JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 3703–3707 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00144-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.
High Prevalence of Clarithromycin-Resistant Helicobacter pylori Strains and Risk Factors Associated with Resistance in Madrid, Spain
1211Sonia Agudo,* Guillermo Pe?rez-Pe?rez,Teresa Alarco?n,and Manuel Lo?pez-Brea
1Hospital de la Princesa, Universidad Auto?noma de Madrid, Madrid, Spain,and Departments of 2Medicine and Microbiology, NYU School of Medicine, New York, New York
Received 22 January 2010/Returned for modi？cation 30 March 2010/Accepted 19 July 2010
Clarithromycin is one of the antibiotics used for the treatment of Helicobacter pylori infections, and clari-
thromycin resistance is the most important factor when it comes to predicting eradication failure. The present study analyzed H. pylori isolates for the presence of 23S rRNA gene mutations and determined the risk factors associated with resistance among H. pylori isolates collected in Madrid, Spain, in 2008. We studied 118 H. pylori strains isolated from the same number of patients. A total of 76.3% of the patients were born in Spain, 52.7% were children, 20.3% had previously been treated, and 66.1% were female. Clarithromycin resistance was
determined by Etest. H. pylori strains were considered resistant if the MIC was >1 mg/liter. DNA extraction was carried out by use of the NucliSens easyMAG platform with NucliSens magnetic extraction reagents (bioMe?rieux). The DNA sequences of the 23S rRNA genes of clarithromycin-resistant and -sensitive strains
were determined to identify speci；c point mutations. The vacA genotype and cagA status were determined by PCR. We found that 42 (35.6%) strains were resistant to clarithromycin by Etest. Etest results were con；rmed by detection of the presence of point mutations in 34 (88.1%) of these strains. Eight H. pylori strains were resistant to clarithromycin by Etest but did not have a point mutation in the 23S rRNA gene. Mutation at A2143G was found in 85.3% of the strains, mutation at A2142G in 8.8%, and mutation at T2182C in 5.9%. Dual mutations were found in 8.8% of the strains. H. pylori clarithromycin-resistant strains were strongly associated with pediatric patients, with patients born in Spain, and with patients who had previously been treated (P < 0.02). In addition, H. pylori strains resistant to clarithromycin more frequently presented the vacA s2/m2 genotype and were more likely to be cagA negative than susceptible strains (39.1% and 11.2%, respectively; P value < 0.001). We concluded that, in the present study, H. pylori clarithromycin-resistant strains are more frequently found in children, in patients mostly born in Spain, and in individuals who were previously treated for H. pylori infection and that these individuals are more likely colonized with a less virulent H. pylori strain.
Helicobacter pylori is a microaerobic, Gram-negative spiral North America, 16% in Japan, and 1.7% to 23.4% in Europe bacterium that colonizes the human stomach and is found in (14, 19, 21). Overall, resistance to clarithromycin has been more than half of the world’s population (32). Infections with detected more in patients living in the south than in those H. pylori are closely associated with chronic gastritis, peptic living in the north of Europe (21). Fewer studies have focused ulcer disease, and the development of gastric cancer (8, 32). on the prevalence of clarithromycin resistance among H. pylori All consensus guidelines recommend eradication of H. pylori strains from children compared with that among strains from for patients with symptoms (9, 28). Standard therapy combines the adult population. These studies will be useful for estimat- a proton pump inhibitor (PPI) or ranitidine bismuth citrate ing the rate of clarithromycin-resistant H. pylori isolates among and two antibiotics, chosen from among amoxicillin, clarithro- children and adults in Spain in the future (1, 23). mycin, and metronidazole (24, 25). However, this therapy has Clarithromycin acts by binding to the peptidyltransferase been questioned because of the increased eradication failure region of 23S rRNA and inhibits protein synthesis (36). The rates. Many factors have been implicated as causes of treat- resistance to clarithromycin in H. pylori has been shown to be ment failure, including ineffective penetration of antibiotics due to point mutations in the peptidyltransferase region of into the gastric mucosa, antibiotic inactivation by the low stom- domain V of the 23S rRNA. Two copies of the 23S rRNA gene ach pH, a lack of patient compliance, and the emergence of are present in H. pylori, and the most common mutation is an acquired resistance to antibiotics by H. pylori (26, 27). A-to-G transition at position 2143 (A2143G) (13, 36), but In many cases, clarithromycin is the key component of these several point mutations, at positions A2142G, A2144G, and combination therapies. However, resistance to clarithromycin T2182C, have been described. Recent reports have indicated has become one of the major reasons for treatment failure that other mutations, such as A2115G, G2141A, C2147G, (13). The prevalence of H. pylori resistance to clarithromycin T2190C, C2195T, A2223G, and C2694A, might also be asso- varies among different countries, such as 10.6% to 25% in ciated with clarithromycin resistance (20, 31). Other mecha- nisms of resistance, such as methylase production, the actions of macrolide-inactivating enzymes, and active ef？ux, have been * Corresponding author. Mailing address: Department of Microbi- described in several bacteria. Active ef？ux has also recently ology, Hospital Universitario de la Princesa, Diego de Leo?n, 62, 28006 been described in H. pylori (22). Madrid, Spain. Phone and fax: 0034915202317. E-mail: soniaagu Since the worldwide increase in the rate of clarithromycin @hotmail.com. resistance represents a problem of relevance, some studies Published ahead of print on 28 July 2010.
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3704 AGUDO ET AL. J. CLIN. MICROBIOL.
Epsilometer test (Etest; AB Biodisk, Solna, Sweden) on Mueller-Hinton sheep have been performed in order to identify its relationship with blood agar plates (BBL). Plates with strips containing clarithromycin were incu- bacterial genetic factors (12, 35, 38). bated for 72 h under microaerophilic conditions. The MIC was considered the Two genes (cytotoxin-associated gene A [cagA] and vacuo- lowest concentration of drug which inhibited visible growth and was read as the lation-associated gene A [vacA]) have been identi？ed to be the intercept of the elliptical zone of inhibition with the graded strip for the Etest. main virulence factors. cagA is located in the cag pathogenicity On the basis of CLSI recommendations (10), strains were resistant if the MIC was 1 mg/liter and intermediate if the MIC was 0.5 mg/liter. Strains with MICs island (PAI), which encodes a type IV secretion system, and below those thresholds were considered susceptible. the presence of cagA is closely associated with more severe Determination of clarithromycin resistance by genotypic methods. To detect gastric diseases (2, 15, 34). The VacA toxin induces vacuole speci？c mutations for clarithromycin resistance in the 23S rRNA gene, PCR formation in the host cells. There is considerable variation in methods were carried out. Primers were designed from the 23S rRNA gene of vacuolation activity among H. pylori strains, primarily due to reference strain of H. pylori 26695. The primers used included 5 -CCACAGCG ATGTGGTCTCAG (sense, positions 1891 to 1911) and 5 -CTCCATAAGAG differences in the vacA gene structure in the signal region (s1 CCAAAGCCC (antisense, positions 2200 to 2220). The numbers represent the and s2) and the middle region (m1 and m2). vacA s1/m1 and positions in the 23S rRNA gene of H. pylori 26695. Ampli？cation was carried out s1/m2 produce high and moderate levels of VacA toxin, re- in a thermal cycler (MBS O.5 S; Thermo Hybaid). PCR cycling conditions spectively, whereas s2/m2 produces little or no toxin (11). A consisted of 35 cycles of 30 s of denaturation at 94?C, 30 s of annealing at 60?C, strong association between clarithromycin susceptibility and and 30 s of extension at 72?C. The PCR products were puri？ed using a QIAquick PCR puri？cation kit (Qiagen, Valencia, CA). Sequencing reactions were per- these virulence factors has been reported (12, 38). formed by the Macrogen USA Company (Baltimore, MD) and were carried out The focus of the present study was to evaluate the distribu- in a BigDye Terminator cycle sequencing kit. Computer sequence alignments tion of clarithromycin-resistant H. pylori strains and their as- were performed with the Clustal program (European Bioinformatics Institute sociation with genotypic markers, such as the cagA gene and [http://www.ebi.ac.uk/clustalw/]), and sequence comparisons were carried out with the Genedoc program (www.nrbsc.org/gfx/genedoc/index.htlm). allelic variants of the vac gene. We also examined the distri- An extra PCR ampli？cation was performed on eight strains that showed bution of H. pylori clarithromycin resistance in relation to the discrepancies between phenotypic and genotypic methods. We designed primers patient’s age, place of birth, and history of treatment. Our derived from the 23S rRNA sequence of the H. pylori 26695 reference strain main goal is to determine potential host and bacterial factors (GenBank accession number U27270; sense primer, 5 -TCCTCTCGTACTA that may help in predicting resistance to clarithromycin among GGGA [positions 2057 to 2074]; antisense primer, 5 -CTGCATGAATGGCGT AACGAG [positions 2731 to 2752]). PCR products were sequenced to identify H. pylori isolates. new mutations or mutations different from those published previously (20, 31). Assessment of cagA and vacA status and gene ampli；cation. The puri？ed DNA from 118 H. pylori strains was subjected to PCR for detection of the H. MATERIALS AND METHODS pylori cagA gene, using the CagA primers described by Panayotopoulou et al. Patients. A total of 118 H. pylori clinical strains were studied, including 61 (29), which ampli？ed a region of between 360 and 700 bp. The signal peptide (s) obtained from children who were 18 years of age (27 males, 34 females) and 57 region of the vacA gene was ampli？ed using the primers described by Atherton obtained from adults 18 years of age (13 males, 44 females). The strains were et al. (6), which evaluated the region encoding the s region of the gene. The isolated from gastric biopsy specimens obtained during upper gastroduodenal midregion (m) was ampli？ed using the primers described by G. Perez-Perez et al. endoscopy from two children’s hospitals (Hospital Infantil Universitario Nin?o (unpublished data; sense primer, 5 -CAATCTGTCCAATCAAGCGAG; anti- Jesu?s and Hospital Universitario Doce de Octubre, Madrid, Spain) and an adult sense primer; 5 -TGAGTTGTTTGATATTGAC). Four different PCR products hospital (Hospital Universitario de la Princesa, Madrid) from May 2008 to were obtained: s1 (176 bp), s2 (200 bp), m1 (290 bp), and m2 (350 bp). December 2008. Most of the patients (76.3%) were born in Spain, and 20.3% of Statistical analysis. Differences between groups were statistically evaluated by them had previously been treated for H. pylori infections. However, the patients using the chi-square test with Yate’s correction. Differences were considered had not received PPIs or antibiotics for at least 2 weeks prior to the endoscopy signi？cant at the 5% probability level. Statistical analysis was performed using and before initiation of any new therapy. Each patient or the patient’s parents speci？c software (Epi Info; www.cdc.gov/epiinfo). signed an informed-consent form that was previously approved by the ethics committee of each hospital. Clinical isolates. Samples for culture were placed in sterile saline solution for RESULTS transport. Biopsy specimens were received at the Department of Microbiology (Hospital Universitario de la Princesa) and processed in less than 3 h from the Clarithromycin resistance. The assessment of clarithromy- time that the biopsy specimens were obtained. Tissue was streaked onto both cin susceptibility by Etest showed that 75 patients (63.6%) nonselective medium (Columbia agar with 5% sheep blood; BioMe`rieux, Marcy l’Etoile, France) and selective medium (Pylori agar; BioMe`rieux) and incubated were infected with H. pylori-susceptible strains and 42 (35.6%) for 10 days at 37?C in a microaerobic atmosphere (5% O, 10% CO, 85% N) 222with H. pylori-resistant strains. To con？rm the high prevalence which was produced by a microaerobic generator envelope (GasPak EZ gas of resistance to clarithromycin, we determined the correlation generator; Becton Dickinson and Company, MD). Isolates were presumptively between the results of Etest and sequence analysis of the 23S identi？ed as H. pylori on the basis of colony morphology, (gray, small, and translucent colonies after 3 to 4 days of incubation on blood agar), positive rRNA gene. There was a strong association between the pres- biochemical reactions (cytochrome oxidase, catalase, and urease), and Gram ence of 23S rRNA gene mutations and macrolide resistance. staining (curved rod), as recommended elsewhere (5, 17, 18). Overall, 34 of the 42 (81%) clarithromycin-resistant strains DNA extraction. The total bacterial genomic DNA of the 118 isolates was contained at least one point mutation in their 23S rRNA se- extracted by using a NucliSens easyMag instrument (BioMe`rieux), according to the manufacturer’s instructions. Brie？y, the bacterial cell suspension was pre- quences. We also tested 20 clarithromycin-susceptible H. pylori treated with lysis buffer. The lysed sample was transferred to a plastic vessel with strains and found no point mutation in any of the 23S rRNA 550 l of silica. This was followed by automatic magnetic separation. Nucleic acid sequences. Thus, using classical and molecular methods, we was recovered in 55 l elution buffer. The DNA concentration was determined demonstrated that 27.2% of H. pylori strains in Madrid were on a NanoDrop instrument (NanoDrop Technologies, Wilmington, DE). Determination of clarithromycin resistance by phenotypic methods. We pre- clarithromycin resistant. There was a discrepancy between the pared 48-h cultures of H. pylori that were suspended in sterile saline and adjusted two methods for eight (19%) strains; the strains were resistant to a density equal to McFarland turbidity standard of 3, as recommended by to clarithromycin by Etest, but no point mutation was identi- CLSI (10). The bacterial suspensions were spread onto Mueller-Hinton blood ？ed in the sequence of the 23S rRNA gene. In addition, for agar plates (BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD) one strain it was not possible to perform the Etest, and the with sterile cotton swabs. The MIC of each isolate was determined by the
VOL. 48, 2010 CLARITHROMYCIN-RESISTANT H. PYLORI STRAINS IN SPAIN 3705
TABLE 1. vacA genotype distribution among 118 cagA-positive and TABLE 2. Association between clarithromycin susceptibility and
cagA-negative H. pylori strains cagA status among 117 H. pylori strains isolated from Spain
No. (%) of strains No. (%) of strains
Genotype P cagA cagA negative Clarithromycin Clarithromycin Total cagA positive Total Discrepant status (n 44) (n 74) susceptible resistant (n 117) (n 8) a(n 34) (n 75) 35 (79.6) 3 (4.0) s1/m1 or s1/m2 38 P 0.0001 Positive 44 36 (81.8) 5 (11.4) 3 (6.8) s2/m2 72 3 (6.8) 69 (93.2) P 0.0001 aNegative 73 39 (53.4) 29 (39.7) 5 (6.8) s2/m1 4 3 (6.8) 1 (1.4) NS Mix 4 3 (6.8) 1 (1.4) NS a For the values of 81.8% versus 53.4%, P 0.001 (chi-square test). a NS, not signi？cant. from patients born in Spain were more often resistant to clari- thromycin than strains isolated from patients born outside results for that strain were excluded from further statistical Spain (33.7% and 14.3%, respectively; P 0.05). analysis. The analysis of patients previously treated and those never The predominant mutations among the 34 H. pylori clari- treated for H. pylori infections con？rmed that H. pylori isolates thromycin-resistant strains were at A2143G in 26 cases from previously treated patients were more often resistant to (76.6%), A2142G in 2 cases (5.8%), and T2182C in 2 cases clarithromycin than H. pylori strains isolated from patients who (5.8%), while double point mutations of A2143G plus T2182C had not received any treatment (58.3% and 21.5%, respec- were found in 3 cases and a double point mutation of A2142G tively; P 0.0002). plus T2182C was found in 1 case. In addition, there were no Analysis of a possible association between H. pylori clari- differences in the MIC values among isolates with a point thromycin resistance and the gender of the patient showed that mutation in A2143G, A2142G, or T2182C. females and males were infected by resistant isolates in almost Clarithromycin resistance relationship with cagA gene and identical proportions (30.8% and 25%, respectively). vacA mosaicism distribution. The cagA and vacA (s and m region) genotypes of the 118 H. pylori strains were assessed.
The cagA gene was detected in 44 strains (37.3%). In the case DISCUSSION of the vacA gene, we found that 38 (32.2%) of the strains had H. pylori eradication usually entails the use of a proton pump the s1 signal. The s1/m1 combination was observed in 22
(18.6%) strains, s1/m2 was found in 16 (13.6%), and s2/m2 was inhibitor or bismuth salts in combination with two antibiotics. found in 72 (61.1%), while the remaining rare combination, Resistance of H. pylori to antimicrobials agents is the main s2/m1, was found in 4 (3.3%) strains. A single mosaic combi- cause of treatment failure. Clarithromycin is recognized as the nation of the s/m region was observed in 114 (96.6%) of the key antibiotic for H. pylori treatment since it has the most
strains studied, while a colonization with a double vacA mosaic powerful bactericidal effect in vitro compared to the effects of
combination was detected in 4 (3.3%) cases. The distribution the other available antibacterial agents. Unfortunately, the of vacA genotypes according to cagA gene status is presented level of primary clarithromycin resistance is increasing world- in Table 1. As expected, the s2/m2 vacA genotype was more wide, and the level of resistance to clarithromycin varies be- frequently detected in H. pylori cagA-negative strains than in tween different geographical regions. The severity of gastric cagA-positive strains (93.2% and 6.8%, respectively; P in？ammation, the dosage of proton pump inhibitor, and the 0.0001). pathology (peptic ulcer disease versus non-peptic ulcer dis- The distribution of H. pylori genotypes in relation to clari- ease) also affect the outcome of therapy (33, 38). thromycin resistance showed that H. pylori strains that were The ？rst relevant ？nding of the present study is that the rate cagA positive were more often susceptible to clarithromycin of H. pylori resistance to clarithromycin is high, occurring in (81.8% versus 53.4% for cagA-negative strains; P 0.001), as nearly 30% of the strains isolated in our geographic area. Spain shown in Table 2. Furthermore, as expected, clarithromycin has one of the highest levels of clarithromycin resistance re- resistance in H. pylori isolates was strongly associated with ported in Europe (1, 21). Such an observation is important in vacA s2/m2 (38.9% and 13.1%, respectively; P 0.001). order to treat patients. Patients born in Spain were more often
colonized with resistant strains than immigrant patients born Relationship of clarithromycin resistance with host factors.
outside Spain. The majority of these patients are from South The results of analysis of H. pylori clarithromycin resistance in
relation to age, place of birth, prior treatment, and gender of America or Eastern European countries, where the level of the patient from whom the H. pylori strain was isolated are resistance to clarithromycin is lower than that in the south of shown in Table 3. Europe, and for this reason they may be infected with less The results for H. pylori isolates with different clarithromycin resistant strains (4, 7, 16, 37).
susceptibility patterns according to patient age showed that H. Clarithromycin resistance in H. pylori mainly results from
pylori strains recovered from children were more often resis- point mutations in the peptidyltransferase loop region of the tant to clarithromycin than strains isolated from adults (40.0% 23S rRNA gene. In this study, the most frequent point muta- and 17.5%, respectively; P 0.003). tion found was at position A2143G in 85% of our strains. This We found a relationship between clarithromycin susceptibil- mutation predominates in H. pylori strains isolated from Eu-
ity in the H. pylori isolates and the place of birth of the patients rope and also from Japan (13, 19). The strong association from whom the bacteria were isolated. H. pylori strains isolated between resistance to macrolides and speci？c mutations in the
3706 AGUDO ET AL. J. CLIN. MICROBIOL.
TABLE 3. Association between clarithromycin susceptibility and cagA status, age, place of birth, and prior treatment
No. (%) of patients
Characteristic Clarithromycin susceptible Clarithromycin resistant Total (n 117) Discrepant (n 8) (n 75) (n 34) Children 30 (50.0) 24 (40.0) 60 6 (10.0) Adults 57 45 (78.9) 10 (17.5) 2 (3.5) Spanish 89 53 (59.6) 30 (33.7) 6 (6.7) Not Spanish 28 22 (78.6) 4 (14.3) 2 (7.1) Prior treatment 24 8 (33.3) 14 (58.3) 2 (8.3) No prior treatment 93 66 (71.0) 20 (21.5) 7 (7.5)
characterize H. pylori virulence in relation to disease severity. 23S rRNA gene was con？rmed in 81% of the cases. However,
Several epidemiological studies have shown geographical vari- there were discrepancies between the results of sequence anal-
ysis of the 23S rRNA gene and clarithromycin resistance tested ations in its virulence factors, such as the cagA locus and the
by Etest for eight strains. All eight strains were resistant by mosaic combination of the vacA gene alleles. The assessment
Etest but did not present any of the point mutations in the 23S of vacA gene mosaicism found all possible combinations; the rRNA gene sequence described above. PCR ampli？cation was s2/m1 mosaicism is rare, but has been reported before (33). performed in order to amplify the conserved 695-bp region of s2/m2 was the vacA mosaicism detected the most often, and H. pylori 23S rRNA to look for a point of mutation in these this combination was detected more frequently in cagA-nega-
strains. tive strains than in cagA-positive strains. The presence of cagA
Recent reports indicated that other mutations, such as is strongly associated with the presence of the vacA s1 allele.
A2115G, G2141A, C2147G, T2190C, C2195T, A2223G, and The prevalence of cagA and the s1 and m1 vacA alleles found
C2694A, might also be associated with clarithromycin resis- in our series was similar to that observed in other studies in our tance (20, 31). For this reason, we performed a second PCR region (3). This distribution differs from that observed in other that ampli？ed a larger region of the 23S rRNA gene in order countries, such as the United States, where s1/m1 and s2/m2
to look for these point mutations, but we did not ？nd any of are equally prevalent, and Germany and the United Kingdom, them. On the basis of those results, we suggested that in these where s1/m1 and s1/m2 are the most frequent combinations, strains other mechanisms which are not related to the 23S respectively (11).
rRNA gene sequence, such as the presence of an ef？ux pump, Our results indicate a difference in sensitivity to clarithro- may play a role in resistance to clarithromycin, as has been mycin between the H. pylori genotypes. The present study in- described by other authors (22). dicated that s2/m2 strains, which are mostly cagA negative,
An expected ？nding of this study was the relationship be- seem to be more resistant to clarithromycin than strains with tween clarithromycin resistance and age. The rate of clarithro- the s1/m1 and s1/m2 mosaic combinations, which are mostly mycin resistance was signi？cantly higher in children than cagA positive. It suggests that these genetic patterns could adults, and this type of association has been reported previ- provide a selective advantage during bacterial replication. Iso- ously (30). This disparity in resistance rates seems to be cor- lates that are cagA positive, a genotype which is thought to be related to the national level of macrolide consumption, since a more virulent, may damage the gastric epithelium more, and it crossover resistance mechanism among different types of mac- is conceivable that antibiotics can reach higher concentrations rolides develops rapidly. New macrolides were marketed in in in？amed mucosa. The presence of s2/m2 strains that induce
Spain at the beginning of the 1990, and clarithromycin began less in？ammation in the host gastric epithelia may be a factor to be marketed in 1991. Children have had more exposure to contributing to reduced antibiotic delivery and could hamper macrolides, and nowadays, respiratory infections in young chil- the eradication of H. pylori. Another possible explanation, ac- dren are very frequently treated with this group of antibiotics cording to the results of other studies, is that antibiotic activity (1, 23). interferes with the metabolism of a dividing cell, and cagA-
The antibiotic resistance of H. pylori is the most important positive strains may proliferate faster than cagA-negative ones
reason for failure of its eradication. Patients who received and would therefore be more susceptible to antibiotics (38). treatment against H. pylori were colonized with a clarithromy- In conclusion, the prevalence of H. pylori clarithromycin
cin-resistant strain more often than patients who did not re- resistance is high in our area. Being born in Spain, being a child ceive treatment. This antibiotic is the one most frequently previously treated for H. pylori infection, or being colonized included in the standard triple therapies for H. pylori eradica- with a cagA-negative strain was correlated with the presence of tion, and treatment failure was most often explained because a clarithromycin-resistant strain.
the original strains were resistant to clarithromycin in many
cases (13). To avoid treatment failure and the consequent ACKNOWLEDGMENTS development of secondary resistance, it is important to choose
the most appropriate ？rst-line treatment regimen. This choice This study was supported in part by Fondo de Investigaciones Sani- should be made on the basis of knowledge of the antimicrobial tatias grants FIS 05/2442 and FIS 05/2452 and an NIH grant (grant resistance peculiar to a given geographical area. p60MD000538-05) to the NYU Center for the Study of Asian Amer- The cagA and vacA genotype markers are widely used to ican Health.
VOL. 48, 2010 CLARITHROMYCIN-RESISTANT H. PYLORI STRAINS IN SPAIN 3707
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