00. editorialv5.qxd

Originales
in Helicobacter pylori clinical isolates 3 Hospital Infantil Universitario Niño Jesús Objective. To determine the primary and secondary re-
Alto porcentaje de resistencia a claritromicina
sistance to several antimicrobial agents in Spanish Helico - y metronidazol en aislamientos clínicos de
bacter pylori clinical isolates obtained from paediatric pa- Helicobacter pylori procedentes de pacientes
tients from January 2002 to June 2006.
pediátricos
Methods. Samples were collected from gastric biopsies
of symptomatic paediatric patients and H. pylori cultured Introducción. Determinar la resistencia primaria y
according to standard microbiological procedures. Resis- secundaria a varios agentes antibióticos en aislamientos tance was determined by E-test. Strains were considered re- clínicos de Helicobacter pylori procedentes de pacientes sistant if minimal inhibitory concentration (MIC) ≥ 2 mg/l pediátricos desde enero de 2002 a junio de 2006.
for amoxycillin, ≥ 4 mg/l for tetracycline, ≥ 8 mg/l formetronidazole, ≥ 1 mg/l for clarithromycin, MIC ≥ 4 mg/l Métodos. Las muestras fueron biopsias gástricas pro-
for ciprofloxacin, MIC ≥ 32 mg/l for rifampicin and interme- cedentes de pacientes pediátricos sintomáticos. H. pylori diate if MIC = 0.5 mg/l for clarithromycin, and MIC = 2 mg/l fue cultivado acorde con los estándares de los procedi- mientos microbiológicos. La resistencia a antibióticos fuedeterminada mediante E-test. Los aislamientos fueron con- Results. A total of 101 patients were included: 38 ma-
siderados resistentes si la CMI ≥ 2 mg/l para amoxicilina, les and 63 females (sex ratio M/F: 0.6). Average age was ≥ 4 mg/l para tetraciclina, ≥ 8 mg/l para metronidazol, 10 years (range: 4-18 years). All strains were susceptible to ≥ 1 mg/l para claritromicina, ≥ 4 mg/l para ciprofloxacino amoxycillin, tetracycline and rifampicin, 35.7% were resis- y ≥ 32 mg/l para rifampicina, y fue considerado intermedio tant to metronidazole, 54.6% to clarithromycin and 1.8% to si la concentración mínima inhibitoria (CMI) = 0,5 mg/l ciprofloxacin. 2.0% were intermediate to clarithromycin para claritromicina, y CMI = 2 mg/l para ciprofloxacino.
and 1.8% to ciprofloxacin. Double resistance to metronida-zole and clarithromycin rated at 17.2%. Thirty-five patients Resultados. De los 101 pacientes incluidos en el estu-
(34.7%) had a history of treatment failure, and were con- dio: 38 fueron hombres y 63 mujeres. La media de edad sidered as secondary H. pylori. Primary resistance rates to fue de 10 años. Todas las cepas fueron sensibles a amoxi- metronidazole and clarithromycin were 32.8% and 49.2%, cilina, tetraciclina y rifampicina. El 35,7% de los aisla- respectively, and secondary resistance rates were 41.2% and mientos fueron resistentes a metronidazol, el 54,6% fue- ron resistentes a claritromicina y el 1,8% a ciprofloxacino.
El 17,2% de los aislamientos tuvieron doble resistencia a Conclusions. Resistance to clarithromycin (56.6%) was
metronidazol y claritromicina. Treinta y cinco pacientes higher than to metronidazole (35.7%) in the H. pylori (34,7%) tuvieron un fallo en el tratamiento frente a strains studied. Clarithromycin resistance was very high H. pylori, previamente. La resistencia primaria a metroni- even in strains from paediatric patients not previously dazol y claritromicina fue del 32,8 y 49,2%, respectiva- treated for H. pylori infection.
mente, y la resistencia secundaria fue de un 41,2 y 70,6%, Key words:
Primary resistance. Paediatrics. Ciprofloxacin. Rifampicin. Treatment failure.
Conclusión. La resistencia a claritromicina (56,6%)
Rev Esp Quimioter 2009;22(2):88-92 fue más alta que a metronidazol (35,7%) en los aislamien-tos clínicos de H. pylori estudiados. La resistencia a clari-tromicina fue alta incluso en los pacientes que no habían tenido tratamientos previos frente a H. pylori. Departamento de MicrobiologíaHospital Universitario de La Princesa Palabras clave:
Resistencia primaria. Pediátricos. Ciprofloxacino. Rifampicina. Tratamiento fracasado.
28006 Madrid (Spain)Correo electrónico: soniaagu@hotmail.com High percentage of clarithromycin and metronidazole resistance in Helicobacter pylori clinical isolatesobtained from Spanish children INTRODUCTION
Samples collection and identification
Helicobacter pylori is a microaerophilic spiral-shaped Biopsies were received at the Department of Microbiolo- gy (Hospital Universitario de La Princesa, Madrid) and about 0.5 µm with 4-6 flagellas and it is found in the gas- processed before 3 h. Samples for culture were placed in ster- tric mucous layer or adherent to the epithelial lining of the ile saline solution for transport. Biopsy tissue was chopped into stomach. The organism has an abundant urease enzyme smaller pieces and homogenised under aseptic conditions. Ho- production, which is important for colonization, due to the mogenised tissue was streaked onto both non-selective (Co- formation of ammonia on the gastric mucosa, increasing lumbia agar, with 5% sheep blood; BioMérieux) and selective the pH of its environment. This enzyme is also important for media (Pylori agar; BioMérieux) incubated 10 days at 37 oC in a microaerophilic atmosphere (5% O , 10% CO , 85% N ). Iso- lates were identified as H. pylori-based on colony morphol- H. pylori has a special affinity for gastric mucosa and is ogy (small, grey and translucent), positive biochemical reac- associated with various digestive diseases, such as peptic ul- tions for urease, catalase and oxidase test, and negative Gram cer (duodenal and gastric), chronic active gastritis and mu- staining. Colonies of 48 h H. pylori cultures were suspended cosa- associated lymphoid tissue and it is considered a risk in sterile saline and adjusted to a density equal to McFarland factor in the development of the gastric cancer.1-3 H. pylori turbidity standard.3 The suspensions were spread onto the plates is most frequently acquired during childhood and usually with sterile cotton swabs. The minimal inhibitory concentra- persists throughout life, causing different digestive diseases tions (MICs) of isolates were determined by the Epsilometer in childhood and adulthood unless a specific treatment is test (E-test; AB Biodisk, Solna, Sweden) on Mueller Hinton sheep given (spontaneous eradication is rare).
blood agar (BBL, Becton Dickinson Microbiology Systems, Cock-eysville, MD, USA). Plates with strips containing amoxycillin, Treatment with antibiotics is widely recommended for clarithromycin, tetracycline, metronidazole ciprofloxacin and several of these diseases, such as peptic ulcer. The Maas- rifampicin were incubated for 72 h under microaerophilic con- tricht III Consensus Report recommended proton pump in- ditions. MIC was considered as the lowest concentration of drug hibitor (PPI) or ranitidine bismuth citrate-based triple regi- which inhibited visible growth and read as the intercept of the men with clarithromycin and amoxycillin or metronidazole elliptical zone of inhibition with the graded strip for the E-test.
as a first-line therapy.4 However, side effects, poor compli- Based on the CLSI6 and other previously published data7,8, strains ance and resistance to the antibiotics used are common were resistant if MIC ≥ 2 mg/l for amoxycillin, MIC ≥ 4 mg/l causes of treatment failure. Resistance to metronidazole for tetracycline, MIC ≥8 mg/l for metronidazole, MIC ≥1 mg/l and clarithromycin is population-dependent, and several for clarithromycin, MIC ≥ 4 mg/l for ciprofloxacin and studies suggest that clarithromycin resistance is higher in MIC ≥32mg/l for rifampicin and intermediate if MIC = 0.5 mg/l strains obtained from children than in those from adults.5 for clarithromycin and MIC = 2 mg/l for ciprofloxacin. They The evaluation of antibiotic resistance in paediatric patients were susceptible below these thresholds. MIC and MIC were from different areas can help in optimizing therapeutic reg- calculated as the MIC value that inhibited 50% or 90% of the The aim of this study was to determine the primary and secondary resistance to several antimicrobial agents in Statistical analysis
Spanish Helicobacter pylori clinical isolates obtained frompaediatric patients from January 2002 to June 2006.
Data were analyzed using Stata® 8.0 (StataCorp, Texas, USA).
Differences between groups were tested with Chisquare(categorical variables) and Student (continuous variables) tests.
MATERIAL AND METHODS
Significance was construed at p ≤ 5%.
Patients
Samples were collected from gastric biopsies of sympto- matic paediatric patients (aged 18 years old and under) at- Description of the population and symptoms
tending to the Gastroenterology Unit from two children’shospitals (Hospital Infantil Universitario Niño Jesús and H. pylori strains were obtained from gastric biopsy speci- Hospital Universitario 12 de Octubre, Madrid). Both hospi- mens of 101 consecutive H. pylori-positive paediatric tals were H. pylori-reference centres and parents signed an patients. There were 38 males and 69 females (sex2 ratio informed consent form for the endoscopy. At the time sam- M/F = 0.6). The average age was 10 years (range: 4-18 ples were taken, patients had not received Proton Pump In- years), with digestive and non-digestive symptoms. 93% hibitors (PPIs) or antibiotics for at least two weeks. Medical of symptoms were digestive-mainly epigastralgia (63%), records were reviewed to obtain treatment history to know perilumbilical pain (17%), vomiting (26%) and pirosis/re- if patients were previously treated for H. pylori infection.
flux (19%). Nondigestive symptoms, including anaemia High percentage of clarithromycin and metronidazole resistance in Helicobacter pylori clinical isolatesobtained from Spanish children (4.4%), halitosis (2.2%), asthma (1.1%), disorder of ali-mentary behaviour (1.1%) and alteration of general status Minimal inhibitory concentration
and range (in mg/l)
of the 6 antibiotics tested against
Helicobacter pylori clinical isolates
Previous medication and treatment failure
obtained from paediatrics patients
34.7% of the patients had been previously treated for H. Antibiotic
pylori infection. They were treated by antibiotics (amoxycillin,metronidazole, clarithromycin) plus antacids (67%) or by Antimicrobial resistance
All strains were susceptible to amoxycillin, tetracycline and rifampicin, 35.7% (35 out of 98 strains tested for thisantibiotic) were resistant to metronidazole, 54.6% (54 out DISCUSSION
of 99) to clarithromycin and 1.8% (1 out of 55) tociprofloxacin. Intermediate strains to clarithromycin and H. pylori is considered the major cause of chronic gastri- ciprofloxacin were 2% (2 out of 99) and 1.8% (1 out of 55) tis and peptic ulcer disease. Additionally, it can increase the respectively. Double resistance to metronidazole and clar- risk of malignancies in adulthood. In general, H. pylori erad- ithromycin rated at 17.2%. Results can be found in the ication usually entails a proton pump inhibitor or bismuth salts in combination with two antibiotics (metronidazole, amoxy-cillin, tetracycline or clarithromycin).
zole, clarithromycin, ciprofloxacin and rifampicin are de- Resistance of H. pylori to antimicrobial agents is the main cause of treatment failure, even a triple regimen (two antibioticsplus antacids). H. pylori antimicrobial resistance varies between Thirty-five patients (34.7%) had a history of treatment different geographical regions. The severity of gastric in- failure, and were considered secondary resistance to flammation, dosage of proton pump inhibitor and the nature H. pylori. Primary resistance rates to metronidazole andclarithromycin were 32.8% and 49.2%, respectively andsecondary resistance rates were 41.2% and 70.6%, respec- Number (n) and percentage (%)
of primary and secondary resistance
to amoxycillin, tetracycline,
Primary and secondary resistance rates to metronidazole clarithromycin, metronidazole,
and clarithromycin (including double resistance) are de- ciprofloxacin and rifampicin in
tailed in the table 3 (intermediate strains to clarithromycin children infected by Helicobacter
pylori. Madrid, 2002-2006*
Secondary
Antibiotic
resistance
resistance
Number and percentage of resistant
Helicobacter pylori clinical isolates
from paediatric children
Antibiotic
resistance
resistance
intermediate intermediate
* Intermediate strains to clarithromycin and ciprofloxacin were considered High percentage of clarithromycin and metronidazole resistance in Helicobacter pylori clinical isolatesobtained from Spanish children of pathology (ulcerative versus non ulcerative disease) also af- treatment is being with two antibiotics plus PPIs. The most use- fect the outcome of therapy. In the last few years, guidelines ful antibiotics were clarithromycin and metronidazole. Although of H. pylori infection in children have been published. They sug- the rate of double resistance is low in Europe, it is high in the gested that children should be treated with a twice-daily triple- drug regimen, with two antibiotics (clarithromycin, amoxy-cillin and metronidazole) plus a PPI during two weeks. These All strains were susceptible to amoxycillin and tetracycline drugs are considered to be most effective against H. pylori, but in our study. We tested the susceptibility to tetracycline in all drug resistance is a growing problem.
H. pylori strains to know the prevalence of resistance in ourarea although this antibiotic should not be advised for chil- Overall, H. pylori resistance to clarithromycin was higher dren. Due to this fact, there are not many studies about the than other antimicrobial agents in the paediatric patients in- susceptibility to tetracycline in H. pylori isolates in children, cluded in this study. It was 49.2% in primary resistance and although in most of them, resistance was not found. In a study 70.6% in secondary resistance. Macrolide resistance is based from Bulgaria 3.4% of resistant strains were found.15 on defined point mutations in the peptidytransferase loop inboth copies of 23 S rRNA genes. Monotherapy with clar- In the rest of Europe the rate of resistance to amoxycillin ithromycin could induce these mutations.9 In Spain and oth- is very low. For example, in a multicentre European study per- er European countries, including France, Portugal, Poland, formed by Glupzcynski, et al.16 Nine resistance isolates to Turkey and Bulgaria an increase of resistance to clar- amoxycillin were described, in Italy, Greece, Denmark and Eng- ithromycin was observed.10,11 In Northern European countries, land. The percentage of amoxycillin resistance is described as this increase was not observed. This difference probably de- less than 1% in many regions but in some other parts, as a study pends on macrolide consumption. New macrolides were of Iran, a 56% of resistance to this antibiotic was described.17 marketed in Spain at the beginning of the 1990, and clar- In an article from Japan, they studied the correlation between ithromycin in 1991; these children have been more exposed substitutions in penicillin-binding protein 1 and amoxycillin to macrolides. It is very frequent nowadays to treat respira- tory infections in young children. Compared to with other stud-ies in our hospital,12 the rate of resistance to clarithromycin As the rates of resistance to clarithromycin and metron- during 1999 and 2000 was 29.16% (28 out of 96) and the re- idazole are high, new antibiotics are investigated for treat- sistance to clarithromycin increased from 29.16% to 49.2% ment of H. pylori. For instance-triple PPI-based regimen, in- in the period 1999-2000 to 2002-2006. Resistance to this an- cluding rifampicin and levofloxacin have recently been used tibiotic has increased considerably during this period. The sec- as alternative regimens to classical therapy.19 ondary resistance to clarithromycin was higher than the pri-mary, because this antibiotic is the most frequently included In our study, only 3.6% were resistant to ciprofloxacin (both in the standard triple therapies for H. pylori eradication and intermediate and fully resistant). Rates of resistance lower than when susceptibility is studied after treatment failure was be- 10% have been reported from several countries such as France, cause the original strains was resistant to clarithromycin in Japan, The Netherlands or Germany.7,20,21 But, there is a study from Korea,22 where they reviewed the susceptibility tociprofloxacin during ten years and they suggested that resis- The resistance to metronidazole was high in our popula- tance to this antibiotic has been increasing in his population, tion too. The primary resistance was 32.8% and the secondary from 13.9% in 1994 to 33.8% in 2003 were resistant strains one was 41.2%. The resistance to metronidazole is very vari- and the resistance was mediated through point mutation in able in different countries; it is high in underdeveloped coun- tries because this antibiotic is widely used to treat parasiticdiseases and gynaecological infections in female patients.13 Rifampicin is normally used in the treatment of mycobac- There was a study in our hospital about the evolution of the terial infections and in severe bone infections due to Staphy- susceptibility to metronidazole during 9 years14 and metron- lococcus aureus or legionnaire’s disease, but rifabutin (a semi- idazole resistance was 7.14% in 1991–1993, 20.25% in synthetic derivative of rifamycin) has shown to have a high 1994–1996 and 43.90% in 1997-1999. Metronidazole resis- efficacy against H. pylori. In our study all strains were sus- tance increased during these nine years and in that period ex- ceptible to rifampicin. A study about the rate of rifampicin re- ceeds clarithromycin resistance. In the present study the per- sistance in H. pylori isolated from patients in Germany centage of resistance to metronidazole was lower than the showed 1.4% of resistance to rifampicin. Resistance is still low percentage of resistance to clarithromycin.
and associated with mutations in the rpo B gene. This antibioticcould be recommended in drug-resistant H. pylori infections.
Clarithromycin and metronidazole are two antibiotics However, the use of rifampicin should be restricted to avoid used in the H. pylori treatment very often; for this reason it increasing the resistance in the future.8 is very important to know the prevalence of resistance to bothantibiotics. Double resistance was 15.4% in primary resistance In conclusion, resistance rates of H. pylori to metronida- and 26.5% in secondary resistance. The secondary resistance zole and clarithromycin in children are critical and induce treat- was higher than primary due to the majority of the primary ment failure although they are associated to other antibiotics.
High percentage of clarithromycin and metronidazole resistance in Helicobacter pylori clinical isolatesobtained from Spanish children Double resistance could become a serious clinical problem. This licobacter pylori strains obtained from children living in Europe.
is particularly important in areas with a high prevalence of pri- mary metronidazole resistance, where more treatment failures 11. Francesco V, Margiotta M, Zullo A, Asan C, Giorgio F, Burattini and more double resistance will occur. It is very important to O, et al. Prevalence of primary clarithromycin resistance in Heli- know local (geographical) prevalence of antimicrobial resis- cobacer pylori strains over a 15 year period in Italy. J Antimi- tance before recommending a treatment for H. pylori. Empirical treatment in areas with identified high resistance rates 12. Alarcón T, Vega E, Domingo D, Martínez MJ, López-Brea M.
should therefore be avoided. Future trials are needed to study Clarithromycin resistance among Helicobacter pylori strains isolated from children: prevalence and study of mechanism ofresistance by PCR-restriction fragment length polymorphismanalysis. J Clin Microbiol 2003;41:486-8. ACKNOWLEDGEMENTS
13. John Albert M, Al-Mekhaizeem K, Neil L, Dhar R, Dhar PM, Al- Ali M, et al. High prevalence and level of resistance to This work was supported by Fondo de Investigación Sani- metronidazole, but lack of resistance to other antimicro- tarias de la Seguridad Social, grant FIS PI 052442 and PI 052452. bials in Helicobacter pylori, isolated from a multiracial population in Kuwait. Aliment Pharmacol Ther 2006;24: Conflict of interests: non declared.
14. López-Brea M, Martínez J, Domingo D, Alarcón T. A 9 year study of clarithromycin and metronidazole resistance in Helicobacter REFERENCES
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