Vigilância da resistência a antibióticos contra o Helicobacter pylori na região do Biobío (Chile) em uma década / Antibiotic resistance surveillance of helicobacter pylori at the biobío region (chile) in a decade

ABSTRACT BACKGROUND: Helicobacter pylori infection in Chile remains as a public and private health-care system's challenge, with a prevalence of the infection over 70%. Nowadays, antibiotic treatment of the infection is mandatory to prevent the arising of severe associated diseases but failures in the eradication therapy mainly due to clarithromycin resistance has been observed worldwide and first line eradication therapy seems to be not effective anymore in several geographical areas. Thus, health-care systems are committed to maintain an epidemiological surveillance upon the evolution of the antibiotic resistance of this priority 2 pathogen. OBJECTIVE: This work reports a 10 years surveillance of the primary antibiotic resistance of H. pylori clinical isolates at the Biobío region-Chile, and the evolution of resistance toward amoxicillin, clarithromycin, levofloxacin, metronidazole, and tetracycline among the species. METHODS: H. pylori strains were investigated during the periods 2005-2007 (1435 patients analysed) and 2015-2017 (220 patients analysed) by inoculating a saline homogenate biopsy onto the surface of Columbia agar (Oxoid, Basingstoke, UK) - supplemented with 7% horse red blood cells plus DENT inhibitor (Oxoid, Basingstoke, UK) - following by incubation at 37ºC under 10% CO2 atmosphere for five days. Antibiotic resistance pattern of the isolates was assessed using the disk diffusion test in Müeller-Hinton agar supplemented with 7% horse red blood cells followed by incubation for further three days under 10% CO2 atmosphere. Statistical analysis was done using the SPSS v22 software and P values <0.05 were considered statistically significant. RESULTS: A total of 41% of 1435 patients were detected to be infected with H. pylori by bacteriological culture in 2005-2007 period, meanwhile 32.7% from 220 patients were also infected in 2015-2017 period. The clinical isolates of H. pylori are mostly susceptible to amoxicillin and tetracycline (both over 98% of strains), but less susceptible to levofloxacin in both periods analysed (over 79% of the strains). On the other hand, metronidazole continuous showing the highest score of resistant isolates (over 40% of resistant strains), although an 18% fewer resistant strains were observed in 2015-2017 period. Clarithromycin, the key antibiotic in eradication therapies, has an increased frequency of resistant strain isolated in the decade (22.5% in 2005-2007 and 29.2% in 2015-2017). Multidrug resistant strains (two, three and four antibiotics) were also detected in both periods with the highest scores for simultaneous resistance to clarithromycin-metronidazole (18%) and clarithromycin-metronidazole-levofloxacin (12.5%) resistant strains. According to gender, the isolates resistant to amoxicillin, clarithromycin and metronidazole were more frequent in female, with a specific increment in amoxicillin and clarithromycin resistance. CONCLUSION: The frequency of clarithromycin resistance (29.2%) detected in 2015-2017 suggests that conventional triple therapy is no longer effective in this region.

RESUMO CONTEXTO: A infecção por Helicobacter pylori no Chile permanece como um desafio do sistema de saúde público e privado, com prevalência da infecção acima de 70%. Hoje em dia, o tratamento antibiótico da infecção é obrigatório para prevenir o surgimento de graves doenças associadas, mas falhas na terapia de erradicação, principalmente devido à resistência à claritromicina, têm sido observadas em todo o mundo, e a terapia de erradicação de primeira linha parece não ser mais eficaz em várias áreas geográficas. Assim, os sistemas de saúde estão comprometidos em manter uma vigilância epidemiológica sobre a evolução da resistência aos antibióticos deste patógeno prioritário tipo 2. OBJETIVO: Este trabalho relata uma vigilância de 10 anos da resistência antibiótica primária de isolados clínicos de H. pylori na região do Biobío-Chile, e a evolução da resistência em relação à amoxicilina, claritromicina, levofloxacina, metronidazol e tetraciclina entre as espécies. MÉTODOS: As cepas de H. pylori foram investigadas durante os períodos 2005-2007 (1435 pacientes analisados) e 2015-2017 (220 pacientes analisados) inoculando uma biópsia de homogeneizado fisiológico na superfície do agar Columbia (Oxoid, Basingstoke, Reino Unido) - suplementado com 7% de glóbulos vermelhos do cavalo mais o inibidor de DENTE (Oxoid, Basingstoke, Reino Unido) - seguindo pela incubação em 37ºC a atmosfera de 10% de CO2 por cinco dias. O padrão de resistência aos antibióticos dos isolados foi avaliado utilizando-se o teste de difusão em disco em agar Müeller-Hinton suplementado com 7% de glóbulos vermelhos de cavalo seguidos de incubação por mais três dias a atmosfera de 10% de CO2. A análise estatística foi realizada utilizando-se o software SPSS V22 e os valores de P<0,5 foram considerados estatisticamente significantes. RESULTADOS: Um total de 41% dos 1435 pacientes foram detectados como contaminados por H. pylori pela cultura bacteriológica no período 2005-2007, ao mesmo tempo 32,7% de 220 pacientes foram contaminados igualmente no período 2015-2017. Os isolados clínicos de H. pylori são principalmente suscetíveis à amoxicilina e tetraciclina (tanto mais de 98% das cepas), mas menos suscetíveis à levofloxacina em ambos os períodos analisados (mais de 79% das cepas). Por outro lado, o metronidazol permaneceu mostrando a maior pontuação de resistentes isolados (mais de 40% de cepas resistentes), embora tenham sido observados 18% menos cepas resistentes no período de 2015-2017. A claritromicina, o antibiótico-chave em terapias de erradicação, tem uma frequência aumentada de cepa resistente isolada na década (22,5% em 2005-2007 e 29,2% em 2015-2017). Cepas multirresistentes (dois, três e quatro antibióticos) também foram detectadas em ambos os períodos com os maiores escores de resistência simultânea à claritromicina-metronidazol (18%) e claritromicina-metronidazol-levofloxacina (12,5%) cepas resistentes. De acordo com o sexo, os isolados resistentes à amoxicilina, claritromicina e metronidazol foram mais frequentes no sexo feminino, com incremento específico em amoxicilina e resistência à claritromicina. CONCLUSÃO: A frequência de resistência à claritromicina (29,2%) detectada em 2015-2017 sugere que a terapia tripla convencional não é mais efetiva nesta região.

Mutaciones del gen ARN ribosómico 23S de Helicobacter pylori asociadas con resistencia a claritromicina en pacientes atendidos en una unidad de endoscopia de Medellín, Colombia / Mutations in the Helicobacter pylori 23S rRNA gene associated with clarithromycin resistance in patients at an endoscopy unit in Medellín, Colombia

Resumen Introducción. La claritromicina es el antibiótico de primera línea para el tratamiento de la infección por Helicobacter pylori. La resistencia bacteriana se produce principalmente por mutaciones puntuales del gen ARN ribosómico 23S (ARNr 23S). Objetivo. Determinar la frecuencia de las mutaciones puntuales A2143G y A2142G del gen ARNr 23S asociadas con la resistencia de H. pylori a la claritromicina en muestras de pacientes con manifestaciones dispépticas en Medellín, región noroccidental de Colombia. Materiales y métodos. Se extrajo ADN a partir de muestras de biopsia gástrica obtenidas de pacientes con manifestaciones dispépticas atendidos en una unidad de endoscopia entre el 2016 y el 2017. Mediante reacción en cadena de la polimerasa (PCR), se amplificaron las regiones s y m del gen vacA y una región del gen ARNr 23S bacteriano. La presencia de las mutaciones A2142G y A2143G se determinó por la técnica de polimorfismos de longitud de fragmentos de restricción (RFLP) con las enzimas BbsI y BsaI, respectivamente. Resultados. Se encontró una prevalencia de infección de 44,2 % (175/396), según el informe de histopatología. En 143 de estas 175 muestras positivas se amplificaron las tres regiones del genoma bacteriano. Se identificaron las mutaciones A2143G y A2142G en 27 muestras (18,8 %; 27/143), la mutación más frecuente fue la A2143G (81,5 %; 22/27). Conclusiones. Hubo una gran prevalencia de mutaciones asociadas con la resistencia de H. pylori a la claritromicina en la población de estudio. Se requieren estudios adicionales para establecer la resistencia bacteriana en la población colombiana y, así, determinar los tratamientos de primera línea y de rescate.

Abstract Introduction: Clarithromycin is the first-line antibiotic for the treatment of Helicobacter pylori infection. Bacterial resistance is mainly due to the presence of specific mutations in the 23S ribosomal RNA (rRNA) gene. Objective: To determine the frequency of A2143G and A2142G specific mutations in the 23S rRNA gene associated with clarithromycin resistance of H. pylori in samples from patients with dyspeptic manifestations in Medellín, northwestern Colombia. Materials and methods: DNA was extracted from gastric biopsy samples of patients with dyspeptic manifestations seen at an endoscopy unit in Medellín between 2016 and 2017. PCR was performed to amplify the bacterial s and m vacA regions, and a region in the 23S rRNA gene. The presence of the A2142G and A2143G mutations was determined using the restriction fragment length polymorphism (RFLP) technique with the BbsI and BsaI enzymes, respectively. Results: The prevalence of infection was 44.2% (175/396), according to the histopathology report. The positive samples were analyzed and the three regions of the bacterial genome were amplified in 143 of the 175 samples. The A2143G and A2142G mutations were identified in 27 samples (18.8%, 27/143). The most frequent mutation was A2143G (81.5%, 22/27). Conclusions: We found a high prevalence of H. pylori mutations associated with clarithromycin resistance in the study population. Further studies are required to determine the bacterial resistance in the Colombian population in order to define first line and rescue treatments.

Helicobacter pylori: resistencia múltiple en pacientes de Bogotá, Colombia / Helicobacter pylori: Multiple resistance in patients from Bogotá, Colombia

Resumen Introducción. La resistencia a los antibióticos es la principal causa del fracaso del tratamiento contra Helicobacter pylori; la claritromicina y el metronidazol son los antibióticos que generan mayor resistencia. En Colombia, la resistencia primaria a estos dos antibióticos y el uso excesivo de levofloxacina han alcanzado los límites aceptados (13,6, 83 y 16 %, respectivamente). A pesar de ello, se usa el tratamiento empírico combinando estos antibióticos en pacientes en los que ha fallado anteriormente. Objetivo. Determinar la resistencia a los antibióticos en pacientes previamente tratados para H. pylori en Bogotá, Colombia. Materiales y métodos. Se llevó a cabo un estudio descriptivo en el que se evaluó mediante dilución en agar la resistencia a la amoxicilina, la claritromicina, la levofloxacina y el metronidazol en 10 aislamientos provenientes de 5 pacientes con tres o cuatro tratamientos fallidos para H. pylori. La resistencia a los antibióticos se confirmó mediante secuenciación de ADN (Magrogen, Korea). Resultados. Ocho de los aislamientos presentaron resistencia a dos o más antibióticos y todos fueron resistentes a la levofloxacina. Los patrones de sensibilidad de los aislamientos provenientes del antro pilórico y del cuerpo del estómago, fueron diferentes en tres de los pacientes. Conclusión. Hasta donde se sabe, esta es la primera evidencia de resistencia múltiple de H. pylori en Colombia en pacientes previamente tratados. Los resultados evidenciaron las consecuencias del uso de un esquema ineficaz de tratamiento antibiótico y la necesidad de evaluar la sensibilidad a los antibióticos en diferentes sitios anatómicos del estómago. La resistencia múltiple limita el número de antibióticos útiles para erradicar H. pylori.

Abstract Introduction: The main cause for Helicobacter pylori infection treatment failure is antibiotic resistance, where clarithromycin and metronidazole play the main role. In Colombia, primary resistance as a consequence of the use of these two antibiotics and excessive levofloxacin use is above the accepted limit (13.6%, 83%, and 16%, respectively). Despite this fact, empirical therapies that include the combination of these antibiotics are used in patients with previous therapeutic failure. Objective: To determine antibiotic resistance in patients previously treated for H. pylori in Bogotá, Colombia. Materials and methods: We conducted a descriptive study that included ten isolates obtained from five patients with three or four previous failed treatments for H. pylori. Antibiotic resistance to amoxicillin, clarithromycin, levofloxacin, and metronidazole was investigated by agar dilution and confirmed by DNA sequencing (Magrogen, Korea). Results: Eight isolates were resistant to two or more antibiotics. All isolates were resistant to levofloxacin. Susceptibility patterns in isolates from the gastric antrum and the body of the stomach were different in three patients. Conclusion: As far as we know, this is the first evidence of multiple H. pylori resistance in Colombia in previously treated patients. Results demonstrated the consequences of using an ineffective antibiotic scheme and the need to assess antibiotic susceptibility in different anatomical sites of the stomach. The consequences of multiple resistance decrease possible antibiotic effectiveness to eradicate H. pylori in the future.

Usefulness of rapid urease test samples for molecular analysis of clarithromycin resistance in Helicobacter pylori / Utilidad de las muestras de test rápido de ureasa para el análisis molecular de resistencia a claritromicina en Helicobacter pylori

Helicobacter pylori is a gastric pathogen that is widely recognized as a causative agent of gastric disease. Its eradication is variable, mainly due to increased resistance to clarithromycin. Our objective was: to evaluate (i) if the biopsy specimen used for the rapid urease test is a useful sample to detect resistance to clarithromycin by PCR-RFLP and (ii) the distribution of A2142G and A2143G point mutations in the 23S rRNA gene, in relation to virulence factors in our region. Gastric specimens were collected from adult dyspeptic patients (n = 141) and H. pylori was investigated by the rapid urease test, histopathological analysis and PCR for the hsp60 gene. Clarithromycin resistance was detected by PCR-RFLP in 62 H. pylori (+) paired biopsy specimens submitted to molecular analysis and the rapid urease test. H. pylori virulence factors were analyzed by multiplex PCR using specific primers for the cagA, vacA and babA2 genes. Thirteen out of 62 strains (20.9%) were resistant to clarithromycin: 6/13 (46.2%) harbored the A2143G mutation whereas 7/13 (53.8%) carried the A2142G point mutation. vacA m1s1 was the most frequent genotype among the resistant strains. In conclusion, the biopsy specimens used for the rapid urease test were suitable samples for clarithromycin resistance detection in patients infected with H. pylori, which became especially useful in cases where the number or size of the biopsies is limited. In addition, this is the first report of a molecular analysis for clarithromycin resistance performed directly from gastric biopsies in our region.

Helicobacter pylori es un patógeno ampliamente reconocido como causante de enfermedad gástrica. Su erradicación es variable, principalmente debido al incremento de la resistencia a claritromicina. Nuestros objetivos fueron evaluar la utilidad de la biopsia usada para realizar el test rápido de ureasa en la detección de resistencia a claritromicina por PCR-RFLP y conocer la distribución de las mutaciones puntuales A2142G y A2143G en el gen ARNr 23S, en relación con los factores de virulencia en nuestra región. Se recolectaron muestras gástricas (n=141) provenientes de pacientes adultos dispépticos y se investigó la presencia de H. pylori mediante el test rápido de ureasa, análisis histopatológico y PCR para el gen hsp60. La resistencia a claritromicina se analizó por PCR-RFLP en 62 muestras pareadas de biopsias gástricas H. pylori+ destinadas al análisis molecular y al test rápido de ureasa. Los factores de virulencia de H. pylori fueron analizados mediante PCR multiplex usando oligonucleótidos específicos para los genes cagA, vacA y babA2. Trece de 62 cepas (20,9%) fueron resistentes a claritromicina, 6/13 (46,2%) llevaron la mutación A2143G, mientras que 7/13 (53,8%) presentaron la mutación A2142G. El genotipo vacA s1m1 fue el más frecuente entre las cepas resistentes a claritromicina. En conclusión, las biopsias destinadas al test rápido de ureasa fueron muestras apropiadas para la detección de la resistencia a claritromicina en pacientes infectados con H. pylori. Esto es especialmente útil en aquellos casos en los que el número o el tamaño de las muestras son limitados. Además, este es el primer reporte de estudio de resistencia a claritromicina (mediante técnicas moleculares), directamente de biopsias gástricas en nuestra región.

Primary antibiotic resistance and its relationship with cagA and vacA genes in Helicobacter pylori isolates from Algerian patients

Abstract The epidemiology of Helicobacter pylori resistance to antibiotics is poorly documented in Africa and especially in Algeria. The aim of our study was to determine the antibiotic resistance rates, as well as its possible relationship with VacA and CagA virulence markers of isolates from Algerian patients. One hundred and fifty one H. pylori isolate were obtained between 2012 and 2015 from 200 patients with upper abdominal pain. Antimicrobial susceptibility testing was performed for amoxicillin, clarithromycin, metronidazole, ciprofloxacin, rifampicin and tetracycline. Molecular identification of H. pylori and the detection of vacA and cagA genes were performed using specific primers. We found that H. pylori was present in 83.5% of collected biopsies, 54.9% of the samples were cagA positive, 49.67% were vacA s1m1, 18.30% were vacA s1m2 and 25.49% were vacA s2m2. Isolates were characterized by no resistance to amoxicillin (0%), tetracycline (0%), rifampicin (0%), a high rate of resistance to metronidazole (61.1%) and a lower rate of resistance to clarithromycin (22.8%) and ciprofloxacin (16.8%). No statically significant relationship was found between vagA and cagA genotypes and antibiotic resistance results (p > 0.5) except for the metronidazole, which had relation with the presence of cagA genotype (p = 0.001).

O mel-derivado de Lactobacillus rhamnosus alivia a infecção gastro-intestinal induzida pelo Helicobacter pylori-e inflamação gástrica em C57Bl/6 ratos: um estudo imuno-histológico / Honey-derived lactobacillus rhamnosus alleviates helicobacter pylori-induced gastro-intestinal infection and gastric inflammation in c57bl/6 mice: an immuno-histologic study

ABSTRACT BACKGROUND: Helicobacter pylori (H. pylori) has been introduced by since 1983 by Marshal and Warren to play the main role in the pathophysiology of gastritis and gastric ulcers. Almost half of the world population1 is infected by H. pylori. Current therapeutic regimen against H. pylori includes the use of a proton pump inhibitor plus two or more antibiotics. However, the efficacy of this regimen is decreasing mainly due to antibiotic resistance and side effects of medications. This fact has resulted in public interest in other therapeutic options and the role of probiotics merits special attention in this regard. OBJECTIVE: This study aims to evaluate the efficacy of honey-derived Lactobacillus rhamnosus on H. pylori-induced gastric inflammation and gastro-intestinal infection in C57BL/6 Mice. METHODS: The 24 C57BL/6 Mice were randomly divided into three groups of eight mice each. All the mice were fed with 1cc suspension containing 5*1010 CFU/ mL of ATCC43504 strains of H. pylori for 3 consecutive days, twice daily via polyethylene gavage tubes. At the end of 4th week, infection with H. pylori was confirmed with stool Ag (ELISA) and following sacrifice of one mouse from each group, histopathologic study confirmed gastritis. The groups were subjected to different therapies as stated, 1: without Bismuth (Bi), Omeprazole (Om) and L. rhamnosus prescription, 2: Bi, Om and Clarithromycin (Cl) and 3: Bi, Om plus 1cc of suspension of 109 CFU/mL of L. rhamnosus. After 2 weeks, the stool was analyzed for Ag and the mice were sacrificed for evaluation of histopathologic changes. RESULTS: Treatment with L. rhamnosus group provided Zero titer of stool Ag and was associated with improved gastric inflammation in all subjects, similar to the clarithromycin group. CONCLUSION: Honey-derived L. rhamnosus probiotics provides similar results as clarithromycin in terms of improvement of H. pylori infection and gastritis in C57BL/6 Mice model, without its cons of antibiotic resistance.

RESUMO CONTEXTO: O Helicobacter pylori (H. pylori) foi reconhecido em 1983 por Marechal e Warren como protagonista principal na fisiopatologia de gastrite e úlceras gástricas. Quase metade da população mundial está infectada por H. pylori. O regime terapêutico atual contra H. pylori inclui o uso de um inibidor da bomba de prótons associada a dois ou mais antibióticos. No entanto, a eficácia deste regime está diminuindo principalmente devido à resistência aos antibióticos e efeitos colaterais de medicamentos. Este fato resultou no interesse público em outras opções terapêuticas e o papel dos probióticos merece atenção especial a este respeito. OBJETIVO: Este estudo visa avaliar a eficácia do mel-derivado do Lactobacillus rhamnosus na inflamação gástrica e infecção gastrointestinal H. pylori-induzida em camundongos C57Bl/6. MÉTODOS: Vinte e quatro camundongos C57Bl/6 foram divididos aleatoriamente em três grupos de oito camundongos cada. Todos os ratos foram alimentados com suspensão de 1cc contendo 5*1010 UFC/mL de cepas ATCC43504 de H. pylori por 3 dias consecutivos, duas vezes por dia através de gavagem por tubos de polietileno. No final da 4ª semana, a infecção com H. pylori foi confirmada pelo antígeno fecal (ELISA) e após o sacrifício de um rato de cada grupo, o estudo histopatológico confirmou gastrite. Os grupos foram submetidos a diferentes terapias, como indicado, 1: sem prescrição de bismuto (BI), Omeprazol (Om) e L. rhamnosus, 2: Bi, Om e claritromicina (CL) e 3: Bi, Om mais 1cc de suspensão de 109 UFC/mL de L. rhamnosus. Após 2 semanas, as fezes foram analisadas para o antígeno e os ratos foram sacrificados para a avaliação das alterações histopatológicas. RESULTADOS: O tratamento com o grupo L. rhamnosus forneceu o título zero de antígeno e foi associado com a inflamação gástrica melhorada em todos os camundongos, similar ao grupo claritromicina. CONCLUSÃO: O probiótico mel-derivado L. rhamnosus fornece resultados semelhantes ao da claritromicina em termos de melhoria da infecção H. pylori e gastrite em C57Bl/6 camundongos modelos, sem os inconvenientes de resistência aos antibióticos.

Lack of association between rrl and erm(41) mutations and clarithromycin resistance in Mycobacterium abscessus complex

BACKGROUND Mycobacterium abscessus complex (MABC) includes species with high resistance rates among mycobacterial pathogens. In fact, MABC infections may not respond to clarithromycin treatment, which has historically been very effective against MABC infection. Molecular markers have been proposed to detect both acquired (rrl polymorphisms) and inducible (erm(41) polymorphisms) clarithromycin resistance in MABC isolates. OBJECTIVES This study aimed to evaluate the susceptibility profile and molecular markers of clarithromycin resistance in MABC. METHODS The clarithromycin susceptibility profile was determined by broth microdilution with reads on days 3, 5, 7 and 14. Mutations in the rrl and erm(41) genes were evaluated by polymerase chain reaction (PCR) using specific primers, followed by sequencing. FINDINGS A total of 14 M. abscessus subsp. abscessus isolates and 28 M. abscessus subsp. massiliense isolates were evaluated, and clarithromycin resistance was observed in all isolates for up to three days of incubation. None of the 42 isolates exhibited a point mutation in the rrl gene, while all the isolates had a T28 polymorphism in the erm(41) gene. Moreover, all 28 M. abscessus subsp. massiliense isolates had a deletion in the erm(41) gene. MAIN CONCLUSIONS While all the MABC isolates exhibited acquired clarithromycin resistance, no isolates exhibited a point mutation in the rrl gene in this study. The M. abscessus subsp. massiliense isolates demonstrated clarithromycin resistance, which is an uncommon phenotype. The molecular data for the rrl and erm(41) genes were not consistent with the phenotypic test results of clarithromycin susceptibility, indicating a lack of correlation between molecular clarithromycin resistance markers for both acquired and inducible resistance.

Sensibilidad a los antimicrobianos de aislamientos de Helicobacter pylori aislados de lesiones gástricas / Antimicrobial susceptibility of Helicobacter pylori strains isolated from gastric lesions

Se evaluó la sensibilidad a los antimicrobianos de 30 aislamientos de Helicobacter pylori aislados de biopsias gástricas mediante los métodos de difusión por discos y tiras de E-test. Los antimicrobianos evaluados fueron amoxicilina, claritromicina, metronidazol y ciprofloxacina. No se encontraron cepas resistentes a amoxicilina, el 17% (5/30) fueron resistentes a claritromicina, el 20% (6/30) a ciprofloxacina por ambos métodos, y el 37% (11/30) a metronidazol por E-test. Si bien el número de cepas estudiadas fue reducido, hubo una sola discrepancia en la interpretación de la sensibilidad cuando se compararon ambos métodos: el metronidazol fue categorizado como sensible por E-test e intermedio por el método de difusión por discos. No pudo determinarse una asociación estadísticamente significativa entre el tipo de lesión histológica y el patrón de resistencia encontrado.

Antimicrobial susceptibility was evaluated by two diffusion methods: E-test strips to determine minimum inhibitory concentration (MIC) and disk diffusion for amoxicillin, clarithromycin, metronidazole and ciprofloxacin in 30 Helicobacter pylori strains isolated from gastric biopsies. No strains were resistant to amoxicillin, 17% (5/30) were resistant to clarithromycin, 20% (6/30) ciprofloxacin by both methods, and 37% (11/30) to metronidazole by the E-test. Although the number of strains studied was reduced, there was a single mismatch in interpreting susceptibility when both methods were compared; the same mismatch was observed for metronidazole, being categorized as sensitive by the E-test and as intermediate by disk diffusion. No association between the histological type of lesion and the resistance pattern found could be determined.

Molecular detection of clarithromycin and fluoroquinolones resistance in helicobacter pylori infection, directly applied to gastric biopsies, in an urban brazilian population

ABSTRACT Background - Antimicrobial resistance is the major factor leading to eradication failure in H. pylori treatment. Molecular tests are useful to detect genetic mutations predictive of clarithromycin and fluoroquinolones resistance. Knowledge of the local prevalence rate of resistance is important to define the best recommended treatment. Objective - To assess the prevalence of primary resistance of H. pylori to clarithromycin and fluoroquinolones, using a molecular test, in a Southeastern urban Brazilian population. Methods - A total of 72 H. pylori seropositive patients [65% female, mean age 39 (19-73) years] never treated before for this infection were studied. All patients underwent gastroscopy in addition to antrum and corpus biopsies and molecular test GenoType HelicoDR (Hain Life Science, Germany) to detect H. pylori and point mutations in genes responsible for clarithromycin and fluoroquinolone resistance. The molecular procedure was divided into three steps: DNA extraction from biopsy samples, a multiplex amplification with biotinylated primers and a reverse hybridization. The most frequent point mutations involved in resistance to the two antibiotics were evaluated. Results - Resistance to clarithromycin was detected in nine (12.5%) patients and to fluoroquinolones in eight (11.1%) patients. The point mutation A2147G was the most common (77.8%) among resistant strains to clarithromycin. In 50% of the resistant strains to fluoroquinolones, the mutant codon couldn't be identified. Conclusion - The resistance rates to clarithromycin and fluorquinolones in a large urban population in the Southeast of Brazil were acceptable, suggesting that these drugs remain appropriate options to first and second-line of H. pylori treatment. The molecular test represents an adequate diagnostic tool for monitoring H. pylori resistance.

RESUMO Contexto - A resistência aos antimicrobianos é o principal fator associado à falha terapêutica no tratamento do H. pylori. Testes moleculares são úteis na detecção das mutações genéticas associadas ao desenvolvimento de resistência à claritromicina e fluorquinolonas. O conhecimento da taxa de prevalência local de resistência é importante na definição do melhor esquema terapêutico. Objetivo - Estimar a prevalência de resistência primária do H. pylori à claritromicina e fluorquinolonas, empregando-se um teste molecular, em uma capital do Sudeste do Brasil. Métodos - Setenta e dois pacientes com sorologia positiva para H. pylori [65% mulheres, idade média 39 (19-73) anos], nunca tratados previamente para essa infecção, foram selecionados. Todos os pacientes submeteram-se à endoscopia digestiva com biópsias de antro e corpo e realização do teste molecular GenoType HelicoDR (Hain Life Science, Alemanha) para a detecção do H. pylori e das mutações pontuais dos genes responsáveis pela resistência à claritromicina e fluorquinolonas. O procedimento molecular constituía-se de três etapas: extração do DNA a partir das amostras endoscópicas, amplificação multiplex com primers biotinilados e hibridização reversa. As mutações pontuais mais frequentemente envolvidas com resistência aos dois antibióticos foram avaliadas. Resultados - Resistência à claritromicina foi detectada em nove (12,5%) pacientes e às fluorquinolonas em oito (11,1%) pacientes. A mutação pontual A2147G foi a mais comum (77,8%) entre as cepas resistentes à claritromicina. Em 50% das cepas resistentes à fluorquinolonas, o códon mutante não pôde ser identificado. Conclusão - As taxas de resistência à claritromicina (12,5%) e às fluorquinolonas (11,1%), em uma importante capital do Sudeste do Brasil, mostraram índices aceitáveis, sugerindo que essas drogas permanecem opções apropriadas para o tratamento de primeira e segunda linha do H. pylori. O teste molecular constitui uma ferramenta diagnóstica adequada para monitorar a resistência do H. pylori.

Antimicrobial resistance of Helicobacter pylori strains to five antibiotics, including levofloxacin, in Northwestern Turkey

INTRODUCTION: Antibiotic resistance is the main factor that affects the efficacy of current therapeutic regimens against Helicobacter pylori. This study aimed to determine the rates of resistance to efficacy clarithromycin, amoxicillin, tetracycline, levofloxacin and metronidazole among H. pylori strains isolated from Turkish patients with dyspepsia. METHODS: H. pylori was cultured from corpus and antrum biopsies that were collected from patients with dyspeptic symptoms, and the antimicrobial susceptibility of H. pylori was determined using the E-test (clarithromycin, amoxicillin, tetracycline, metronidazole and levofloxacin) according to the EUCAST breakpoints. Point mutations in the 23S rRNA gene of clarithromycin-resistant strains were investigated using real-time PCR. RESULTS: A total of 98 H. pylori strains were isolated, all of which were susceptible to amoxicillin and tetracycline. Of these strains, 36.7% (36/98) were resistant to clarithromycin, 35.5% (34/98) were resistant to metronidazole, and 29.5% (29/98) were resistant to levofloxacin. Multiple resistance was detected in 19.3% of the isolates. The A2143G and A2144G point mutations in the 23S rRNA-encoding gene were found in all 36 (100%) of the clarithromycin-resistant strains. Additionally, the levofloxacin MIC values increased to 32 mg/L in our H. pylori strains. Finally, among the clarithromycin-resistant strains, 27.2% were resistant to levofloxacin, and 45.4% were resistant to metronidazole. CONCLUSIONS: We conclude that treatment failure after clarithromycin- or levofloxacin-based triple therapy is not surprising and that metronidazole is not a reliable agent for the eradication of H. pylori infection in Turkey. .

RESISTANCE TO AMOXICILLIN, CLARITHROMYCIN AND CIPROFLOXACIN OF Helicobacter pylori ISOLATED FROM SOUTHERN BRAZIL PATIENTS / Resistência à amoxicilina, claritromicina e ciprofloxacina de Helicobacter pylori isolados de pacientes do Sul do Brasil

Introduction: Helicobacter pylori is a bacteria which infects half the world population and is an important cause of gastric cancer. The eradication therapy is not always effective because resistance to antimicrobials may occur. The aim of this study was to determine the susceptibility profile of H. pylori to amoxicillin, clarithromycin and ciprofloxacin in the population of Southern Brazil. Material and methods: Fifty four samples of H. pylori were evaluated. The antibiotics susceptibility was determined according to the guidelines of the British Society for Antimicrobial Chemotherapy and the Comité de l'Antibiogramme de la Société Française de Microbiologie. Results: Six (11.1%) H. pylori isolates were resistant to clarithromycin, one (1.9%) to amoxicillin and three (5.5%) to ciprofloxacin. These indices of resistance are considered satisfactory and show that all of these antibiotics can be used in the empirical therapy. Conclusion: The antibiotics amoxicillin and clarithromycin are still a good option for first line anti-H. pylori treatment in the population of Southern Brazil.

Introdução: Helicobacter pylori é uma bactéria que infecta metade da população mundial e é considerada importante causa de câncer gástrico. A terapia de erradicação nem sempre é eficaz, pois pode ocorrer a resistência aos antimicrobianos. Este estudo determinou a sensibilidade de H. pylori frente à amoxicilina, claritromicina e ciprofloxacina na população do Sul do Brasil. Material e métodos: Foram avaliadas 54 amostras de H. pylori. A sensibilidade aos antibióticos foi determinada segundo as orientações da British Society for Antimicrobial Chemotherapy e do Comité de l'Antibiogramme de la Société Française de Microbiologie. Resultados e discussão: Sete (13%) isolados de H. pylori foram resistentes à claritromicina, um (1,9%) à amoxicilina e três (5,5%) à ciprofloxacina. Estes índices são satisfatórios e demonstram que esses antibióticos podem ser utilizados na terapia empírica. Conclusão: Os antibióticos amoxicilina e claritromicina ainda são uma boa opção no tratamento de primeira linha anti-H. pylori na população do Sul do Brasil.

Determinación de mutaciones de un solo nucleótido en el gen 23S rRNA de Helicobacter pylori relacionadas con resistencia a claritromicina en una población del departamento del Cauca, Colombia / Determination of single nucleotide mutations in the 23S rRNA gene of Helicobacter pylori related to clarithromycin resistance in a population from Cauca, Colombia

Introducción. La terapia antibiótica combinada para la erradicación de Helicobacter pylori debería basarse en los patrones locales de resistencia. Objetivo. Determinar la resistencia de H. pylori a claritromicina en una población del departamento del Cauca mediante la identificación de mutaciones en el gen 23S r RNA en ADN obtenido de biopsias gástricas. Materiales y métodos. Se incluyeron en el estudio 162 pacientes con dispepsia funcional. El gen 23S rRNA se amplificó por PCR y el patrón de mutaciones se identificó por secuenciación directa. Resultados. La frecuencia de resistencia a claritromicina fue de 4 %. La mutación A2143G del gen se encontró en cuatro pacientes (2,46 %) y la mutación A2142G, en tres pacientes (1,85 %). Conclusiones. El estudio encontró que el genotipo más frecuente en los especímenes positivos para H. pylori fue 2143G, seguido por A2142G. La prevalencia observada de resistencia de H. pylori fue baja; por lo tanto, se considera que el tratamiento con claritromicina es una opción válida para la erradicación de H. pylori en la población objeto de estudio.

Introduction: Antibiotic combination therapy for the eradication of Helicobacter pylori should be based on local resistance patterns. Objective: To d etermine the resistance of H. pylori to clarithromycin in a population from Cauca province, through the identification of mutations in the 23S rRNA gene in DNA from gastric biopsies. Materials and methods: A total of 162 patients with functional dyspepsia were included in the study. The 23S rRNA gene and the DNA from 162 gastric specimens were amplified by PCR, and the mutation pattern was identified by direct sequencing. Results: The frequency of clarithromycin resistance was 4%. A2143G mutation was found in four patients (2.46%) and A2142G mutation was found in three patients (1.85%). Conclusions: Our study shows that the most frequent genotype in H. pylori -positive specimens was A2143G, followed by A2142G. The observed resistance prevalence of H. pylori was low; thus, we consider that clarithromycin treatment is a valid option for H. pylori eradication in the study population.

The Drug Resistance Profile of Mycobacterium abscessus Group Strains from Korea

BACKGROUND: Bacteria of the Mycobacterium abscessus group are the second most common pathogens responsible for lung disease caused by nontuberculous mycobacteria in Korea. There is still a lack of studies investigating the genetic mechanisms involved in M. abscessus resistance to antibiotics other than clarithromycin. This study investigated the characteristics of drug resistance exhibited by M. abscessus clinical isolates from Korea. METHODS: We performed drug susceptibility testing for a total of 404 M. abscessus clinical strains. Subspecies were differentiated by molecular biological methods and examined for mutations in drug resistance-related genes. RESULTS: Of the 404 strains examined, 202 (50.00%), 199 (49.26%), and 3 (0.74%) strains were identified as M. abscessus, M. massiliense, and M. bolletii, respectively. Of the 152 clarithromycin-resistant strains, 6 possessed rrl mutations, while 4 of the 30 amikacin-resistant strains contained rrs mutations, and 5 of the 114 quinolone-resistant strains had gyr mutations. All mutant strains had high minimal inhibitory concentration values for the antibiotics. CONCLUSIONS: Our results showed the distribution of the strains with mutations in drug resistance-related genes was low in the M. abscessus group. Furthermore, we performed drug susceptibility testing and sequence analyses to determine the characteristics of these genes in the M. abscessus group.

Changes in the Eradication Rate of Conventional Triple Therapy for Helicobacter pylori Infection in Korea / 대한소화기학회지

Although, the prevalence of Helicobacter pylori infection in Korea has declined owing to the eradication therapy, recent seroprevalence of H. pylori infection is still reported to be as high as 54.4%. Until now, "standard regimen" for eradication of H. pylori has been conventional triple therapy consisting of proton pump inhibitor, amoxicillin, and clarithromycin. However, with the increase in antibiotic resistance, especially against clarithromycin, the eradication rate of conventional triple therapy has steadily declined during the past 13 years in Korea. Present eradication rate of standard triple therapy is reported to be less than 80%, which is the Maginot line of efficacy for the currently available regimen. Therefore, new first line eradication regimen is needed to enhance the eradication rate of H. pylori infection.

New Therapeutic Strategies against Helicobacter pylori / 대한소화기학회지

The standard therapy for Helicobacter pylori infection in Korea is a triple-drug regimen consisting of a proton pump inhibitor with two antibiotics such as clarithromycin, amoxicillin, and metronidazole. However, as the eradication rate of this regimen has declined over the past decade, this prompted the formulation of new therapeutic regimens. New therapeutic strategies against H. pylori infection that had been tried all over the world include sequential therapy, concomitant therapy, and tailored therapy This article will review the basic concepts and the results of previous clinical trials on the aforementioned new therapeutic regiments.

Future Trends of Helicobacter pylori Eradication Therapy in Korea / 대한소화기학회지

The prevalence of Helicobacter pylori infection in Korea shows a decreasing trend and has changed to that of developed country, especially for those below 30 years old. However, the primary antibiotic resistance rates are higher than those of developed countries. The reason for the decrease in the efficacy of standard triple therapy is mainly due to the increase in the resistance against clarithromycin. Sequential therapy seems to be more effective than the standard triple therapy, but the intention-to-treat eradication rate of sequential therapy in Korea, which is mostly under 80.0%, is still not satisfactory. Therefore, a promising regimen is needed. Recently, the Japanese health insurance system admitted 'H. pylori-infected gastritis' as an indication of eradication. Furthermore, the Kyoto Consensus Meeting on H. pylori Gastritis held from January 30th to February 1st, 2014, proposed that 'all H. pylori positive patients should be offered to receive H. pylori eradication'. This suggests that the concept of eradication has been changed from 'treatment' to 'prevention'. Various individualized tailored therapy based on the polymorphism, age and other demographic factors and antibiotic resistance has been attempted to maximize H. pylori eradication therapy. The aim of this article is to review the current epidemiology, H. pylori resistance state, treatment guideline, and to assess the possible future strategy and treatment for H. pylori infection in Korea.

Sensibilidad in vitro a amoxicilina y claritromicina de Helicobacter pylori obtenido de biopsias gástricas de pacientes en zona de bajo riesgo para cáncer gástrico / Antimicrobial susceptibility of Helicobacter pylori with chronic gastritis

Introducción. La infección por Helicobacter pylori prevalece en más de la mitad de la población mundial. Es prioritario evaluar la sensibilidad actual in vitro de H. pylori a los antimicrobianos usados en los protocolos de erradicación, fundamentalmente para determinar los patrones y resolver la infección. Objetivo. Determinar la prevalencia de la infección y la sensibilidad antibiótica de H. pylori en biopsias gástricas. Materiales y métodos. Se investigan la prevalencia de la infección y la sensibilidad antibiótica de H. pylori de 203 pacientes con gastritis crónica procedentes de Tumaco (Nariño), mediante pruebas histopatológicas (escala Dixon) y microbiológicas (cultivo en agar sangre con suplemento de antibióticos) y, además, su resistencia a amoxicilina y claritromicina mediante el método de dilución en agar. Resultados. La prevalencia de la infección para H. pylori con pruebas histopatológicas y microbiológicas es de 88,7 % y 84,7 % respectivamente; la prevalencia de resistencia de H. pylori a amoxicilina y claritromicina y a ambos antibióticos, fue de 20,5 %, 19,8 % y 10,96 %, respectivamente. Conclusiones. Los resultados de este estudio indican que es alta la incidencia de H. pylori resistente a claritromicina y amoxicilina en pacientes de Tumaco con gastritis crónica. También, se encontraron cepas multirresistentes a claritromicina y amoxicilina.

Introduction. Infection by Helicobacter pylori is prevalent in approximately half the world´s population.However, the susceptibility of H. pylori to antimicrobial agents must be evaluated by in vitro methods in order to determine the susceptibility levels and to guide treatment regimes. Objective. The prevalence of infection and antibiotic susceptibility was evaluated in gastric biopsies containing H. pylori. Materials and methods. The prevalence of infection and antibiotic susceptibility was investigated in 203 patients with chronic gastritis from Tumaco, Colombia. Diagnosis was confirmed by histopathological evidence (Dixon scale) and microbiological (culture on blood agar supplemented with antibiotics).The antibiotic resistance of H. pylori was measured by its response toantimicrobial amoxicillin and clarithromycin using the agar dilution method. Results. The prevalence of infection for H. pylori with histopathological and microbiological tests was88.7% and 84.7% respectively. The prevalence of resistance of H. pylori to antimicrobial amoxicillin was 20.5%, to clarithromycin 19.8%, and to both antibiotics concurrently, 10.9%. Conclusions. A high incidence of clarithromycin-resistant and amoxicillin-resistant H. pylori was discovered in patients from Tumaco with chronic gastritis. Dual drug-resistant strains of H. pylori to clarithromycin and amoxicillin were also present.

Regional Difference of Antibiotic Resistance of Helicobacter pylori Strains in Korea / 대한소화기학회지

BACKGROUND/AIMS: This study was performed to compare the prevalence rates of primary antibiotic resistance in Helicobacter pylori (H. pylori) isolates among different regions of Korea. METHODS: H. pylori were isolated from gastric mucosal biopsy specimens of 99 Koreans who lived in Gyeonggi (n=40), Kangwon province (n=40) and Busan (n=19) from April to August in 2008. All the patients had no history of H. pylori eradication therapy. The susceptibilities of the H. pylori isolates to amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, ciprofloxacin, levofloxacin, and moxifloxacin were tested according to the agar dilution method. RESULTS: There was a difference in resistance to clarithromycin in three institutes located among Gyeonggi (32.5%), Kangwon province (12.5%) and Busan (42.1%) by One way ANOVA test (p=0.027) and nonparametric Kruskal Wallis test (p=0.027). However, by post-hoc analysis, there was no statistically significant difference among three regions. Similarly, the other 7 antibiotics (amoxicillin, metronidazole, tetracycline, azithromycin, ciprofloxacin, levofloxacin and moxifloxacin) did not show any significant difference. CONCLUSIONS: There was no significant regional difference of the primary antibiotic resistance of H. pylori. However, the included patient number might not be enough for this conclusion demanding further evaluations.

Regional Difference of Antibiotic Resistance of Helicobacter pylori Strains in Korea / 대한소화기학회지

BACKGROUND/AIMS: This study was performed to compare the prevalence rates of primary antibiotic resistance in Helicobacter pylori (H. pylori) isolates among different regions of Korea. METHODS: H. pylori were isolated from gastric mucosal biopsy specimens of 99 Koreans who lived in Gyeonggi (n=40), Kangwon province (n=40) and Busan (n=19) from April to August in 2008. All the patients had no history of H. pylori eradication therapy. The susceptibilities of the H. pylori isolates to amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, ciprofloxacin, levofloxacin, and moxifloxacin were tested according to the agar dilution method. RESULTS: There was a difference in resistance to clarithromycin in three institutes located among Gyeonggi (32.5%), Kangwon province (12.5%) and Busan (42.1%) by One way ANOVA test (p=0.027) and nonparametric Kruskal Wallis test (p=0.027). However, by post-hoc analysis, there was no statistically significant difference among three regions. Similarly, the other 7 antibiotics (amoxicillin, metronidazole, tetracycline, azithromycin, ciprofloxacin, levofloxacin and moxifloxacin) did not show any significant difference. CONCLUSIONS: There was no significant regional difference of the primary antibiotic resistance of H. pylori. However, the included patient number might not be enough for this conclusion demanding further evaluations.

Clarithromycin-resistant Helicobacter pylori in Recife, Brazil, directly identified from gastric biopsies by polymerase chain reaction / Helicobacter pylori resistente à claritromicina no Recife, Brasil, identificada diretamente de biopsias gástricas pela reação em cadeia de polimerase

CONTEXT: Clarithromycin is the most effective drug used in the eradication of infection by Helicobacter pylori. Due to worldwide increase in resistance, pre-treatment susceptibility testing for clarithromycin is recommended. OBJECTIVES: To evaluate the prevalence of clarithromycin resistance of H. pylori in Recife, a city in Northeast Brazil. METHODS: From January 2006 to December 2007, 114 gastric biopsy samples positive for H. pylori at culture were directly assayed by polymerase chain reaction (PCR) to detect the most frequent point mutations involved in clarithromycin resistance. Results were compared with those obtained by Etests. RESULT: Molecular and phenotypic methods showed 111 (97.4 percent) susceptible or resistant concordant results. PCR detected 3 (2.6 percent) biopsy specimens with H. pylori-resistant genotypes, which were misdiagnosed as susceptible by Etests. In Recife, based on PCR results, primary clarithromycin resistance was found in 15 (16.5 percent) patients, prevalence close to that observed in Southeast Brazil. Resistance increased to 52 percent among previously treated patients. The point mutation A2143G was present in 20 (71.4 percent) of specimens and A2142G, in 8 (28.6 percent) of specimens. A2142C was not found. CONCLUSION: In Recife, the prevalence of primary clarithromycin resistance, 16.5 percent, showed the need for pretreatment susceptibility testing in H. pylori infections.

CONTEXTO: Claritromicina é a droga mais eficaz usada na erradicação da infecção pelo H. pylori. Devido ao aumento mundial da resistência, o teste de susceptibilidade à claritromicina pré-tratamento é recomendado. OBJETIVO: Determinar a prevalência de H. pyloriclaritromicina resistente em Recife, PE, Brasil. MÉTODO: De janeiro de 2006 a dezembro de 2007, 114 biopsias gástricas positivas à cultura para H. pylori foram diretamente analisadas pela reação em cadeia da polimerase (PCR), para detectar a frequência das mutações em ponto que envolvem a resistência à claritromicina. Os resultados foram comparados com os obtidos pelos E-testes. RESULTADO: Os métodos molecular e fenótipo fenotípico mostraram 111 (97.4 por cento) resultados concordantes, sensíveis ou resistentes. A PCR detectou três (2.6 por cento) espécimes de H. pyloricom genótipo resistente, diagnosticados erroneamente como sensíveis pelo E-teste. No Recife, baseando-se nos resultados da PCR, a resistência primária à claritromicina foi encontrada em 15 (16.5 por cento) pacientes, esta prevalência também foi observada no sudeste do Brasil. Entre os pacientes previamente tratados, a resistência elevou-se para 52 por cento. A mutação em ponto A2143G foi observada em 20 (71.4 por cento) dos espécimes e a A2142G, em 8 (28.6 por cento). A mutação A2142C não foi encontrada. CONCLUSÃO: No Recife, a prevalência da resistência a claritromicina, 16.5 por cento, mostrou a necessidade de realização dos testes de susceptibilidade prétratamento nas infecções por H. pylori.