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/genéticaRESUMEN
The accurate and rapid identification of bacteria isolated from the respiratory tract of patients with cystic fibrosis (CF) is critical in epidemiological studies, during intrahospital outbreaks, for patient treatment, and for determination of therapeutic options. While the most common organisms isolated from sputum samples are Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in recent decades an increasing fraction of CF patients has been colonized by other nonfermenting (NF) gram-negative rods, such as Burkholderia cepacia complex (BCC) bacteria, Stenotrophomonas maltophilia, Ralstonia pickettii, Acinetobacter spp., and Achromobacter spp. In the present study, we developed a novel strategy for the rapid identification of NF rods based on Fourier transform infrared spectroscopy (FTIR) in combination with artificial neural networks (ANNs). A total of 15 reference strains and 169 clinical isolates of NF gram-negative bacteria recovered from sputum samples from 150 CF patients were used in this study. The clinical isolates were identified according to the guidelines for clinical microbiology practices for respiratory tract specimens from CF patients; and particularly, BCC bacteria were further identified by recA-based PCR followed by restriction fragment length polymorphism analysis with HaeIII, and their identities were confirmed by recA species-specific PCR. In addition, some strains belonging to genera different from BCC were identified by 16S rRNA gene sequencing. A standardized experimental protocol was established, and an FTIR spectral database containing more than 2,000 infrared spectra was created. The ANN identification system consisted of two hierarchical levels. The top-level network allowed the identification of P. aeruginosa, S. maltophilia, Achromobacter xylosoxidans, Acinetobacter spp., R. pickettii, and BCC bacteria with an identification success rate of 98.1%. The second-level network was developed to differentiate the four most clinically relevant species of BCC, B. cepacia, B. multivorans, B. cenocepacia, and B. stabilis (genomovars I to IV, respectively), with a correct identification rate of 93.8%. Our results demonstrate the high degree of reliability and strong potential of ANN-based FTIR spectrum analysis for the rapid identification of NF rods suitable for use in routine clinical microbiology laboratories.
Asunto(s)
Fibrosis Quística/microbiología , Bacterias Aerobias Gramnegativas/clasificación , Bacterias Aerobias Gramnegativas/aislamiento & purificación , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Esputo/microbiología , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Dermatoglifia del ADN , ADN Bacteriano/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Humanos , Redes Neurales de la Computación , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/genética , Rec A Recombinasas/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
This work describes the application of several analytical techniques to characterize the development of Bordetella pertussis biofilms and to examine, in particular, the contribution of virulence factors in this development. Growth of surface-attached virulent and avirulent B. pertussis strains was monitored in continuous-flow chambers by techniques such as the crystal violet method, and nondestructive methodologies like fluorescence microscopy and Fourier transform (FT) IR spectroscopy. Additionally, B. pertussis virulent and avirulent strains expressing green fluorescent protein were grown adhered to the base of a glass chamber of 1-microm thickness. Three-dimensional images of mature biofilms, acquired by confocal laser scanning microscopy, were quantitatively analysed by means of the computer program COMSTAT. Our results indicate that only the virulent (Bvg(+)) phase of B. pertussis is able to attach to surfaces and develop a mature biofilm. In the virulent phase these bacteria are capable of producing a biofilm consisting of microcolonies of approximately 200 microm in diameter and 24 microm in depth. FTIR spectroscopy allowed us not only to follow the dynamics of biofilm growth through specific biomass and biofilm marker absorption bands, but also to monitor the maturation of the biofilm by means of the increase of the carbohydrate-to-protein ratio.