RESUMEN

Burkholderia contaminans, a species of the Burkholderia cepacia complex-prevalent in certain Latin-American and European countries-can cause chronic pulmonary infection in persons with cystic fibrosis. Our aim was to gain insights into long-term lung infections with a focus on correlating how bacterial phenotypic traits in the chronic infection impact on patients' clinical outcome. Genotypic characteristics of 85 B. contaminans isolates recovered from 70 patients were investigated. For 16 of those patients, the clinical status and bacterial phenotypic characteristics, e.g. several virulence factors, phenotypic variants, and the antimicrobial susceptibility pattern, were evaluated. Two clones were found in the whole bacterial population: (i) the multiresistant ST 872 PCR-recA-RFLP-HaeIII-K-pattern clone, which carries a pathogenic island homologous to BcenGI11 of B. cenocepacia J2315, and (ii) the ST 102 PCR-recA-RFLP-HaeIII-AT-pattern clone. The emergence of certain bacterial phenotypes in the chronic infection such as the nonmucoid phenotype, small colony variants, brownish pigmented colonies, and hypermutators, proved to be, together with coinfection with Pseudomonas aeruginosa, the possible markers of more challenging infections and poor prognosis. The presence of cocolonizers and the bacterial phenotypes that are especially adapted to persist in long-term respiratory tract infections have a crucial role in patients' clinical outcomes.

Asunto(s)

Infecciones por Burkholderia , Complejo Burkholderia cepacia , Fibrosis Quística , Neumonía , Humanos , Infección Persistente , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Pulmón/microbiología , Fenotipo , Infecciones por Burkholderia/microbiología

RESUMEN

INTRODUCTION: The Burkholderia cepacia complex (BCC) bacteria are opportunistic pathogens that cause nosocomial infections and are especially dangerous for cystic fibrosis (CF) patients. Burkholderia contaminans is an emerging BCC species isolated from CF patients that also occurs as a contaminant in pharmaceutical and personal care products, sometimes linking it with outbreaks. METHODOLOGY: A total of 55 B. contaminans isolates from CF and non-CF patients in Argentina were identified by recA sequencing and MALDI TOF MS. A standardized Pulsed Field Gel Electrophoresis (PFGE) protocol was set up in order to assess genetic diversity, outbreak investigations, and possible clone persistence. RESULTS: All isolates were identified as B. contaminans by both MALDI-TOF MS and recA sequence analysis. PFGE has enabled us to compare and determine the genetic relationship between B. contaminans isolates. Isolates were distributed in different PFGE clusters with evidence of the presence and persistence of a clone, over a period of 3 years, in the same hospital. This large hospital outbreak involved CF and non-CF patients. Moreover, PFGE results showed a good correlation between sporadic or outbreak-related isolates and the available epidemiological information. CONCLUSIONS: These findings highlight the importance of B. contaminans in Argentina and provide evidence for encouraging the surveillance of highly transmissible clones. The study also contributes to global knowledge about B. contaminans infections.

Asunto(s)

Infecciones por Burkholderia , Complejo Burkholderia cepacia , Fibrosis Quística , Argentina/epidemiología , Burkholderia , Infecciones por Burkholderia/epidemiología , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/genética , Fibrosis Quística/complicaciones , Humanos

RESUMEN

Burkholderia cenocepacia complex is associated with high transmissibility, virulence, and poor prognosis in cystic fibrosis (CF) patients. However, extrapulmonary infections are rare. We investigated the genome of a B. cenocepacia IIIA isolated from a liver abscess in a Brazilian CF patient and compared it to strain J2315. The whole genome was sequenced, and contigs were annotated by Rapid Annotation using Subsystem Technology. The Pathosystems Resource Integration Center was used to map antimicrobial and virulence genes. The genomic island (GIs) analysis was performed using two prediction methods, and the presence of putative plasmids and insertion sequences (ISs) was investigated. The isolate was confirmed as B. cenocepacia IIIA to ST-28 (ET12 lineage). A total of 64 genes for antimicrobial resistance and 47 genes related to virulence were identified. Among the virulence factors, there was a predominance of factors related to the invasion mechanism, to the flagellar biosynthesis protein, and to the RNA polymerase sigma factor for flagellar operon (cdpA). Two IS families (IS3 and IS5) and only one plasmid were found. On average 56 GIs were predicted by at least one of the methods applied. Comparative analysis showed resistance mechanisms and virulence factors revealing invasive determinants used by B. cenocepacia IIIA (ET12) in the process of disease spread to other infection sites (extrapulmonary) of highly virulent strains in CF patients.

Asunto(s)

Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/genética , Fibrosis Quística/microbiología , Genoma Bacteriano/genética , Absceso Hepático/microbiología , Adolescente , Brasil , Infecciones por Burkholderia/complicaciones , Burkholderia cenocepacia/clasificación , Burkholderia cenocepacia/aislamiento & purificación , Fibrosis Quística/complicaciones , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana/genética , Femenino , Genes Bacterianos/genética , Islas Genómicas/genética , Humanos , Absceso Hepático/complicaciones , Plásmidos/genética , Factores de Virulencia/genética

RESUMEN

BACKGROUND: Burkholderia sensu stricto is comprised mainly of opportunistic pathogens. This group is widely distributed in the environment but is especially important in clinical settings. In Mexico, few species have been correctly identified among patients, most often B. cepacia is described. METHODOLOGY/PRINCIPAL FINDINGS: In this study, approximately 90 strains identified as B. cepacia with the VITEK2 system were isolated from two medical centers in Mexico City and analyzed by MLSA, BOX-PCR and genome analysis. The initial identification of B. cepacia was confirmed for many strains, but B. contaminans, B. multivorans and B. vietnamiensis were also identified among clinical strains for the first time in hospitals in Mexico. Additionally, the presence of B. pseudomallei was confirmed, and a novel species within the B. cepacia complex was documented. Several strains misidentified as B. cepacia actually belong to the genera Pseudomonas, Stenotrophomonas and Providencia. CONCLUSIONS/SIGNIFICANCE: The presence of different Burkholderia species in Mexico was confirmed. Correct identification of Burkholderia species is important to provide accurate treatment for immunosuppressed patients.

Asunto(s)

Infecciones por Burkholderia/epidemiología , Burkholderia/clasificación , Burkholderia/genética , Burkholderia/aislamiento & purificación , Infecciones por Burkholderia/microbiología , ADN Bacteriano/análisis , Genoma Bacteriano , Humanos , México , Tipificación de Secuencias Multilocus , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética

RESUMEN

Low molecular weight protein tyrosine phosphatases (LMW-PTP) are ubiquitous enzymes found across a spectrum of genera from prokaryotes to higher eukaryotes. LMW-PTP belong to the Cys-based PTP class II protein family. Here, we show that LMW-PTP can be categorized into two different groups, referred as class II subdivision I (class II.I) and subdivision II (class II.II). Using BPtpA from the opportunistic pathogen Burkholderia cenocepacia, as a representative member of the LMW-PTP class II.I, we demonstrated that four conserved residues (W47, H48, D80, and F81) are required for enzyme function. Guided by an in silico model of BPtpA, we show that the conserved residues at α3-helix (D80 and F81) contribute to protein stability, while the other conserved residues in the W-loop (W47 and H48) likely play a role in substrate recognition. Overall, our results provide new information on LMW-PTP protein family and establish B. cenocepacia as a suitable model to investigate how substrates are recognized and sorted by these proteins.

Asunto(s)

Proteínas Bacterianas/metabolismo , Burkholderia cenocepacia/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/química , Humanos , Modelos Moleculares , Fosforilación , Proteínas Tirosina Fosfatasas/química

RESUMEN

INTRODUCTION: Cystic fibrosis patients develop pulmonary exacerbations (PEs) that require intravenous treatment. The objective of this study was to determine the risk factors associated with PEs and establish the percentage of patients who failed to recover their lung function. POPULATION AND METHODS: Observational, retrospective, cohort study. The medical records of cystic fibrosis patients seen at Hospital de Niños Ricardo Gutiérrez in 2013 were reviewed. Patients were divided into group 1, with PE (Fuchs criteria), and group 2, without PE. Age, sex, p.F508del mutation, percentage of baseline forced expiratory volume in the first second, baseline body mass index Z-score, chronic Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and Burkholderia cepacia complex colonization (Leeds criteria), percentage of cystic fibrosis-related diabetes, and recovery of baseline forced expiratory volume in the first second were recorded. RESULTS: A total of 117 patients were included. Group 1: 50, group 2: 67 patients. PEs were associated with a lower body mass index Z-score (RR: 1.45; p = 0.002), p.F508del mutation (RR: 3.23; p = 0.05), and chronic Burkholderia cepacia complex (RR: 3.69; p = 0.002), Pseudomonas aeruginosa (RR: 1.89; p = 0.01) and methicillinresistant Staphylococcus aureus colonization (RR: 2.32; p = 0.002). Twenty-four percent of patients failed to recover their lung function. CONCLUSIONS: The presence of the p.F508del mutation, a poor nutritional status, and chronic colonization were the risk factors for exacerbation. A fourth of patients failed to recover their lung function.

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Introducción. Los pacientes con fibrosis quística presentan exacerbaciones respiratorias (ER) que requieren tratamiento endovenoso. El objetivo fue determinar los factores de riesgo asociados a ER y obtener porcentaje de pacientes que no recuperaban su función pulmonar previa. Población y métodos. Observacional, de cohorte, retrospectivo. Se revisaron las historias clínicas de los pacientes con fibrosis quística atendidos en el Hospital de Niños Ricardo Gutiérrez durante 2013. Se dividieron en: grupo 1, con ER (criterios de Fuchs), y grupo 2, sin ER. Se registró edad, género, mutación p.F508del, porcentaje del volumen espiratorio forzado en el primer segundo basal, puntaje Z de índice de masa corporal basal, colonización crónica (criterios de Leeds) por Pseudomonas aeruginosa, Staphylococcus aureus meticilino resistente y complejo Burkholderia cepacia, porcentaje de diabetes relacionada con fibrosis quística y recuperación del volumen espiratorio forzado en el primer segundo basal. Resultados. Se incluyeron 117 pacientes. Grupo 1: 50; y grupo 2: 67 pacientes. Se asociaron a las ER: el menor puntaje Z de IMC (RR: 1,45; p = 0,002), p.F508del (RR: 3,23; p = 0,05) y colonización crónica por el complejo Burkholderia cepacia (RR: 3,69; p = 0,002), Pseudomonas aeruginosa (RR: 1,89; p = 0,01) y Staphylococcus aureus meticilino resistente (RR: 2,32; p = 0,002). El 24 % no recuperó su función pulmonar. Conclusiones. p.F508del, el bajo estado nutricional y la colonización crónica fueron factores de riesgo para exacerbación. Una cuarta parte de los pacientes no recuperó su función pulmonar previa.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/complicaciones , Enfermedades Pulmonares/etiología , Estado Nutricional , Adolescente , Infecciones por Burkholderia/epidemiología , Infecciones por Burkholderia/microbiología , Niño , Preescolar , Estudios de Cohortes , Fibrosis Quística/genética , Femenino , Volumen Espiratorio Forzado , Humanos , Lactante , Enfermedades Pulmonares/epidemiología , Masculino , Infecciones por Pseudomonas/epidemiología , Infecciones por Pseudomonas/microbiología , Estudios Retrospectivos , Factores de Riesgo , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología

RESUMEN

La espectrometría de masas (EM) (matrix assisted laser desorption ionization-time of flight) MALDI-TOF demostró ser una herramienta robusta para la identificación de numerosos grupos taxonómicos. No obstante, presenta limitaciones. Una ventaja clave de la técnica es la flexibilidad para la incorporación de espectros proteicos de microorganismos ausentes en la base de datos comercial. Dada la prevalencia de Burkholderia contaminans en los pacientes fibroquísticos en Argentina, y a que en ellos es crucial el diagnóstico microbiológico rápido y confiable, la EM MALDI-TOF surge como una herramienta estratégica. El objetivo del trabajo fue desarrollar una base de datos adicional con espectros peptídicos de aislamientos de referencia de B. contaminans. La misma demostró ser exitosa para la identificación del 97% de los aislamientos analizados. Por lo cual la EM MALDI-TOF con la base de datos extendida resultó ser una herramienta útil para la identificación y diferenciación de otras especies relacionadas a B. contaminans.

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MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometry (MS) proved to be a robust tool for the identification of numerous taxonomic groups. However, it has limitations. A key advantage of this technique is the flexibility for the incorporation of protein profiles of microorganisms not included in the commercial database. Due to the prevalence of Burkholderia contaminans in fibrocystic patients in Argentina and the fact that rapid and reliable microbiological diagnosis is crucial in them, MALDI-TOF MS emerges as a strategic tool. The aim of this work was to develop an additional database with peptide spectra of reference isolates of B. contaminans. This database demonstrated to be successful for the identification of 97% of the isolates analyzed. Therefore, MALDI-TOF MS with the extended database was a useful tool for the identification and differentiation of other related species to B. contaminans.

Asunto(s)

Humanos , Bases de Datos Factuales , Técnicas Bacteriológicas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Burkholderia/aislamiento & purificación , Especificidad de la Especie , Proteínas Bacterianas/análisis , Algoritmos , Reproducibilidad de los Resultados , Infecciones por Burkholderia/complicaciones , Infecciones por Burkholderia/microbiología , Burkholderia/clasificación , Burkholderia/química , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología

RESUMEN

The correct identification of different genera and bacterial species is essential, especially when these bacteria cause infections and appropriate therapies need to be chosen. Bacteria belonging to the Burkholderia cepacia complex are considered important opportunistic pathogens, causing different types of infections in immunocompromised, principally in patients with cystic fibrosis. Twenty-one isolates were obtained from different soil samples and identified by sequencing of 16S rRNA, 23S rRNA, recA gene, MLST and by VITEK 2 and MALDI-TOF MS systems. Then, statistical analyses were performed. VITEK 2 and MALDI-TOF MS systems showed different bacterial genera. Sequencing of the 16S rRNA, 23S rRNA gene and amplification of recA gene showed that all the isolates belong to the B. cepacia complex. Sequencing of the recA gene showed a predominance of B. cenocepacia. The PCR of the recA gene showed a high specificity when it is necessary to identify the bacteria belonging to the B. cepacia complex in comparison with 16S and 23S rRNA genes sequencing. MLST analyzes showed a diversity of STs, which have not yet been correlated to the species. Phenotypic identification was not suitable for the identification of these pathogens since in many cases different genera have been reported, including identification by using MALDI-TOF MS.

Asunto(s)

Técnicas de Tipificación Bacteriana/métodos , Complejo Burkholderia cepacia/clasificación , Complejo Burkholderia cepacia/genética , Complejo Burkholderia cepacia/aislamiento & purificación , Filogenia , Microbiología del Suelo , Proteínas Bacterianas/genética , Infecciones por Burkholderia/microbiología , Fibrosis Quística/microbiología , ADN Bacteriano/genética , Tipificación de Secuencias Multilocus/métodos , Infecciones Oportunistas/microbiología , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Rec A Recombinasas/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos

RESUMEN

Burkholderia contaminans is a member of the Burkholderia cepacia complex (Bcc), a pathogen with increasing prevalence among cystic fibrosis (CF) patients and the cause of numerous outbreaks due to the use of contaminated commercial products. The antibiotic resistance determinants, particularly ß-lactamases, have been poorly studied in this species. In this work, we explored the whole genome sequence (WGS) of a B. contaminans isolate (FFH 2055) and detected four putative ß-lactamase-encoding genes. In general, these genes have more than 93% identity with ß-lactamase genes found in other Bcc species. Two ß-lactamases, a class A (Pen-like, suggested name PenO) and a class D (OXA-like), were further analyzed and characterized. Amino acid sequence comparison showed that Pen-like has 82% and 67% identity with B. multivorans PenA and B. pseudomallei PenI, respectively, while OXA-like displayed strong homology with class D enzymes within the Bcc, but only 22-44% identity with available structures from the OXA family. PCR reactions designed to study the presence of these two genes revealed a heterogeneous distribution among clinical and industrial B. contaminans isolates. Lastly, blaPenO gene was cloned and expressed into E. coli to investigate the antibiotic resistance profile and confers an extended-spectrum ß-lactamase (ESBL) phenotype. These results provide insight into the presence of ß-lactamases in B. contaminans, suggesting they play a role in antibiotic resistance of these bacteria.

Asunto(s)

Proteínas Bacterianas/genética , Complejo Burkholderia cepacia/enzimología , Complejo Burkholderia cepacia/genética , Genoma Bacteriano/genética , beta-Lactamasas/genética , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/efectos de los fármacos , Fibrosis Quística/microbiología , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Homología de Secuencia de Aminoácido , beta-Lactamasas/química , beta-Lactamasas/metabolismo

RESUMEN

MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometry (MS) proved to be a robust tool for the identification of numerous taxonomic groups. However, it has limitations. A key advantage of this technique is the flexibility for the incorporation of protein profiles of microorganisms not included in the commercial database. Due to the prevalence of Burkholderia contaminans in fibrocystic patients in Argentina and the fact that rapid and reliable microbiological diagnosis is crucial in them, MALDI-TOF MS emerges as a strategic tool. The aim of this work was to develop an additional database with peptide spectra of reference isolates of B. contaminans. This database demonstrated to be successful for the identification of 97% of the isolates analyzed. Therefore, MALDI-TOF MS with the extended database was a useful tool for the identification and differentiation of other related species to B. contaminans.

Asunto(s)

Técnicas Bacteriológicas , Burkholderia/aislamiento & purificación , Bases de Datos Factuales , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Algoritmos , Proteínas Bacterianas/análisis , Burkholderia/química , Burkholderia/clasificación , Infecciones por Burkholderia/complicaciones , Infecciones por Burkholderia/microbiología , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Humanos , Reproducibilidad de los Resultados , Especificidad de la Especie

RESUMEN

INTRODUCTION: Burkholderia cepacia (B. cepacia) complex is composed of 20 phylogenetically closely related bacterial species. Some species have emerged as opportunistic pathogens in immunocompromised patients and are responsible for nosocomial outbreaks. The B. cepacia complex is a recognized respiratory pathogen in patients with cystic fibrosis. Burkholderia cenocepacia and Burkholderia multivorans (B. multivorans) are the most prevalent species in the world, according to the literature. However, research groups in Argentina have described a particular local epidemiology, with prevalence of Burkholderia contaminans (B. contaminans). METHODS: A total of 68 isolates of B. cepacia complex recovered of 46 cystic fibrosis patients attended at 14 hospitals distributed in 9 provinces of the country were studied. Identification was carried out by conventional phenotypic methods and was confirmed by recA gene sequencing. Sequences were analysed using the BLASTN program and comparing with B. cepacia complex type strains sequences deposited in GenBank. Antibiotic susceptibility tests were performed on isolates of the most prevalent species according to CLSI M45 guidelines. RESULTS: The prevalent specie was B. contaminans (49%, n = 33) followed by B. cenocepacia (25%; n = 17). The remaining species were Burkholderia seminalis (B. seminalis) (7%, n = 5), B. cepacia (7%, n = 5), B. multivorans (6%, n = 4), Burkholderia vietnamensis (5%, n=3) and Burkholderia pyrrocinia (1%; n = 1). The 46% of B. contaminans isolates were resistant to SXT and 76% sensitive to MIN, MEM and CAZ. The isolates of B. cenocepacia were 100% resistant to SXT and MIN and 47% to CAZ and MEM. B. seminalis showed high levels of resistance to TMS (80%), CAZ (60%) and MIN (60%), and 60% of the isolates showed intermediate sensitivity to MEM. CONCLUSION: Previous reports have described the prevalence of B. contaminans isolation from cystic fibrosis patients in Argentina, Spain and Portugal, and a case of two patients with cystic fibrosis in Ireland has recently been reported. Due to the high frequency with which B. contaminans is isolated in our country, it is necessary to promote the investigation of possible sources of infection and to understand the factors and mechanisms involved in the apparent greater transmissibility of this species. Different antimicrobial resistance profiles were detected between the species.

Asunto(s)

Infecciones por Burkholderia/epidemiología , Complejo Burkholderia cepacia/aislamiento & purificación , Infección Hospitalaria/epidemiología , Fibrosis Quística/complicaciones , Argentina/epidemiología , Proteínas Bacterianas/genética , Infecciones por Burkholderia/complicaciones , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/efectos de los fármacos , Complejo Burkholderia cepacia/genética , Infección Hospitalaria/microbiología , Fibrosis Quística/microbiología , Susceptibilidad a Enfermedades , Farmacorresistencia Microbiana , Genes Bacterianos , Humanos , Prevalencia , Rec A Recombinasas/genética , Estudios Retrospectivos , Especificidad de la Especie

RESUMEN

The Burkholderia cepacia complex (BCC) can cause a severe decline in lung function in cystic fibrosis (CF). Our objective was to determine the BCC prevalence and to evaluate its clinical impact on CF. Clinical and laboratory variables were determined for CF patients with BCC (Group-A = 50 patients) and without BCC (Group-B = 134 patients). The microorganisms were identified by biochemical tests, the Vitek2®Compact test, recA-PCR and recA-nested-PCR with species-specific primers and DNA sequencing. The patients were evaluated by the Shwachman-Kulczycki score (SKCS), Bhalla score (BS), spirometry and body mass index (BMI). The BCC prevalence was 22.5%. The most common species were Burkholderia multivorans (30%), Burkholderia cepacia (24%), Burkholderia cenocepacia IIIA (10%), B. cenocepacia IIIB (2%) and Burkholderia vietnamiensis (2%). There was difference between the groups in nutritional status (p = 0.02) and general activity (p = 0.026). There was difference in total BS points (p = 0.04) and the following parameters: bronchiectasis severity (p = 0.007), peribronchial thickening (p = 0.013), bronchiectasis extent (p = 0.01) and general aspects of the affected bronchial zone (p = 0.02). The respiratory disorder classifications were as follows: obstructive-4.8% (Group-A) and 23.8% (Group-B); restrictive-9.5% (Group-A and Group-B); obstructive + restrictive-19% (Group-A) and 1.6% (Group-B); and obstructive + restrictive with a decreased forced expiratory flow-47.6% (Group-A) and 30.2% (Group-B) (p = 0.02). Nutritional status was a minor contributing factor to weight, height and BMI in the Group-A (p = 0.02). The BCC prevalence, particularly the prevalence of B. multivorans, was higher in this study. The SKCS, BS, spirometry and nutritional status results showed that BCC has a negative impact on clinical status. Phenotypic methods are useful for the identification of presumptive BCC. The Vitek2®Compact test showed accuracy in BCC identification. PCR, nested-PCR, and recA sequencing showed specificity in BCC species identification.

Asunto(s)

Infecciones por Burkholderia/epidemiología , Infecciones por Burkholderia/microbiología , Burkholderia/clasificación , Burkholderia/aislamiento & purificación , Fibrosis Quística/complicaciones , Adolescente , Adulto , Técnicas de Tipificación Bacteriana , Brasil/epidemiología , Burkholderia/genética , Burkholderia/fisiología , Infecciones por Burkholderia/patología , Niño , Preescolar , Estudios Transversales , Fibrosis Quística/patología , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Técnicas de Diagnóstico Molecular , Reacción en Cadena de la Polimerasa , Prevalencia , Análisis de Secuencia de ADN , Adulto Joven

RESUMEN

The bacterium Burkholderia ubonensis is commonly co-isolated from environmental specimens harbouring the melioidosis pathogen, Burkholderia pseudomallei. B. ubonensis has been reported in northern Australia and Thailand but not North America, suggesting similar geographic distribution to B. pseudomallei. Unlike most other Burkholderia cepacia complex (Bcc) species, B. ubonensis is considered non-pathogenic, although its virulence potential has not been tested. Antibiotic resistance in B. ubonensis, particularly towards drugs used to treat the most severe B. pseudomallei infections, has also been poorly characterised. This study examined the population biology of B. ubonensis, and includes the first reported isolates from the Caribbean. Phylogenomic analysis of 264 B. ubonensis genomes identified distinct clades that corresponded with geographic origin, similar to B. pseudomallei. A small proportion (4%) of strains lacked the 920kb chromosome III replicon, with discordance of presence/absence amongst genetically highly related strains, demonstrating that the third chromosome of B. ubonensis, like other Bcc species, probably encodes for a nonessential pC3 megaplasmid. Multilocus sequence typing using the B. pseudomallei scheme revealed that one-third of strains lack the "housekeeping" narK locus. In comparison, all strains could be genotyped using the Bcc scheme. Several strains possessed high-level meropenem resistance (≥32 µg/mL), a concern due to potential transmission of this phenotype to B. pseudomallei. In silico analysis uncovered a high degree of heterogeneity among the lipopolysaccharide O-antigen cluster loci, with at least 35 different variants identified. Finally, we show that Asian B. ubonensis isolate RF23-BP41 is avirulent in the BALB/c mouse model via a subcutaneous route of infection. Our results provide several new insights into the biology of this understudied species.

Asunto(s)

Antibacterianos/farmacología , Burkholderia/clasificación , Burkholderia/efectos de los fármacos , Microbiología Ambiental , Variación Genética , Filogeografía , Tienamicinas/farmacología , Animales , Australia , Burkholderia/genética , Burkholderia/aislamiento & purificación , Infecciones por Burkholderia/microbiología , Infecciones por Burkholderia/patología , Modelos Animales de Enfermedad , Genotipo , Meropenem , Ratones Endogámicos BALB C , Tipificación de Secuencias Multilocus , Antígenos O/genética , Papúa Nueva Guinea , Puerto Rico , Tailandia , Virulencia

RESUMEN

A nosocomial polyclonal outbreak associated to bacteremia caused by different Burkholderia cepacia complex (BCC) species and clones is reported. Molecular characterization identified Burkholderia stabilis, Burkholderia contaminans, and Burkholderia ambifaria among BCC isolates obtained from patients in neonatal and adult intensive care units. BCC was also isolated from an intrinsically contaminated ultrasound gel, which constituted the presumptive BCC source. Prior BCC outbreak related to contaminated ultrasound gels have been described in the setting of transrectal prostate biopsy. Outbreak caused strains and/or clones of BCC have been reported, probably because BCC are commonly found in the natural environment; most BCC species are biofilm producers, and different species may contaminate an environmental source. The finding of multiple species or clones during the analysis of nosocomial BCC cases might not be enough to reject an outbreak from a common source.

Asunto(s)

Bacteriemia/microbiología , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/aislamiento & purificación , Infección Hospitalaria/microbiología , Geles/efectos adversos , Ultrasonografía/efectos adversos , Adulto , Bacteriemia/diagnóstico , Infecciones por Burkholderia/diagnóstico , Complejo Burkholderia cepacia/clasificación , Infección Hospitalaria/diagnóstico , Brotes de Enfermedades , Humanos , Recién Nacido , Unidades de Cuidados Intensivos , Ultrasonografía/enfermería

RESUMEN

The Burkholderia cepacia complex (Bcc) represents an important group of pathogens involved in long-term lung infection in cystic fibrosis (CF) patients. A positive selection of hypermutators, linked to antimicrobial resistance development, has been previously reported for Pseudomonas aeruginosa in this chronic infection setting. Hypermutability, however, has not yet been systematically evaluated in Bcc species. A total of 125 well characterized Bcc isolates recovered from 48 CF patients, 10 non-CF patients and 15 environmental samples were analyzed. In order to determine the prevalence of mutators their spontaneous mutation rates to rifampicin resistance were determined. In addition, the genetic basis of the mutator phenotypes was investigated by sequencing the mutS and mutL genes, the main components of the mismatch repair system (MRS). The overall prevalence of hypermutators in the collection analyzed was 13.6%, with highest occurrence (40.7%) among the chronically infected CF patients, belonging mainly to B. cenocepacia, B. multivorans, B. cepacia, and B. contaminans -the most frequently recovered Bcc species from CF patients worldwide. Thirteen (76.5%) of the hypermutators were defective in mutS and/or mutL. Finally, searching for a possible association between antimicrobial resistance and hypermutability, the resistance-profiles to 17 antimicrobial agents was evaluated. High antimicrobial resistance rates were documented for all the Bcc species recovered from CF patients, but, except for ciprofloxacin, a significant association with hypermutation was not detected. In conclusion, in the present study we demonstrate for the first time that, MRS-deficient Bcc species mutators are highly prevalent and positively selected in CF chronic lung infections. Hypermutation therefore, might be playing a key role in increasing bacterial adaptability to the CF-airway environment, facilitating the persistence of chronic lung infections.

Asunto(s)

Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/genética , Fibrosis Quística/complicaciones , Reparación de la Incompatibilidad de ADN , Tasa de Mutación , Infecciones del Sistema Respiratorio/microbiología , Antibacterianos/farmacología , Complejo Burkholderia cepacia/aislamiento & purificación , Enfermedad Crónica , Estudios de Cohortes , Enzimas Reparadoras del ADN/deficiencia , Enzimas Reparadoras del ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Farmacorresistencia Bacteriana , Microbiología Ambiental , Humanos , Datos de Secuencia Molecular , Rifampin/farmacología , Análisis de Secuencia de ADN

RESUMEN

AIM: The objective was to describe an outbreak of bloodstream infections by Burkholderia cepacia complex (Bcc) in bone marrow transplant and hematology outpatients. METHODS: On February 15, 2008 a Bcc outbreak was suspected. 24 cases were identified. Demographic and clinical data were evaluated. Environment and healthcare workers' (HCW) hands were cultured. Species were determined and typed. Reinforcement of hand hygiene, central venous catheter (CVC) care, infusion therapy, and maintenance of laminar flow cabinet were undertaken. 16 different HCWs had cared for the CVCs. Multi-dose heparin and saline were prepared on counter common to both units. FINDINGS: 14 patients had B. multivorans (one patient had also B. cenopacia), six non-multivorans Bcc and one did not belong to Bcc. Clone A B. multivorans occurred in 12 patients (from Hematology); in 10 their CVC had been used on February 11/12. Environmental and HCW cultures were negative. All patients were treated with meropenem, and ceftazidime lock-therapy. Eight patients (30%) were hospitalized. No deaths occurred. After control measures (multidose vial for single patient; CVC lock with ceftazidime; cleaning of laminar flow cabinet; hand hygiene improvement; use of cabinet to store prepared medication), no new cases occurred. CONCLUSIONS: This polyclonal outbreak may be explained by a common source containing multiple species of Bcc, maybe the laminar flow cabinet common to both units. There may have been contamination by B. multivorans (clone A) of multi-dose vials.

Asunto(s)

Bacteriemia/microbiología , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/aislamiento & purificación , Infecciones Relacionadas con Catéteres/microbiología , Brotes de Enfermedades , Adolescente , Adulto , Bacteriemia/epidemiología , Trasplante de Médula Ósea , Infecciones por Burkholderia/epidemiología , Infecciones Relacionadas con Catéteres/epidemiología , Niño , Preescolar , Femenino , Enfermedades Hematológicas , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven

RESUMEN

Aim: The objective was to describe an outbreak of bloodstream infections by Burkholderia cepacia complex (Bcc) in bone marrow transplant and hematology outpatients. Methods: On February 15, 2008 a Bcc outbreak was suspected. 24 cases were identified. Demographic and clinical data were evaluated. Environment and healthcare workers' (HCW) hands were cultured. Species were determined and typed. Reinforcement of hand hygiene, central venous catheter (CVC) care, infusion therapy, and maintenance of laminar flow cabinet were undertaken. 16 different HCWs had cared for the CVCs. Multi-dose heparin and saline were prepared on counter common to both units. Findings: 14 patients had B. multivorans (one patient had also B. cenopacia), six non-multivorans Bcc and one did not belong to Bcc. Clone A B. multivorans occurred in 12 patients (from Hematology); in 10 their CVC had been used on February 11/12. Environmental and HCW cultures were negative. All patients were treated with meropenem, and ceftazidime lock-therapy. Eight patients (30%) were hospitalized. No deaths occurred. After control measures (multidose vial for single patient; CVC lock with ceftazidime; cleaning of laminar flow cabinet; hand hygiene improvement; use of cabinet to store prepared medication), no new cases occurred. Conclusions: This polyclonal outbreak may be explained by a common source containing multiple species of Bcc, maybe the laminar flow cabinet common to both units. There may have been contamination by B. multivorans (clone A) of multi-dose vials.

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O objetivo foi descrever um surto de infecções da corrente sanguínea por complexo B. cepacia (Bcc) nos ambulatórios de hematologia e transplante de medula óssea. Métodos: Em 15/02/2008, um surto de Bcc foi suspeitado. 24 casos foram identificados. Os dados demográficos e clínicos foram avaliados. Mãos de profissionais da saúde e ambiente foram cultivadas. Espécies foram determinadas e tipadas. Reforço da higiene das mãos, cuidados com cateteres, terapia de infusão e manutenção da câmara de fluxo laminar foram realizadas. 16 profissionais de saúde (PS) diferentes manipularam os cateteres. Heparina multidoses e soro eram preparadas em um balcão comum a ambas as unidades. Resultados: 14 pacientes tiveram B. multivorans (um paciente teve também B. cenopacia), 6 Bcc não-multivorans e um teve um agente não pertencente a Bcc. Clone A de B. multivorans ocorreu em 12 pacientes (da Hematologia), em 10 o cateter havia sido utilizado nos dias 11 ou 12 de fevereiro. Culturas ambientais e de PS foram negativos. Todos os pacientes foram tratados com meropenem e selo de ceftazidima. Oito pacientes (30%) foram hospitalizados. Não ocorreram mortes. Após as medidas de controle, nenhum novo caso ocorreu. Conclusões: Este surto policlonal pode ser explicado por uma fonte comum contendo várias espécies de Bcc, talvez a câmara de fluxo laminar comum a ambas as unidades. Pode ter havido contaminação por B. multivorans (clone A) de frascos multi-dose.

Asunto(s)

Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Bacteriemia/microbiología , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/aislamiento & purificación , Infecciones Relacionadas con Catéteres/microbiología , Brotes de Enfermedades , Trasplante de Médula Ósea , Bacteriemia/epidemiología , Infecciones por Burkholderia/epidemiología , Infecciones Relacionadas con Catéteres/epidemiología , Enfermedades Hematológicas

RESUMEN

Burkholderia cenocepacia may cause serious infections in patients with cystic fibrosis, and this microorganism can be highly transmissible. Pulsed-field gel electrophoresis is widely used to study the dynamics of strain spread in cystic fibrosis patients. The aim of this work was to perform pulsed-field gel electrophoresis-based molecular typing of B. cenocepacia isolates to evaluate the epidemiology of this species at our hospital. A total of 28 isolates from 23 cystic fibrosis patients were analyzed. Initially, we compared isolates obtained from the same patient at different periods of time. We then compared the pulsed-field gel electrophoresis profiles of 15 IIIA isolates, and in a third analysis, evaluated the genetic profile of 8 IIIB isolates from different patients. The pulsed-field gel electrophoresis profiles of isolates from the same patient indicated that they are genetically indistinguishable. Analysis of isolates from different patients revealed the presence of multiple clonal groups. These results do not indicate cross-transmission of a unique clone of B. cenocepacia among cystic fibrosis patients, although this has been observed in some patients. Our findings highlight the importance of adequate patient follow-up at cystic fibrosis centers and adherence to management and segregation measures in cystic fibrosis patients colonized with B. cenocepacia.

Asunto(s)

Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/genética , Fibrosis Quística/microbiología , Técnicas de Tipificación Bacteriana/métodos , Burkholderia cenocepacia/aislamiento & purificación , ADN Bacteriano/análisis , Electroforesis en Gel de Campo Pulsado , Genotipo , Humanos , Fenotipo , Reacción en Cadena de la Polimerasa

RESUMEN

A total of 120 Burkholderia cepacia complex isolates collected during 2004-2010 from 66 patients in two cystic fibrosis reference centers in Argentina were analyzed. Burkholderia contaminans was the species most frequently recovered (57.6%), followed by Burkholderia cenocepacia (15%), a species distribution not reported so far. The recA-PCR-based techniques applied to the B. contaminans isolates revealed that 85% of the population carried the recA-ST-71 allele. Our results showed the utility of BOX-PCR genotyping in analyzing B. contaminans diversity. This approach allowed us to address clonal transmission during an outbreak and the genetic changes occurring in infecting bacteria over the course of chronic infection.

Asunto(s)

Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/genética , Complejo Burkholderia cepacia/aislamiento & purificación , Fibrosis Quística/complicaciones , Variación Genética , Argentina , Técnicas de Tipificación Bacteriana , Complejo Burkholderia cepacia/clasificación , Genotipo , Humanos , Tipificación Molecular , Reacción en Cadena de la Polimerasa , Rec A Recombinasas/genética

RESUMEN

Las especies del complejo Burkholderia cepacia (CBC) son capaces de causar infecciones crónicas del tracto respiratorio en pacientes con fibrosis quística y en otros individuos inmunocomprometidos. La mayoría de estas especies exhiben alta resistencia a la terapia antibiótica, lo que genera la necesidad de una detección rápida y precisa para poder implementar estrategias de control adecuadas. En este trabajo se utilizó la técnica de reacción en cadena de la polimerasa (PCR) para amplificar el gen recA (PCR-recA), con el fin de identificar microorganismos pertenecientes al CBC. Con este método molecular como referencia, se evaluó la sensibilidad (S) y la especificidad (E) de dos sistemas de identificación comerciales automatizados, VITEK 2 y API 20NE (bioMérieux®), así como también el valor de las pruebas bioquímicas manuales más representativas para la identificación de estos microorganismos. El método VITEK 2 presentó una S del 71,1 % y una E del 100 %; para el método API 20NE, estos valores fueron 69,7 % y 90,2 %, respectivamente. En cuanto a las pruebas fenotípicas manuales, los resultados obtenidos fueron más heterogéneos, lo que posiblemente se deba a que estas bacterias podrían sufrir presión selectiva para sobrevivir en pacientes crónicos y perder factores fenotípicos característicos. La técnica de PCR-recA resultó de fácil implementación, por lo que cabe considerar a esta técnica de identificación como una opción viable, aun en laboratorios de diagnóstico clínico de mediana complejidad.

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Species belonging to the Burkholderia cepacia complex (BCC) are capable of causing chronic respiratory tract infections in patients suffering from cystic fibrosis as wel as in immunocompromised individuals. Most of these species are highly resistant to antibiotic therapy, generating the need for their rapid and accurate detection for the proper treatment and clinical management of these patients. In this wok, the polymerase chain reaction (PCR) technique based on the amplification of the recA gene (PCR-recA) was applied for an accurate identification of bacteria belonging to the BCC. Sensitivity (S) and specificity (E) of two biochemically-based commercial automated systems, API 20NE and VITEK 2 (bioMérieux®), and of the most representative biochemical manual tests for the identification of the Burkholderia cepacia complex were herein evaluated. The commercial systems VITEK 2 and API 20NE showed the following sensitivity and specificity vaues for identification to the species level, S: 71.1 %, E: 100 %, S: 69.7 %, E: 90.2 %, respectively. More complex results were observed for phenotypic manual tests, since BCC bacteria can undergo selective pressure to survive in chronic patients causing the loss of their typical phenotypic characteristics. The PCR-recA technique was easy to implement even in medium-complexity clinical diagnostic laboratories.

Asunto(s)

Humanos , Técnicas de Tipificación Bacteriana/métodos , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/aislamiento & purificación , Juego de Reactivos para Diagnóstico , Infecciones del Sistema Respiratorio/microbiología , Automatización , Proteínas Bacterianas/genética , Infecciones por Burkholderia/diagnóstico , Infecciones por Burkholderia/etiología , Colorimetría/métodos , Fibrosis Quística/complicaciones , Susceptibilidad a Enfermedades , ADN Bacteriano/genética , Genes Bacterianos , Genotipo , Reacción en Cadena de la Polimerasa/métodos , Estándares de Referencia , Reproducibilidad de los Resultados , Rec A Recombinasas/genética , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/etiología , Sensibilidad y Especificidad , Programas Informáticos