Antimicrobial susceptibility of 6 antimicrobial agents in Helicobacter pylori clinical isolates by using EUCAST breakpoints compared with previously used breakpoints / Sensibilidad de aislamientos clínicos de Helicobacter pylori a seis antimicrobianos utilizando los criterios EUCAST y comparando los resultados con criterios utilizados anteriormente

INTRODUCTION: The aim of this study was to determine the differences in percentage resistance in H. pylori clinical isolates using EUCAST breakpoints compared with previously used breakpoints. MIC value distribution in H. pylori clinical isolates was also studied. METHODS: Susceptibility to amoxicillin, tetracycline, metronidazole, clarithromycin, rifampicin and levofloxacin was performed by E-test in 824 H. pylori clinical isolates. EUCAST and previous breakpoints defined resistance as follows: MIC > 0.12 mg/L and ≥ 2 mg/L for amoxicillin, > 8 mg/L and ≥ 8 mg/L for metronidazole, > 0.5 mg/L and ≥ 1 mg/L for clarithromycin, >1mg/L and ≥ 32 mg/L for rifampicin, and > 1 mg/L and ≥ 4 mg/L for tetracycline and >1mg/L levofloxacin. RESULTS: Overall resistance rate by EUCAST and by previous breakpoints was 8.5% and 3.2% for amoxicillin, 0.6% and 0.1% for tetracycline, 39.2% and 39.7% for metronidazole, 51.2% and 51.2% for clarithromycin, 32% and 3.1% for rifampicin, and 6.7% and 6.7% for levofloxacin. CONCLUSIONS: When using the different breakpoints for antimicrobial susceptibility testing, similar results were found with most antibiotics tested (tetracycline, metronidazole, clarithromycin, and levofloxacin), except for amoxicillin and rifampicin

INTRODUCCIÓN: El objetivo de este estudio era determinar las diferencias en el porcentaje de resistencia de aislamientos clínicos de H. pylori usando los puntos de corte de EUCAST comparado con los puntos de corte usados anteriormente. También se estudió la distribución de los valores de CMI en los aislamientos de H. pylori. MÉTODOS: La sensibilidad de amoxicilina, tetraciclina, metronidazol, claritromicina, rifampicina y levo-floxacina se determinó mediante E-test en 824 aislamientos clínicos de H. pylori. Los puntos de corte utilizados fueron EUCAST: CMI > 0,12 mg/L para amoxicilina, > 8 mg/L para metronidazol, >0,5mg/L para claritromicina y > 1 mg/L para rifampicina, tetraciclina y levofloxacina. Los puntos de corte que se habían utilizado antes de EUCAST fueron: CMI ≥ 2 mg/L para amoxicilina, ≥ 8 mg/L para metronidazol, ≥ 1 mg/L para claritromicina, ≥ 32 mg/L para rifampicina, ≥ 4 mg/L para tetraciclina y > 1 mg/L para levofloxacina. RESULTADOS: La resistencia global con los puntos de corte EUCAST y con los puntos de corte anteriores fue: 8,5% y 3,2% para amoxicilina, 0,6% y 0,1% para tetraciclina, 39,2% y 39,7% para metronidazol, 51,2% y 51,2% para claritromicina, 32% y 3,1% para rifampicina y 6,7% y 6,7% para levofloxacina. CONCLUSIÓN: A pesar de la utilización de diferentes puntos de corte, se obtuvieron resultados de resistencia similares para la mayoría de los antibióticos probados (tetraciclina, metronidazol, claritrnnñomicina, y levofloxacino), con la única excepción de amoxicilina y rifampicina

Antimicrobial susceptibility of 6 antimicrobial agents in Helicobacter pylori clinical isolates by using EUCAST breakpoints compared with previously used breakpoints.

INTRODUCTION: The aim of this study was to determine the differences in percentage resistance in H. pylori clinical isolates using EUCAST breakpoints compared with previously used breakpoints. MIC value distribution in H. pylori clinical isolates was also studied. METHODS: Susceptibility to amoxicillin, tetracycline, metronidazole, clarithromycin, rifampicin and levofloxacin was performed by E-test in 824 H. pylori clinical isolates. EUCAST and previous breakpoints defined resistance as follows: MIC >0.12mg/L and ≥2mg/L for amoxicillin, >8mg/L and ≥8mg/L for metronidazole, >0.5mg/L and ≥1mg/L for clarithromycin, >1mg/L and ≥32mg/L for rifampicin, and >1mg/L and ≥4mg/L for tetracycline and >1mg/L levofloxacin. RESULTS: Overall resistance rate by EUCAST and by previous breakpoints was 8.5% and 3.2% for amoxicillin, 0.6% and 0.1% for tetracycline, 39.2% and 39.7% for metronidazole, 51.2% and 51.2% for clarithromycin, 32% and 3.1% for rifampicin, and 6.7% and 6.7% for levofloxacin. CONCLUSIONS: When using the different breakpoints for antimicrobial susceptibility testing, similar results were found with most antibiotics tested (tetracycline, metronidazole, clarithromycin, and levofloxacin), except for amoxicillin and rifampicin.

Burkholderia cepacia complex infection in an Adult Cystic Fibrosis unit in Madrid

Introduction: Burkholderia cepacia complex have emerged as significant pathogens in cystic fibrosis (CF) patients due to the risk of cepacia syndrome and the innate multi-resistance of the microorganisms to antibiotics. The aim of this study was to describe the antimicrobial susceptibility profiles, the genotypes and subtypes of BCC, and the clinical evolution of CF patients with BCC. Methods The lung function and Brasfield and Shwachman score were assessed in 12 patients. BCC were identified and susceptibility was studied by MicroScan (Siemens). Species and genospecies of BCC were confirmed by molecular methods in a Reference Centre (Majadahonda).Result sBCC were identified in 12 of 70 patients (17.1%) over a ten year period. The mean age to colonization by BCC was 24.4 years (SD: 7.71). B. cenocepacia was isolated in 4 patients (33.3%), B. contaminans was isolated in 3 patients (25%), both B. vietnamiensis and B. stabilis were isolated in 2 patients (16.7%), and B. cepacia, B. multivorans and B. late were isolated in one patient (8.3%). Among the B. cenocepacia, subtype IIIa was identified in two strains, and subtype IIIb was identified in the other two strains. There was susceptibility to meropenem in 90% of BCC, 80% to cotrimoxazole, 60% to minocycline, 50% to ceftazidime, and 40% to levofloxacin. Conclusions B. cenocepacia was the most prevalent species among the BCC isolated in CF adult patients, and subtypes IIIa and IIIb were identified in the 50% of the strains. Meropenem and cotrimoxazole showed the best activity

Introduction: Burkholderia cepacia complex have emerged as significant pathogens in cystic fibrosis (CF)patients due to the risk of cepacia syndrome and the innate multi-resistance of the microorganisms to antibiotics. The aim of this study was to describe the antimicrobial susceptibility profiles, the genotypes and subtypes of BCC, and the clinical evolution of CF patients with BCC. Methods: The lung function and Brasfield and Shwachman score were assessed in 12 patients.BCC were identified and susceptibility was studied by MicroScan (Siemens). Species and genospecies of BCC were confirmed by molecular methods in a Reference Centre (Majadahonda).Results: BCC were identified in 12 of 70 patients (17.1%) over a ten year period. The mean age to colonization by BCC was 24.4 years (SD: 7.71). B. cenocepacia was isolated in 4 patients (33.3%), B. contaminans was isolated in 3 patients (25%), both B. vietnamiensis and B. stabilis were isolated in 2 patients (16.7%), and B. cepacia, B. multivorans and B. late were isolated in one patient (8.3%). Among the B. cenocepacia, subtype IIIa was identified in two strains, and subtype IIIb was identified in the other two strains. There was susceptibility to meropenem in 90% of BCC, 80% to cotrimoxazole, 60% to minocycline, 50% to ceftazidime, and 40% to levofloxacin. Conclusions: B. cenocepacia was the most prevalent species among the BCC isolated in CF adult patients, and subtypes IIIa and IIIb were identified in the 50% of the strains. Meropenem and cotrimoxazole showed the best activity

Burkholderia cepacia complex infection in an Adult Cystic Fibrosis unit in Madrid.

INTRODUCTION: Burkholderia cepacia complex have emerged as significant pathogens in cystic fibrosis (CF) patients due to the risk of cepacia syndrome and the innate multi-resistance of the microorganisms to antibiotics. The aim of this study was to describe the antimicrobial susceptibility profiles, the genotypes and subtypes of BCC, and the clinical evolution of CF patients with BCC. METHODS: The lung function and Brasfield and Shwachman score were assessed in 12 patients. BCC were identified and susceptibility was studied by MicroScan (Siemens). Species and genospecies of BCC were confirmed by molecular methods in a Reference Centre (Majadahonda). RESULTS: BCC were identified in 12 of 70 patients (17.1%) over a ten year period. The mean age to colonization by BCC was 24.4 years (SD: 7.71). B. cenocepacia was isolated in 4 patients (33.3%), B. contaminans was isolated in 3 patients (25%), both B. vietnamiensis and B. stabilis were isolated in 2 patients (16.7%), and B. cepacia, B. multivorans and B. late were isolated in one patient (8.3%). Among the B. cenocepacia, subtype IIIa was identified in two strains, and subtype IIIb was identified in the other two strains. There was susceptibility to meropenem in 90% of BCC, 80% to cotrimoxazole, 60% to minocycline, 50% to ceftazidime, and 40% to levofloxacin. CONCLUSIONS: B. cenocepacia was the most prevalent species among the BCC isolated in CF adult patients, and subtypes IIIa and IIIb were identified in the 50% of the strains. Meropenem and cotrimoxazole showed the best activity.

Helicobacter pylori resistance to antibiotics in Europe and its relationship to antibiotic consumption.

OBJECTIVE: Resistance to antibiotics is the major cause of treatment failure of Helicobacter pylori infection. A study was conducted to assess prospectively the antibacterial resistance rates of H pylori in Europe and to study the link between outpatient antibiotic use and resistance levels in different countries. DESIGN: Primary antibiotic resistance rates of H pylori were determined from April 2008 to June 2009 in 18 European countries. Data on yearly and cumulative use over several years of systemic antibacterial agents in ambulatory care for the period 2001-8 were expressed in Defined Daily Doses (DDD) per 1000 inhabitants per day. The fit of models and the degree of ecological association between antibiotic use and resistance data were assessed using generalised linear mixed models. RESULTS: Of 2204 patients included, H pylori resistance rates for adults were 17.5% for clarithromycin, 14.1% for levofloxacin and 34.9% for metronidazole, and were significantly higher for clarithromycin and levofloxacin in Western/Central and Southern Europe (>20%) than in Northern European countries (<10%). Model fit improved for each additional year of antibiotic use accumulated, but the best fit was obtained for 2005. A significant association was found between outpatient quinolone use and the proportion of levofloxacin resistance (p=0.0013) and between the use of long-acting macrolides only and clarithromycin resistance (p=0.036). CONCLUSION: In many countries the high rate of clarithromycin resistance no longer allows its empirical use in standard anti-H pylori regimens. The knowledge of outpatient antibiotic consumption may provide a simple tool to predict the susceptibility of H pylori to quinolones and to macrolides and to adapt the treatment strategies.

[Pneumococcal bacteremia in adult patients at a hospital in Madrid: serotypes and susceptibility]. / Bacteriemia neumocócica en pacientes adultos de un hospital de Madrid: serotipos y sensibilidad antibiótica.

INTRODUCTION: The aim of this study is to describe the distribution of Streptococcus pneumoniae serotypes, its antimicrobial susceptibility profiles and the relation with vaccines in pneumococcal invasive strains isolated from blood cultures of adult patients. METHODS: All pneumococci isolated (67 strains) from blood cultures were serotyped by latex agglutination (Pneumotest latex) and Quellung reaction (Statens Serum Institut, Denmark). Antimicrobial susceptibility testing to penicillin (PEN), cefotaxime (CT), erythromycin (ERY) and levofloxacin (LEV) was performed by the E-test method (Biomèrieux, France). RESULTS: Among the 67 strains isolated, the most prevalent serotypes were 22F (11.9%) and 3 (11.9%), the second most frequent were 7F (7.5%) and 19A (7.5%). The coverage of the strains by the pneumococcal 7-valent conjugate vaccine (VNC7V), pneumococcal 13-valent conjugate vaccine (VNC13V) and pneumococcal 23-valent polysaccharide (VNP23V) were 16, 49 and 82%, respectively. Serotypes 22F and 3 were responsible for 14 of the 48 episodes of pneumonia with bacteremia (29.2%) and only 2 of the 19 episodes (10.5%) of bacteremia without pneumonia. According to the 2007 CLSI criteria, 12 strains (17.9%) were non-susceptible to penicillin. Eleven of this 12 strains (91.7%) were resistant to erythromycin, simultaneously. CONCLUSIONS: The most common serotypes were 22F, 3, 7F y 19A. Three of them (3, 7F y 19A) are serotypes that are covered by the new VNC13V but not by VNC7V. Serotype 22F is a serotype emergent that is not covered by the VNC7V. The percentage of non-susceptibility to penicillin and resistance to erythromycin was comparable to the percentage reported in our country.

Bacteriemia neumocócica en pacientes adultos de un hospital de Madrid: serotipos y sensibilidad antibiótica / Pneumococcal bacteremia in adult patients at a hospital in Madrid: serotypes and susceptibility

Introducción. El objetivo de este estudio es describir la distribución de los serotipos de Streptococcus pneumoniae, su sensibilidad antimicrobiana y su relación con las vacunas en cepas de neumococo aisladas en hemocultivo de pacientes adultos. Métodos. Se estudiaron un total de 67 cepas de S. pneumoniae. El serotipado se realizó mediante aglutinación con partículas de látex sensibilizadas y la reacción de Quellung. La concentración mínima inhibitoria (CMI) frente a penicilina, eritromicina, cefotaxima y levofloxacino se determinó mediante E-test. Resultados. Los serotipos más prevalentes entre las 67 cepas fueron 22F (11,9%) y 3 (11,9%), seguidos de 7F (7,5%) y 19A (7,5%). Los porcentajes de cepas cubiertas por la vacuna neumocócica 7-valente (VNC7V), vacuna neumocócica 13-valente (VNC13V) y vacuna polisacarida 23-valente (VNP23V) fueron respectivamente de 16, 49 y 82%. Los serotipos 22F y 3 ocasionaron en conjunto 14 de los 48 episodios de neumonía bacteriémica (29,2%) y sólo 2 de los de los 19 episodios (10,5%) de bacteriemia sin neumonía. Según el criterio del CLSI del 2007, 12 cepas (17,9%) fueron no sensibles a penicilina. Once de estas 12 cepas (91,7%) fueron simultáneamente resistentes a eritromicina. Conclusiones. Los serotipos más comunes fueron 22F, 3, 7F y 19A. Tres de ellos (3, 7F y 19A) son serotipos cubiertos por la VNC13V pero no por la VNC7V. El serotipo 22F es un serotipo emergente no cubierto por la VNC7V. Los porcentajes de no sensibilidad a penicilina y resistencia a eritromicina se encontraron en márgenes comparables a los informados en nuestro país(AU)

Introduction. The aim of this study is to describe the distribution of Streptococcus pneumoniae serotypes, its antimicrobial susceptibility profiles and the relation with vaccines in pneumococcal invasive strains isolated from blood cultures of adult patients. Methods. All pneumococci isolated (67 strains) from blood cultures were serotyped by latex agglutination (Pneumotest latex) and Quellung reaction (Statens Serum Institut, Denmark). Antimicrobial susceptibility testing to penicillin (PEN), cefotaxime (CT), erythromycin (ERY) and levofloxacin (LEV) was performed by the E-test method (Biomèrieux, France). Results. Among the 67 strains isolated, the most prevalent serotypes were 22F (11.9%) and 3 (11.9%), the second most frequent were 7F (7.5%) and 19A (7.5%). The coverage of the strains by the pneumococcal 7-valent conjugate vaccine (VNC7V), pneumococcal 13-valent conjugate vaccine (VNC13V) and pneumococcal 23-valent polysaccharide (VNP23V) were 16, 49 and 82%, respectively. Serotypes 22F and 3 were responsible for 14 of the 48 episodes of pneumonia with bacteremia (29.2%) and only 2 of the 19 episodes (10.5%) of bacteremia without pneumonia. According to the 2007 CLSI criteria, 12 strains (17.9%) were non-susceptible to penicillin. Eleven of this 12 strains (91.7%) were resistant to erythromycin, simultaneously. Conclusions. The most common serotypes were 22F, 3, 7F y 19A. Three of them (3, 7F y 19A) are serotypes that are covered by the new VNC13V but not by VNC7V. Serotype 22F is a serotype emergent that is not covered by the VNC7V. The percentage of non-susceptibility to penicillin and resistance to erythromycin was comparable to the percentage reported in our country(AU)

Abdominal abscess due to NDM-1-producing Klebsiella pneumoniae in Spain.

We describe a clinical case of an abdominal abscess due to NDM-1-producing Klebsiella pneumoniae in a 35-year-old Spanish patient after hospitalization in India for perforated appendicitis and peritonitis. The strain belonged to the MLST type 231 and had multiple additional antibiotic resistance genes such as bla(CTX-M-15), armA methylase, aac(6')-Ib-cr, dfrA12, sul1 and qnrB and lack of porin genes ompK35 and ompK36. The patient was cured after abscess drainage.

Rapid detection of clarithromycin resistant Helicobacter pylori strains in Spanish patients by polymerase chain reaction-restriction fragment length polymorphism

Objetivo. Determinar la presencia de mutaciones en el gen 23S rRNA que dan lugar a resistencia a claritromicina en aislamientos clínicos de Helicobacter pylori y evaluar un método nuevo de PCR-RFLP para detectar la mutación más frecuente en nuestra población. Métodos. A partir de biopsias gástricas obtenidas de pacientes sintomáticos, H. pylori fue cultivado acorde con los procedimientos microbiológicos establecidos. La resistencia a claritromicina fue determinada fenotípicamente mediante E-test. La extracción de DNA fue realizada con la plataforma NucliSens, que se basa en la extracción de DNA mediante partículas de sílice magnética siguiendo las instrucciones del fabricante. Para detectar las mutaciones puntuales en el gen 23S rRNA se analizó la secuencia de los productos amplificados por PCR. La PCRRFLP fue realizada usando la enzima BsaI para detectar la mutación en la posición 2143 que da lugar a resistencia a claritromicina. Resultados. Un total de 42 cepas fueron resistentes a claritromicina. Los resultados de E-test fueron confirmados por PCR en 34 (88.1%) de las cepas. Hubo 8 cepas de H. pylori resistentes a claritromicina por E-test donde no se encontró ningún punto de mutación en la secuencia del gen 23S rRNA. La mutación A2143G fue encontrada en el 85.3%. La enzima BsaI fue capaz de detectar esta mutación en todas las cepas que la tenían. Conclusiones. PCR-RFLP es un método fiable para detectar la resistencia a claritromicina en cepas de H. pylori, en especial en países con alta prevalencia como España. Este estudio sugiere que esta prueba puede ser útil antes de elegir el tratamiento erradicador(AU)

Introduction. The aim of this study was to characterize the mutations types present in the 23S rRNA gene related to H. pylori clarithromycin-resistance strains in Spain and evaluate a novel PCR-RFLP method for detection of the most frequent point mutation in our population. Methods. Gastric biopsies were obtained by endoscopy from patients with gastric symptoms. H. pylori was cultured according to standard microbiological procedures and clarithromycin resistance was determined by E-test. DNA extraction was performed by NucliSens platform with the NucliSens magnetic extraction reagents (bioMérieux) according to the manufacturer instructions. Analyses for point mutations in 23S rRNA gene strains were performed by sequence analysis of amplified polymerase chain reaction products. Restriction fragment length polymorphism was performed using BsaI enzyme to detect restriction sites that correspond to the mutation (A2143G). Results. We found 42 out of 118 (35.6%) strains resistant to clarithromycin by E-test. E-test results were confirmed for the presence of point mutation in 34 (88.1%) of these strains. Mutation A2143G was found in 85.3% of the strains. Analyses with the restriction enzyme BsaI was able to confirm the presence of A2143G mutation. There were 8 H. pylori strains resistant to clarithromycin by E-test but without any point mutation in the 23 rRNA gene. Conclusions. We conclude that PCR-RFLP is a reliable method to detect clarithromycin-resistance H. pylori strains in countries with a high prevalence of clarithromycin-resistance as Spain It may be useful before choosing regimens of H. pylori eradication(AU)

[Antibiotic susceptibility of Corynebacterium macginleyi strains causing conjunctivitis]. / Sensibilidad antibiótica de cepas de Corynebacterium macginleyi productoras de conjuntivitis.

INTRODUCTION: Lipophilic corynebacteria are part of the saprophytic skin flora and they rarely cause infection. C. macginleyi is an exception since it has been involved in conjunctivitis and other ocular surface affections. MATERIAL AND METHODS: Thirty three C. macginleyi strains were obtained from conjunctival swabs from patients with conjunctivitis symptoms. The minimum inhibitory concentration (MIC) for 7 antibiotics (penicillin, gentamycin, ciprofloxacin, tetracycline, vancomycin, rifampicin and linezolid) was tested by broth microdilution method. RESULTS: One strain was resistant to ciprofloxacin (MIC=16 mg/L) and two were resistant to tetracycline (MIC= 64 y 16 mg/L). The rest of the strains were susceptible to all the antibiotics tested. CONCLUSIONS: At the moment, C. macginleyi does not present a major problem due to the low resistance rates shown in the present and other studies. However, epidemiological surveillance of its susceptibility pattern is needed as well as an appropriate use of topical antibiotics in order to achieve a good infection control.

Sensibilidad antibiótica de cepas de Corynebacterium macginleyi productoras de conjuntivitis / Antibiotic susceptibility of Corynebacterium macginleyi strains causing conjunctivitis

Introducción. Las corinebacterias lipofílicas forman parte de la flora saprófita de la piel y mucosas del ser humano y raramente son productoras de infección. C. macginleyi es una excepción, ya que se ha visto implicada en casos de conjuntivitis y otras afecciones oculares. Material y métodos. Se obtuvieron 33 cepas de C. macginleyi procedentes de exudados conjuntivales de pacientes con síntomas de conjuntivitis. La concentración mínima inhibitoria (CMI) para 7 antibióticos de uso oftálmico (penicilina, gentamicina, ciprofloxacino, tetraciclina, vancomicina, rifampicina y linezolid) fue probada mediante microdilución en caldo. Resultados. Sólo se detectó una cepa resistente a ciprofloxacino (CMI = 16 mg/L) y dos cepas resistentes a tetraciclina (CMI= 64 y 16 mg/L). El resto de cepas fueron sensibles a todos los antibióticos probados. Conclusiones. C. macginleyi por el momento no parece presentar un problema importante por las bajas tasas de resistencia que el microorganismo ha mostrado en éste y otros estudios, si bien es necesario la vigilancia epidemiológica de las resistencias de la bacteria así como un buen uso de los antibióticos en colirio con el objeto de realizar un tratamiento adecuado de la infección(AU)

Introduction. Lipophilic corynebacteria are part of the saprophytic skin flora and they rarely cause infection. C. macginleyi is an exception since it has been involved in conjunctivitis and other ocular surface affections. Material and methods. Thirty three C. macginleyi strains were obtained from conjunctival swabs from patients with conjunctivitis symptoms. The minimum inhibitory concentration (MIC) for 7 antibiotics (penicillin, gentamycin, ciprofloxacin, tetracycline, vancomycin, rifampicin and linezolid) was tested by broth microdilution method. Results. One strain was resistant to ciprofloxacin (MIC= 16 mg/L) and two were resistant to tetracycline (MIC= 64 y 16 mg/L). The rest of the strains were susceptible to all the antibiotics tested. Conclusions. At the moment, C. macginleyi does not present a major problem due to the low resistance rates shown in the present and other studies. However, epidemiological surveillance of its susceptibility pattern is needed as well as an appropriate use of topical antibiotics in order to achieve a good infection control(AU)

High prevalence of clarithromycin-resistant Helicobacter pylori strains and risk factors associated with resistance in Madrid, Spain.

Clarithromycin is one of the antibiotics used for the treatment of Helicobacter pylori infections, and clarithromycin 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 (bioMérieux). The DNA sequences of the 23S rRNA genes of clarithromycin-resistant and -sensitive strains were determined to identify specific 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 confirmed 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.

Detection of Helicobacter pylori and clarithromycin resistance in gastric biopsies of pediatric patients by using a commercially available real-time polymerase chain reaction after NucliSens semiautomated DNA extraction.

UNLABELLED: The aim of this study was to evaluate a commercially available kit, MutaREAL Helicobacter pylori (Inmundiagnostik, Bensheim, Germany) real-time polymerase chain reaction (PCR), for detection of H. pylori infection and point mutations in the 23S rRNA genes responsible for clarithromycin resistance in gastric biopsies. METHODS: Gastric biopsies were obtained by endoscopy from pediatric patients with gastric symptoms, cultured according to standard microbiologic procedures, and clarithromycin resistance was determined by E-test. DNA extraction was performed by NucliSens platform with the NucliSens magnetic extraction reagents (bioMérieux, Marcy-l'Etoile, France) according to the manufacturer's instructions. MutaREAL kit was used according to manufacturer recommendations in a LightCycler (Roche Diagnostics Gmbh, Mannheim, Germany) for the detection of H. pylori infection and clarithromycin susceptibility. RESULTS: Amplification was positive for H. pylori in 62 and negative in 44 biopsies out of 106 biopsies. All negative biopsies were positive for human beta-globin gene. This real-time PCR assay showed sensitivity of 93.33% (negative predictive value, 90.90%) and specificity of 86.95% (positive predictive value, 90.32%) for H. pylori detection. Clarithromycin resistance was detected in 26 cases by PCR with a sensitivity and specificity of 90.62 and 95.83, respectively. CONCLUSIONS: MutaREAL kit was able to detect H. pylori and its clarithromycin susceptibility with high efficacy. This method is quicker than culture and is suitable to be done in 1 h after DNA extraction. The new system of automatic extraction will lead to reduction in the total time.

Inhibitory effect of Gram-negative and Gram-positive microorganisms against Helicobacter pylori clinical isolates.

OBJECTIVES: To determine the in vitro inhibitory effect of several Gram-negative and Gram-positive microorganisms against Helicobacter pylori clinical isolates. METHODS: The in vitro effect of 32 microorganisms against H. pylori clinical isolates was determined by a diffusion method. Time-kill assay was performed with two Staphylococcus spp. strains. RESULTS: Anti-H. pylori activity was detected with Saccharomyces cerevisiae, Bacillus spp., 1 Enterococcus faecium and 2 Lactobacillus spp. against 7, 11, 1, 5 and 6 H. pylori strains tested. All Staphylococcus spp. showed an anti-H. pylori effect: one Staphylococcus auricularis and two Staphylococcus epidermidis against all H. pylori tested; Staphylococcus aureus, Staphylococcus hominis and S. auricularis against six, five and seven H. pylori strains; and two other coagulase-negative Staphylococcus against one H. pylori strain. An inhibitory effect was detected with one Escherichia coli against one H. pylori. Klebsiella pneumoniae, Salmonella spp. and Acinetobacter baumannii showed activity against four H. pylori strains, and Enterobacter cloacae and Stenotrophomonas maltophilia showed activity against 14 H. pylori isolates. No anti-H. pylori activity was detected with one Lactobacillus spp., two Lactococcus lactis, four Streptococcus spp., one Bacillus cereus, one E. faecium, one Enterococcus faecalis, one E. coli, Pseudomonas aeruginosa and Klebsiella oxytoca. Time-kill assay showed bactericidal activity at 24 h with the two Staphylococcus spp. strains tested. CONCLUSIONS: Several strains of human pathogens or commensal bacteria are able to inhibit H. pylori growth in vitro and it is a strain-dependent phenomenon.

Detection of clarithromycin-resistant Helicobacter pylori in frozen gastric biopsies from pediatric patients by a commercially available fluorescent in situ hybridization.

Clarithromycin resistance is an important factor of eradication failure. A commercially available fluorescent in situ hybridization (FISH) kit (creaFAST) was used to detect H. pylori infection and the resistance to clarithromycin in frozen biopsies. A total of 33 biopsies, H. pylori culture-positive, obtained from pediatric patients were retrospectively studied. Clarithromycin resistance was compared with MICs detected by E-test from H. pylori clinical isolates. All culture-positive biopsies were positive by FISH. Detection of clarithromycin resistance showed sensitivity of 90%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 86.7% compared with results obtained by E-test. Discrepant results were 2 biopsies, clarithromycin-susceptible by FISH but intermediate by E-test. In conclusion, FISH technology is a rapid, easy-to-implement, and reliable cultivation-independent method for routine application; however, when frozen biopsies are studied, some modification of the recommended procedure should be used to obtain better results.

Aislamiento de Pandoraea pulmonicola en paciente con fibrosis quística / Pandoraea pulmonicola isolation in patients with cystic fibrosis

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Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin.

Adherence by Helicobacter pylori increases the risk of gastric disease. Here, we report that more than 95% of strains that bind fucosylated blood group antigen bind A, B, and O antigens (generalists), whereas 60% of adherent South American Amerindian strains bind blood group O antigens best (specialists). This specialization coincides with the unique predominance of blood group O in these Amerindians. Strains differed about 1500-fold in binding affinities, and diversifying selection was evident in babA sequences. We propose that cycles of selection for increased and decreased bacterial adherence contribute to babA diversity and that these cycles have led to gradual replacement of generalist binding by specialist binding in blood group O-dominant human populations.

Clarithromycin resistance among Helicobacter pylori strains isolated from children: prevalence and study of mechanism of resistance by PCR-restriction fragment length polymorphism analysis.

Clarithromycin and metronidazole resistance was 29.1 and 23.9%, respectively, in 96 Helicobacter pylori strains obtained from pediatric patients. No resistance to amoxicillin was observed. Resistance according to patients' ages to clarithromycin and metronidazole was 45.4 and 18.2% in 22 patients from 4 to 8 years old, 30.2 and 20.7% in 53 patients from 9 to 13 years old, and 9.5 and 38.1% in 21 patients from 14 to 18 years old, respectively. The A2143G mutation was the most prevalent (82.1%) among clarithromycin-resistant strains.