Population structure and antimicrobial susceptibility of both nonpersistent and persistent Pseudomonas aeruginosa isolates recovered from cystic fibrosis patients.

Seventy-six Pseudomonas aeruginosa isolates recovered from chronically (n=18) and nonchronically (n=18) colonized cystic fibrosis (CF) patients (2002 to 2009) were grouped in separate polyclonal populations. International CF epidemic clones were not identified, but the high-risk clone ST274, also found circulating in Spanish hospitals, was present. Persistent isolates were more resistant to antibiotics than nonpersistent isolates.

In vitro prevention of Pseudomonas aeruginosa early biofilm formation with antibiotics used in cystic fibrosis patients.

The ability of antibiotics used in bronchopulmonary infections in cystic fibrosis (CF) patients to prevent Pseudomonas aeruginosa early biofilm formation was studied using a biofilm microtitre assay with 57 non-mucoid P. aeruginosa isolates (44 first colonisers and 13 recovered during the initial intermittent colonisation stage) obtained from 35 CF patients. Minimum biofilm inhibitory concentrations (BICs) of levofloxacin, ciprofloxacin, imipenem, ceftazidime, tobramycin, colistin and azithromycin were determined by placing a peg lid with a formed biofilm onto microplates containing antibiotics. A modification of this protocol consisting of antibiotic challenge during biofilm formation was implemented in order to determine the biofilm prevention concentration (BPC), i.e. the minimum concentration able to prevent biofilm formation. The lowest BPCs were for fluoroquinolones, tobramycin and colistin and the highest for ceftazidime and imipenem. The former antibiotics had BPCs identical to or only slightly higher than their minimum inhibitory concentrations (MICs) determined by standard Clinical and Laboratory Standards Institute (CLSI) microdilution and were also active on formed biofilms as reflected by their low BIC values. In contrast, ceftazidime and imipenem were less effective for prevention of biofilm formation and on formed biofilms. In conclusion, the new BPC parameter determined in non-mucoid P. aeruginosa isolates recovered during early colonisation stages in CF patients supports early aggressive antimicrobial treatment guidelines in first P. aeruginosa-colonised CF patients.

Emergence of a mutL mutation causing multilocus sequence typing-pulsed-field gel electrophoresis discrepancy among Pseudomonas aeruginosa isolates from a cystic fibrosis patient.

A multilocus sequence type (MLST) shift (from ST242 to ST996) was detected in Pseudomonas aeruginosa isolates with a uniform pulsed-field gel electrophoresis (PFGE) pattern obtained from a chronically colonized patient. MLST mutational change involved the mutL gene with the consequent emergence of a hypermutable phenotype. This observation challenges the required neutrality of mutL as an appropriate marker in MLST and alerts researchers to the limitations of MLST-only-based population studies in chronic infections under constant antibiotic selective pressure.

MALDI-TOF MS improves routine identification of non-fermenting Gram negative isolates from cystic fibrosis patients.

Identification of non-fermenting Gram-negative bacteria (NFGNB) from cystic fibrosis (CF) patients is often limited. A collection of stored NFGNB isolates (n=182) recovered from CF patients over a 15 year period was examined. The routinely reported identification during this period was compared with that obtained by MALDI-TOF MS. Isolates giving discrepant identification at the genus level were further analyzed by 16S rDNA sequencing. The MALDI-TOF MS system identified 94% of the isolates, including Burkholderia cepacia and Pandoraea spp. isolates, the latter previously misidentified as other NFGNB by conventional microbiological methods. Lack of identification by MALDI-TOF MS was associated with the absence of entries in the database.

Métodos de identificación bacteriana en el laboratorio de microbiología / Bacterial identification methods in the microbiology laboratory

Con el objetivo de identificar el agente etiológico responsable del proceso infeccioso y para conocer las implicaciones patogénicas/patológicas, la evolución clínica, y aplicar una terapia antimicrobiana eficaz, un pilar fundamental en la práctica de la microbiología clínica lo constituye la asignación de especie a un aislamiento microbiano. Dentro de la práctica rutinaria diaria, el laboratorio de microbiología aplica técnicas fenotípicas que permiten lograr este objetivo. Sin embargo, muestran algunas limitaciones que se observan de manera más evidente para algún tipo de microorganismo. Los métodos moleculares permiten soslayar algunas de estas limitaciones, si bien su implementación no es universal. Este hecho se debe a un coste más elevado y al grado de especialización que se requiere para su aplicación, por lo que los métodos moleculares suelen estar centralizados en laboratorios o centros de referencia. Recientemente los métodos basados en proteómica han irrumpido de manera importante en el campo del diagnóstico microbiológico y sin duda va a tener un gran impacto en la organización futura de los servicios de microbiología. Este manuscrito revisa de manera concisa los aspectos más reseñables de los tres métodos de identificación bacteriana arriba descritos que se usan en los laboratorio de microbiol (AU)

In order to identify the agent responsible of the infectious process and understanding the pathogenic/pathological implications, clinical course, and to implement an effective antimicrobial therapy, a mainstay in the practice of clinical microbiology is the allocation of species to a microbial isolation. In daily routine practice microbiology laboratory phenotypic techniques are applied to achieve this goal. However, they have some limitations that are seen more clearly for some kinds of microorganism. Molecular methods can circumvent some of these limitations, although its implementation is not universal. This is due to higher costs and the level of expertise required for thei implementation, so molecular methods are often centralized in reference laboratories and centers. Recently, proteomics-based methods made an important breakthrough in the field of diagnostic microbiology and will undoubtedly have a major impact on the future organization of the microbiology services. This paper is a short review of the most noteworthy aspects of the three bacterial identification methods described above used in microbiology laboratories (AU)

[Bacterial identification methods in the microbiology laboratory]. / Métodos de identificación bacteriana en el laboratorio de microbiología.

In order to identify the agent responsible of the infectious process and understanding the pathogenic/pathological implications, clinical course, and to implement an effective antimicrobial therapy, a mainstay in the practice of clinical microbiology is the allocation of species to a microbial isolation. In daily routine practice microbiology laboratory phenotypic techniques are applied to achieve this goal. However, they have some limitations that are seen more clearly for some kinds of microorganism. Molecular methods can circumvent some of these limitations, although its implementation is not universal. This is due to higher costs and the level of expertise required for thei implementation, so molecular methods are often centralized in reference laboratories and centers. Recently, proteomics-based methods made an important breakthrough in the field of diagnostic microbiology and will undoubtedly have a major impact on the future organization of the microbiology services. This paper is a short review of the most noteworthy aspects of the three bacterial identification methods described above used in microbiology laboratories.

[Chronic bronchial infection: the problem of Pseudomonas aeruginosa]. / Infección bronquial crónica: el problema de Pseudomonas aeruginosa.

Pathogenic bronchopulmonary colonizations and the exacerbations produced are among the most important causes of reduced pulmonary function in patients with bronchiectasis. The most frequent pathogens in these patients are Haemophilus influenzae and Pseudomonas aeruginosa. Lesions are produced by the local inflammatory process and the vicious circle developed by antigen stimulation, the release of inflammatory mediators, the presence of neutrophils, the increase of bacterial inoculum and the release of bacterial exoproducts. P. aeruginosa has been demonstrated to affect the patients with bronchiectasis and poorest quality of life and to colonize those with the poorest pulmonary function and the highest number of antimicrobial treatments. In bronchiectasis, as in chronic obstructive pulmonary disease (COPD) or cystic fibrosis, P. aeruginosa is able to colonize the respiratory mucosa chronically. Due to the ecological niche occupied by P. aeruginosa and the multitude of cycles with antimicrobial agents to which these patients are subjected, the development of antimicrobial resistance is highly likely, encouraged by the high proportion of hypermutation variants in existence. Likewise, P. aeruginosa naturally grows in the form of biofilms on the mucosal surface, greatly contributing to its persistence. Antimicrobial treatment in patients with bronchiectasis and P. aeruginosa colonization should be based on antimicrobial agents, alone or in combination, that do not lose activity when acting on biofilms.

Infección bronquial crónica: el problema de Pseudomonas aeruginosa / Chronic bronchial infection: the problem of Pseudomonas aeruginosa

La colonización patogénica broncopulmonar y las exacerbaciones que se derivan de ella constituyen las causasmás importantes del deterioro de la función pulmonar en los pacientes con bronquiectasias. Haemophilusinfluezae y Pseudomonas aeruginosa son los patógenos más frecuentes en estos pacientes. El efecto lesivo seproduce por el proceso de inflamación local y el círculo vicioso que se desarrolla por el estímulo antigénico, laliberación de mediadores de la inflamación, la presencia de neutrófilos, el aumento del inóculo bacteriano yla liberación de exoproductos bacterianos. Se ha demostrado que P. aeruginosa afecta a los pacientes con bronquiectasiascon peor calidad de vida, coloniza a los que tienen peor funcionalidad pulmonar y mayor númerode tratamientos antimicrobianos. En las bronquiectasias, al igual que en la enfermedad pulmonar obstructivacrónica (EPOC) o fibrosis quística, P. aeruginosa es capaz de colonizar crónicamente la mucosa respiratoria.Debido al nicho ecológico donde se sitúa P. aeruginosa y a la multitud de ciclos con antimicrobianos a los queson sometidos estos pacientes es fácil que se desarrollen resistencias a los antimicrobianos, favorecidas por laelevada proporción de variantes hipermutadoras que existen. Asimismo, hay que resaltar la forma natural decrecimiento en biopelículas de P. aeruginosa en la superficie mucosa y la contribución que ejerce para su persistencia.El tratamiento antimicrobiano en los pacientes con bronquiectasas con colonización por P.aeruginosa ha de basarse en antimicrobianos o asociaciones de éstos que no pierdan actividad al actuar sobrelas biopelículas(AU)

Pathogenic bronchopulmonary colonizations and the exacerbations produced are among the most importantcauses of reduced pulmonary function in patients with bronchiectasis. The most frequent pathogens in thesepatients are Haemophilus influenzae and Pseudomonas aeruginosa. Lesions are produced by the localinflammatory process and the vicious circle developed by antigen stimulation, the release of inflammatorymediators, the presence of neutrophils, the increase of bacterial inoculum and the release of bacterialexoproducts. P. aeruginosa has been demonstrated to affect the patients with bronchiectasis and poorestquality of life and to colonize those with the poorest pulmonary function and the highest number ofantimicrobial treatments. In bronchiectasis, as in chronic obstructive pulmonary disease (COPD) or cysticfibrosis, P. aeruginosa is able to colonize the respiratory mucosa chronically. Due to the ecological nicheoccupied by P. aeruginosa and the multitude of cycles with antimicrobial agents to which these patients aresubjected, the development of antimicrobial resistance is highly likely, encouraged by the high proportion ofhypermutation variants in existence. Likewise, P. aeruginosa naturally grows in the form of biofilms on themucosal surface, greatly contributing to its persistence. Antimicrobial treatment in patients withbronchiectasis and P. aeruginosa colonization should be based on antimicrobial agents, alone or incombination, that do not lose activity when acting on biofilms(AU)

[Polymorphism of mucA and fpvA genes in Pseudomonas aeruginosa isolates from cystic fibrosis patients: co-existence of genetically different variants]. / Polimorfismo de los genes mucA y fpvA en Pseudomonas aeruginosa de pacientes con fibrosis quística: coexistencia de variantes genéticas en el mismo paciente.

INTRODUCTION: Pseudomonas aeruginosa is able to colonize the lungs of cystic fibrosis patients (CF) in an adaptive process that results in the selection of a dominant strain through a process of genetic variation. METHODS: One hundred and twenty tree isolates of P. aeruginosa were sequentially recovered from 6 CF patients during the routine follow-up or exacerbations over periods of 2 to 12 years in the Ramon y Cajal University Hospital (Madrid, Spain). Another 13 isolates were obtained from a single CF patient in a short-term study. They were analysed by restriction fragment length polymorphism (RFLP) and sequencing of mucA and fpvA genes, which code for the alginate biosynthesis regulator and a pyoverdin receptor, respectively, and their antibiotic susceptibility was studied by microdilution. RESULTS: A dominant colonising strain was found in each patient based on the RFLP profile. The polymorphisms of mucA and fpvA genes correlated well with these profiles, but suggested a relationship between strains isolated from two brothers, not inferred by RFLP. Stop codon mutations in mucA were unique to each dominant strain, indicating the adaptive process suffered. The alternate detection of the same mucA and/or fpvA genotypic variants suggested the coexistence of several subpopulations. This hypothesis was confirmed in a prospective study in which 6 variants were isolated in 7 days from the same patient. CONCLUSIONS: Genotypic variants of the P. aeruginosa dominant strains can coexist in the chronic colonization in CF patients. These variants can be undetected by RFLP and they might present variable antibiotic susceptibility.

Polimorfismo de los genes mucA y fpvA en Pseudomonas aeruginosa de pacientes con fibrosis quística: coexistencia de variantes genéticas en el mismo paciente / Polymorphism of mucA and fpvA genes in Pseudomonas aeruginosa isolates from cystic fibrosis patients: co-existence of genetically different variants

Introducción Pseudomonas aeruginosa coloniza la mucosa respiratoria del paciente con fibrosis quística (FQ), seleccionándose en general una única cepa dominante en un proceso de variación genética. Métodos Se obtuvieron secuencialmente 123 aislados de P. aeruginosa en 6 pacientes con FQ atendidos durante su seguimiento habitual o en las exacerbaciones a lo largo de 2-12 años en el Hospital Universitario Ramón y Cajal (Madrid). Otros 13 aislados se obtuvieron de un sólo paciente en un estudio a corto plazo (7 días). Se estudió su sensibilidad antimicrobiana por microdilución y caracterizaron mediante polimorfismo de fragmentos de restricción (RFLP) y secuenciación de los genes mucA y fpvA, que codifican respectivamente el regulador de la síntesis de alginato y un receptor de pioverdina. Resultados Se identificó en cada paciente una cepa dominante según el perfil de RFLP. Los polimorfismos de los genes mucA y fpvA se correlacionaron bien con dicho perfil, pero en dos pacientes hermanos se observó una relación entre cepas no evidente por RFLP. Las mutaciones sin sentido en mucA fueron exclusivas de la cepa dominante de cada paciente, reflejando el proceso adaptativo. La detección alternativa de los mismos polimorfismos en mucA o fpvA mostró la coexistencia de subpoblaciones en cada paciente. Esta hipótesis se confirmó en el estudio prospectivo de 7 días al aislar 6 variantes en un único paciente. Conclusiones En la colonización crónica por P. aeruginosa en pacientes con FQ coexisten variantes genotípicas no siempre detectadas por RFLP y con diferentes perfiles de sensibilidad (AU)

Introduction Pseudomonas aeruginosa is able to colonize the lungs of cystic fibrosis patients (CF) in an adaptive process that results in the selection of a dominant strain through a process of genetic variation. Methods One hundred and twenty tree isolates of P. aeruginosa were sequentially recovered from 6 CF patients during the routine follow-up or exacerbations over periods of 2 to 12 years in the Ramon y Cajal University Hospital (Madrid, Spain). Another 13 isolates were obtained from a single CF patient in a short-term study. They were analysed by restriction fragment length polymorphism (RFLP) and sequencing of mucA and fpvA genes, which code for the alginate biosynthesis regulator and a pyoverdin receptor, respectively, and their antibiotic susceptibility was studied by microdilution. Results A dominant colonising strain was found in each patient based on the RFLP profile. The polymorphisms of mucA and fpvA genes correlated well with these profiles, but suggested a relationship between strains isolated from two brothers, not inferred by RFLP. Stop codon mutations in mucA were unique to each dominant strain, indicating the adaptive process suffered. The alternate detection of the same mucA and/or fpvA genotypic variants suggested the coexistence of several subpopulations. This hypothesis was confirmed in a prospective study in which 6 variants were isolated in 7 days from the same patient. Conclusions Genotypic variants of the P. aeruginosa dominant strains can coexist in the chronic colonization in CF patients. These variants can be undetected by RFLP and they might present variable antibiotic susceptibility (AU)

Unorthodox long-term aerosolized ampicillin use for methicillin-susceptible Staphylococcus aureus lung infection in a cystic fibrosis patient.

Staphylococcus aureus is a significant cause of pulmonary colonization in cystic fibrosis (CF) patients. The optimal strategy of therapy in chronically infected patients with this pathogen is not yet established. We report a successful long-term aerosolized ampicillin treatment of a 14-year-old girl with chronic symptomatic S. aureus lung infection.

Detection of high-risk human papillomavirus by two molecular techniques: hybrid capture and linear array.

A number of human papillomaviruses (HPV) are the etiological agents of cervical cancer. The present study compared the performance of the hybrid capture method with that of linear array for the detection of high-risk HPV in 218 cervical samples. For the linear array technique, the DNA was extracted using two different procedures, one manual and the other automated. There was no difference in high-risk HPV (HR-HPV) detectability between the two extraction procedures but the automated procedure had the advantages of simplicity, time and efficiency. There was agreement in 199 (91.3%) of the results. The K value for the two assays was 0.81 indicative of "near perfect" agreement. Both methods, hybrid capture and linear array, are sensitive options for detection of HPV in cervical samples. Linear array enables the identification of the genotype present in the sample and the presence of multiple infections.

Evaluation of COBAS Ampliprep automated nucleic acid extraction for genotyping human papillomavirus (HPV) by the Roche linear array HPV test

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