), were recently detected.¹⁰

In our area in Southern Italy, 54.8% of naïve H. pylori infected pediatric patients were infected with a clarithromycin-resistant strain and the point mutations detected were A2143G followed by A2142G. The A2143G mutation was significantly associated with a lower cure rate.³² In a previous study by our group the A2143G mutation, followed by A2142G and 2142C, were found in H. pylori infected patients.¹²

In Sicily, South Italy, the predominant point mutation observed was A2143G (80%), followed by A2142G (20%). No differences in the MIC values of the isolated mutation and type of mutation were observed.³³ High MIC values have been alternatively associated with either the A2143G or the A2142G point mutation.¹⁰ As found in our study, the 2142 mutation was associated with high MIC values.³⁴ It has been suggested that MIC levels to clarithromycin or metronidazole may play a role in H. pylori therapy outcome.²⁴

Currently, in many places in the world an increase in H. pylori resistance to levofloxacin is being observed.⁷ In Europe, the range of resistance to this antibiotic was 16–25%.^35,36 In a previous study by our group, only 6.0% of strains were resistant to levofloxacin and this may be related to the pediatric population considered, in which fluoroquinolones are very rarely prescribed.²⁰ In our study, a mutation in the 87 codon was the most frequently detected in H. pylori. Other mutations responsible for levofloxacin resistance in H. pylori have been described.^14,37 Interestingly, higher MIC values were found in strains with the 87 mutation.³⁷ This was confirmed also in our study: all strains with mutations at position 87 were characterized by high MIC values.

In our study, high rates of resistance to metronidazole were found, as reported by other Italian authors. Our results highlight also the concomitant resistance to clarithromycin, levofloxacin, and metronidazole particularly in treated patients. This is due to the use/abuse of these antibiotics for the treatment of H. pylori and other infections. This may pose a problem because metronidazole, together with tetracycline, are the antibiotics present in the last generation eradicating therapies.^3,5,38,39

Conclusions

H. pylori antibiotic resistance is a constantly evolving process and local surveillance is warranted to guide clinicians in the choice of therapy. In our opinion a further increase of rates of resistance can be expected, considering that these classes of antibiotics are widely used for the treatment of other infections. The choice of therapy should be based not only on the local pattern of resistance but also, if possible, on the genotype responsible for this resistance. The application of new technologies such as Next Generation Sequencing to studies of resistance in H. pylori will open new scenarios revealing the mechanisms of resistance in this bacterium, bearing in mind that new mechanisms of resistance to all antibiotics have been proposed and warrant extensive studies, also to ensure a better knowledge of the association between the type of mutations and MIC values.⁴⁰ The eradication of H. pylori infection remains a goal to decrease the burden of diseases caused by this bacterium also in our area. While waiting for new effective antibiotics, the H. pylori resistance pattern evaluated with phenotypic and molecular methods may allow the planning of personalized tailored therapy. The possibility to detect the mutations responsible for resistance also in material such as stool should reduce the time needed to detect the resistance of this bacterium.⁴¹

Authors' Contributions

V.P., R.M., and A.L. made substantial contributions to the conception and design of the study, experiments, and coordination; they supervised the article, isolation of H. pylori, and genotyping analysis of antimicrobial resistance. L.F. and A.P. contributed to bibliographic research, performed data analysis, and phenotypic analysis of resistance to antibiotics. A.P., R.C., O.B., S.M., and M.C. contributed to collect biological samples, to perform EGDS and RUT, and clinical diagnosis. A.P., L.C., and A.B. contributed to Library preparation, WGS, and in silico analysis of H. pylori strains. All authors read and approved the final article.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

Grant support of Ministero Della Salute (Ministry of Health) Ricerca Corrente 2019 No. Project: Line 3, Prog No. 20.

References

Montecucco

, and Rappuoli

. 2001. Living dangerously: how Helicobacter pylori survives in the human stomach. Nat. Rev. Mol. Cell. Biol. 2:457–466.

Malfertheiner

, Megraud

, O'Morain

C.A

, Gisbert

J.P

, Kuipers

E.J

, Axon

A.T

, Bazzoli

, Gasbarrini

, Atherton

, Graham

D.Y

, Hunt

, Moayyedi

, Rokkas

, Rugge

, Selgrad

, Suerbaum

, Sugano

, and El-Omar; European Helicobacter and Microbiota Study Group

E.M

and Panel

Consensus

. 2017. Management of Helicobacter pylori infection the Maastricht V/Florence Consensus Report. European Helicobacter and Microbiota Study Group and Consensus Panel. Gut, 66:6–30.

Zullo

, De Francesco

, Bellesia

, Vassallo

, D'Angelo

, Scaccianoce

, Sacco

, Bresci

, Eramo

, Tanzilli

, Ridola

, Alvaro

, Londoni

, Brambilla

, Manta

, Di Ciaula

, and Portincasa

. 2017. Bismuth-based quadruple therapy following H. pylori eradication failures: a multicenter study in clinical practice. J. Gastrointestin. Liver. Dis. 26:225–229.

Fiorini

, Saracino

I.M

, Zullo

, Gatta

, Pavoni

, and Vaira

. 2017. Rescue therapy with bismuth quadruple regimen in patients with Helicobacter pylori-resistant strains. Helicobacter, 22. DOI:10.1111/hel.12448.

De Francesco

, Pontone

, Bellesia

, Serviddio

, Panetta

, Palma

, and Zullo

. 2018. Quadruple, sequential, and concomitant first-line therapies for H. pylori eradication: a prospective, randomized study. Dig. Liver Dis. 50:139–141.

De Francesco

, Giorgio

, Hassan

, Manes

, Vannella

, Panella

, Ierardi

, and Zullo

. 2010. Worldwide H. pylori antibiotic resistance: a systematic review. J. Gastrointestin. Liver Dis. 19:409–414.

Savoldi

, Carrara

, Graham

D.Y

, Conti

, and Tacconelli

. 2018. Prevalence of antibiotic resistance in Helicobacter pylori: a systematic review and meta-analysis in World Health Organization regions. Gastroenterology, 155:1372–1382.

Mégraud

, Coenen

, Versporten

, Kist

, Lopez-Brea

, Hirschl

A.M

, Andersen

L.P

, Goossens

, and Glupczynski; Study Group Participants

. 2013. Helicobacter pylori resistance to antibiotics in Europe and its relationship to antibiotic consumption. Gut, 62:34–42.

Fontana

, Favaro

, Minelli

, Criscuolo

A.A

, Pietroiusti

, Galante

, and Favalli

. 2002. New site of modification of 23S rRNA associated with clarithromycin resistance of clinical isolates. Antimicrob. Agents Chemother. 46:3765–3769.

10.

De Francesco

, Zullo

, Giorgio

, Saracino

, Zaccaro

, Hassan

, Ierardi

, Di Leo

, Fiorini

, Castelli

, Lo Re

, and Vaira

. 2014. Change of point mutations in Helicobacter pylori rRNA associated with clarithromycin resistance in Italy. J. Med. Microbiol. 63:453–457.

11.

Mégraud

, Bénéjat

, Ontsira Ngoyi

E.N

, and Lehours

. 2015. Molecular approaches to identify Helicobacter pylori antimicrobial resistance. Gastroenterol. Clin. North Am. 44:577–596.

12.

Monno

, Giorgio

, Carmine

, Soleo

, Cinquepalmi

, and Ierardi

. 2012. Helicobacter pylori clarithromycin resistance detected by Etest and TaqMan real-time polymerase chain reaction: a comparative study. APMIS, 120:712–717.

13.

The European Committee on Antimicrobial Susceptibility Testing.. 2017. Breakpoint tables for interpretation of MICs and zone diameters. Version 7.1. Available at www.eucast.org/clinical_breakpoints (accessed November 1, 2018).

14.

Cambau

, Allerheiligen

, Coulon

, Corbel

, Lascols

, Deforges

, Soussy

C.J

, Delchier

J.C

, and Megraud

. 2009. Evaluation of a new test, genotype HelicoDR, for molecular detection of antibiotic resistance in Helicobacter pylori. J. Clin. Microbiol. 47:3600–3607.

15.

Gupta

S.K.

, Padmanabhan

B.R

, Diene

S.M

, Lopez-Rojas

, Kempf

, Landraud

, and Rolain

J.M.

. 2014. ARG-ANNOT, a new bioinformatic tool to discover antibiotic resistance genes in bacterial genomes. Antimicrob. Agents Chemother. 58:212–220.

16.

Abadi, A.T.B. 2017. Resistance to clarithromycin and gastroenterologist's persistence roles in nomination for Helicobacter pylori as high priority pathogen by World Health Organization. World J. Gastroenterol. 23:6379–6384.

17.

De Francesco

, Zullo

, Ierardi

, Giorgio

, Perna

, Hassan

, Morini

, Panella

, Vaira

. 2010. Phenotypic and genotypic Helicobacter pylori clarithromycin resistance and therapeutic outcome: benefits and limits. J. Antimicrob. Chemother. 65:327–332.

18.

Park

C.G.

, Kim

, Lee

E.J

, Jeon

H.S

, and Han

. 2018. Clinical relevance of point mutations in the 23S rRNA gene in Helicobacter pylori eradication: a prospective, observational study. Medicine (Baltimore). 97:e11835.

19.

Thung

, Aramin

, Vavinskaya

, Gupta

, Park

J.Y

, Crowe

S.E

, and Valasek

M.A.

. 2016. Review article: the global emergence of Helicobacter pylori antibiotic resistance. Aliment. Pharmacol. Ther. 43:514–533.

20.

Monno

, Fumarola

, Capolongo

, Calia

, Pazzani

, Ierardi

, and Miragliotta

. 2015. Susceptibility of Helicobacter pylori to antibiotics including tigecycline. J. Med. Microbiol. Diagn. Suppl. 5:005.

21.

De Francesco

, Margiotta

, Zullo

, Hassan

, Giorgio

, Burattini

, Stoppino

, Cea

, Pace

, Zotti

, Morini

, Panella

, and Ierardi

. 2007. Prevalence of primary clarithromycin resistance in Helicobacter pylori strains over a 15 year period in Italy. J. Antimicrob. Chemother. 59:783–785.

22.

De Francesco

, Giorgio

, Ierardi

, Zotti

, Neri

, Milano

, Varasano

, Luzza

, Suraci

, Marmo

, Marone

, Manta

, Mirante

V.G

, de Matthaeis

, Pedroni

, Manes

, Pallotta

, Usai

, Liggi

, Gatto

, Peri

, Sacco

, Bresci

, Monica

, Hassan

, and Zullo

. 2011. A Primary clarithromycin resistance in Helicobacter pylori: the Multicentric Italian Clarithromycin Resistance Observational (MICRO) study. J. Gastrointestin. Liver Dis. 20:235–239.

23.

Di Giulio

, Di Campli

, Di Bartolomeo

, Cataldi

, Marzio

, Grossi

, Ciccaglione

A.F

, Nostro

, and Cellini

. 2016. In vitro antimicrobial susceptibility of Helicobacter pylori to nine antibiotics currently used in Central Italy. Scand. J. Gastroenterol. 51:263–269.

24.

De Francesco

, Zullo

, Fiorini

, Saracino

I.M

, Pavoni

, and Vaira

. 2019. Role of MIC levels of resistance to clarithromycin and metronidazole in Helicobacter pylori eradication. J. Antimicrob. Chemother. 74:772–774.

25.

Gatta

, Scarpignato

, Fiorini

, Belsey

, Saracino

I.M

, Ricci

, and Vaira

. 2018. Impact of primary antibiotic resistance on the effectiveness of sequential therapy for Helicobacter pylori infection: lessons from a 5-year study on a large number of strains. Aliment. Pharmacol. Ther. 47:1261–1269.

26.

Fiorini

, Zullo

, Saracino

I.M

, Pavoni

, and Vaira

. 2018. Antibiotic resistance pattern of Helicobacter pylori strains isolated in Italy during 2010–2016. Scand. J. Gastroenterol. 53:661–664.

27.

Aguilera-Correa

J.J.

, Urruzuno

, Barrio

, Martinez

M.J

, Agudo

, Somodevilla

, Llorca

, and Alarcón

. 2017. Detection of Helicobacter pylori and the genotypes of resistance to clarithromycin and the heterogeneous genotype to this antibiotic in biopsies obtained from symptomatic children. Diagn. Microbiol. Infect. Dis. 87:150–153.

28.

Blümel

, Goelz

, Kist

, and Glocker

E.O.

. 2018. Retrospective study on outcome of salvage Helicobacter pylori eradication therapies based on molecular genetic susceptibility testing. Helicobacter, 23:e12494.

29.

Bilgilier

, Stadlmann

, Makristathis

, Thannesberger

, Kastner

M.T

, Knoflach

, Steiner

, Schöniger-Hekele

, Högenauer

, Blesl

, Datz

, Huber-Schönauer

, Schöfl

, Wewalka

, Püspök

, Mitrovits

, Leiner

, Tilg

, Effenberger

, Moser

, Siebert

, Hinterberger

, Wurzer

, Stupnicki

, Watzinger

, Gombotz

, Hubmann

, Klimpel

, Biowski-Frotz

, Schrutka-Kölbl

, Graziadei

, Ludwiczek

, Kundi

, Hirschl

A.M

, and Steininger; Austrian Helicobacter Study Group of the Austrian Society of Gastroenterology and Hepatology

. 2018. Prospective multicentre clinical study on inter- and intrapatient genetic variability for antimicrobial resistance of Helicobacter pylori. Clin. Microbiol. Infect. 24:267–272.

30.

Ducournau

, Bénéjat

, Sifré

, Bessède

, Lehours

, and Mégraud

. 2016. Helicobacter pylori resistance to antibiotics in 2014 in France detected by phenotypic and genotypic methods. Clin. Microbiol. Infect. 22:715–718.

31.

Toracchio

, Aceto

G.M

, Mariani-Costantini

, Battista

, and Marzio

. 2004. Identification of a novel mutation affecting domain V of the 23S rRNA gene in Helicobacter pylori. Helicobacter, 9:396–399.

32.

Francavilla

, Lionetti

, Castellaneta

, Margiotta

, Piscitelli

, Lorenzo

, Cavallo

, and Ierardi

. 2010. Clarithromycin-resistant genotypes and eradication of Helicobacter pylori. J. Pediatr. 157:228–232.

33.

Fasciana

, Calà

, Bonura

, Di Carlo

, Matranga

, Scarpulla

, Manganaro

, Camilleri

, and Giammanco

. 2015. Resistance to clarithromycin and genotypes in Helicobacter pylori strains isolated in Sicily. J. Med. Microbiol. 64:1408–1414.

34.

Klesiewicz

, Nowak

, Karczewska

, Skiba

, Wojtas-Bonior

, Sito

, and Budak

. 2014. PCR-RFLP detection of point mutations A2143G and A2142G in 23S rRNA gene conferring resistance to clarithromycin in Helicobacter pylori strains. Acta Biochim. Pol. 61:311–315.

35.

Morilla

A.M.

, M.E. Álvarez-Argüelles, Duque

J.M

, Armesto

, Villar

, and Melón

. 2019. Primary antimicrobial resistance rates and prevalence of Helicobacter pylori infection in the north of Spain. A 13-year retrospective study. Gastroenterol. Hepatol. 42:476–485.

36.

Delchier

J.C.

, Bastuji-Garin

, Raymond

, Megraud

, Amiot

, Cambau

, and Burucoa

; HELICOSTIC Study Group. 2019. Efficacy of a tailored PCR-guided triple therapy in the treatment of Helicobacter pylori infection. Med. Mal. Infect. pii: S0399-077X(18)30185-9.

37.

Bińkowska

, Biernat

M.M

, Ł. Łaczmański, and Gościniak

. 2018. Molecular patterns of resistance among Helicobacter pylori. strains in South-Western Poland. Front. Microbiol. 9: , 3154.

38.

Di Ciaula

, Scaccianoce

, Venerito

, Zullo

, Bonfrate

, Rokkas

, and Portincasa

. 2017. Eradication rates in Italian subjects heterogeneously managed for Helicobacter pylori infection. Time to abandon empiric treatments in Southern Europe. J. Gastrointestin. Liver Dis. 26:129–137.

39.

Fiorini

, Zullo

, Saracino

I.M

, Gatta

, Pavoni

, and Vaira

. 2018. Pylera and sequential therapy for first-line Helicobacter pylori eradication: a culture-based study in real clinical practice. Eur. J. Gastroenterol. Hepatol. 30:621–625.

40.

Zanotti

, and Cendron

. 2019. Structural aspects of Helicobacter pylori antibiotic resistance. Adv. Exp. Med. Biol. 1149:227–241.

41.

Iannone

, Giorgio

, Russo

, Riezzo

, Girardi

, Pricci

, Palmer

S.C

, Barone

, Principi

, Strippoli

G.F.M

, Di Leo

, and Ierardi

. New fecal test for non-invasive Helicobacter pylori detection: a diagnostic accuracy study. 2018. World J. Gastroenterol. 24:3021–3029.