Structural Analysis of Bordetella Pertussis Biofilms by Confocal Laser Scanning Microscopy

Biofilms are sessile communities of microbial cells embedded in a self-produced or host-derived exopolymeric matrix. Biofilms can both be beneficial or detrimental depending on the surface. Compared to their planktonic counterparts, biofilm cells display enhanced resistance to killing by environmental threats, chemicals, antimicrobials and host immune defenses. When in biofilms, the microbial cells interact with each other and with the surface to develop architecturally complex multidimensional structures. Numerous imaging techniques and tools are currently available for architectural analyses of biofilm communities. This allows examination of biofilm development through acquisition of three-dimensional images that can render structural features of the sessile community. A frequently utilized tool is Confocal Laser Scanning Microscopy. We present a detailed protocol to grow, observe and analyze biofilms of the respiratory human pathogen, Bordetella pertussis in space and time.Fil: Cattelan, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Yantorno, Osvaldo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Rajendar, Deora. Ohio State University; Estados Unido

Structural Analysis of Bordetella pertussis Biofilms by Confocal Laser Scanning Microscopy

Biofilms are sessile communities of microbial cells embedded in a self-produced or host-derived exopolymeric matrix. Biofilms can both be beneficial or detrimental depending on the surface. Compared to their planktonic counterparts, biofilm cells display enhanced resistance to killing by environmental threats, chemicals, antimicrobials and host immune defenses. When in biofilms, the microbial cells interact with each other and with the surface to develop architecturally complex multidimensional structures. Numerous imaging techniques and tools are currently available for architectural analyses of biofilm communities. This allows examination of biofilm development through acquisition of three-dimensional images that can render structural features of the sessile community. A frequently utilized tool is Confocal Laser Scanning Microscopy. We present a detailed protocol to grow, observe and analyze biofilms of the respiratory human pathogen, Bordetella pertussis in space and time.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Hyperbiofilm formation by <i>Bordetella pertussis</i> strains correlates with enhanced virulence traits

Pertussis, or whooping cough, caused by the obligate human pathogen Bordetella pertussis is undergoing a worldwide resurgence. The majority of studies of this pathogen are conducted with laboratory-adapted strains which may not be representative of the species as a whole. Biofilm formation by B. pertussis plays an important role in pathogenesis. We conducted a side-by-side comparison of the biofilm-forming abilities of the prototype laboratory strains and the currently circulating isolates from two countries with different vaccination programs. Compared to the reference strain, all strains examined herein formed biofilms at high levels. Biofilm structural analyses revealed country-specific differences, with strains from the United States forming more structured biofilms. Bacterial hyperaggregation and reciprocal expression of biofilm-promoting and -inhibitory factors were observed in clinical isolates. An association of increased biofilm formation with augmented epithelial cell adhesion and higher levels of bacterial colonization in the mouse nose and trachea was detected. To our knowledge, this work links for the first time increased biofilm formation in bacteria with a colonization advantage in an animal model. We propose that the enhanced biofilm-forming capacity of currently circulating strains contributes to their persistence, transmission, and continued circulation.Centro de Investigación y Desarrollo en Fermentaciones IndustrialesFacultad de Ciencias Exacta

Hyperbiofilm formation by <i>Bordetella pertussis</i> strains correlates with enhanced virulence traits

Pertussis, or whooping cough, caused by the obligate human pathogen Bordetella pertussis is undergoing a worldwide resurgence. The majority of studies of this pathogen are conducted with laboratory-adapted strains which may not be representative of the species as a whole. Biofilm formation by B. pertussis plays an important role in pathogenesis. We conducted a side-by-side comparison of the biofilm-forming abilities of the prototype laboratory strains and the currently circulating isolates from two countries with different vaccination programs. Compared to the reference strain, all strains examined herein formed biofilms at high levels. Biofilm structural analyses revealed country-specific differences, with strains from the United States forming more structured biofilms. Bacterial hyperaggregation and reciprocal expression of biofilm-promoting and -inhibitory factors were observed in clinical isolates. An association of increased biofilm formation with augmented epithelial cell adhesion and higher levels of bacterial colonization in the mouse nose and trachea was detected. To our knowledge, this work links for the first time increased biofilm formation in bacteria with a colonization advantage in an animal model. We propose that the enhanced biofilm-forming capacity of currently circulating strains contributes to their persistence, transmission, and continued circulation.Centro de Investigación y Desarrollo en Fermentaciones IndustrialesFacultad de Ciencias Exacta

Hyperbiofilm formation by Bordetella pertussis strains correlates with enhanced virulence traits

Pertussis, or whooping cough, caused by the obligate human pathogen Bordetella pertussis is undergoing a worldwide resurgence. The majority of studies of this pathogen are conducted with laboratory-adapted strains which may not be representative of the species as a whole. Biofilm formation by B. pertussis plays an important role in pathogenesis. We conducted a side-by-side comparison of the biofilm-forming abilities of the prototype laboratory strains and the currently circulating isolates from two countries with different vaccination programs. Compared to the reference strain, all strains examined herein formed biofilms at high levels. Biofilm structural analyses revealed country-specific differences, with strains from the United States forming more structured biofilms. Bacterial hyperaggregation and reciprocal expression of biofilm-promoting and -inhibitory factors were observed in clinical isolates. An association of increased biofilm formation with augmented epithelial cell adhesion and higher levels of bacterial colonization in the mouse nose and trachea was detected. To our knowledge, this work links for the first time increased biofilm formation in bacteria with a colonization advantage in an animal model. We propose that the enhanced biofilm-forming capacity of currently circulating strains contributes to their persistence, transmission, and continued circulation.Fil: Cattelan, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Jennings Gee, Jamie. Wake Forest School of Medicine; Estados UnidosFil: Dubey, Purnima. Wake Forest School of Medicine; Estados Unidos. Ohio State University; Estados UnidosFil: Yantorno, Osvaldo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Deora, Rajendar. Wake Forest School of Medicine; Estados Unidos. Ohio State University; Estados Unido

Effect of hydromechanical stress on cellular antigens of Bordetella pertussis

Cells of Bordetella pertussis grown in a bioreactor under stirring conditions were studied to investigate the effect of shear stress on cellular-bound filamentous haemagglutinin (FHA). FHA attached to the bacterial surface, unlike extracellular FHA, was not affected at the shear levels tested. Moreover, no other cellular immunogen involved in the whole-cell protective activity seemed to be affected by hydromechanical forces.Centro de Investigaciones en Fitopatologí

Identificación rápida de bacterias aisladas de pacientes fibroquísticos mediante espectroscopía infrarroja y análisis multivariante

A partir del año 2004 se ha registrado en el Hospital de Niños de La Plata, un aumento en el número de aislamientos de B. cepacia en pacientes fibroquísticos (FQ), así como también en niños con otras patologías. La identificación rápida y precisa de estas bacterias resulta esencial para la iniciación del tratamiento adecuado. Los métodos bioquímicos empleados de rutina para la identificación microbiológica, no brindan por lo general resultados certeros, demandan al menos 5 días de análisis y sólo permiten discriminar hasta el nivel de especie. Sin embargo, las bacterias identificadas como "B. cepacia" comprenden un grupo de 9 especies o genomovares altamente relacionados, denominado colectivamente el Complejo B. cepacia. El objetivo de este trabajo fue desarrollar una estrategia que permitiera la identificación y caracterización rápida de organismos aislados de muestras de esputo de pacientes FQ a nivel de subtipo de genomovar, basada en el empleo de espectroscopia infrarroja con transformada de Fourier (FTIR) combinada con técnicas de análisis multivariante. Se obtuvieron espectros de 14 aislamientos hospitalarios y 16 cepas de referencia previamente confirmadas por técnicas de biología molecular (PCR y corte con enzimas de restricción) en un espectrómetro Perkin Elmer (Spectrum One). Para ello se utilizaron colonias obtenidas en medio sólido en condiciones estandarizadas, proveniente de un cultivo confluente resuspendidas en H2O, transferidas a una celda de ZnSe y llevadas a sequedad hasta obtener un film homogéneo y transparente. Los espectros se normalizaron y sus derivadas primeras fueron sometidas a diferentes análisis multivariantes para su diferenciación y construcción de librerías espectrales. Se desarrollaron y compararon dos sistemas de caracterización, diferenciación e identificación basados en: 1) análisis de clusters (AC) empleando el coeficiente de Pearson y el algoritmo de Ward (software OPUS V.4.0, Bruker Optics, USA) y 2) un sistema de redes neuronales artificiales (ANNs) que discrimina en tres diferentes niveles (género, genomovar y subtipo), entrenado aplicando diferentes algoritmos (NeuroDeveloper© software, Germany). Se demostró que FT-IR combinada con AC y/o ANNs permite la identificación rápida (menos de 36 horas), precisa y a bajo costo de organismos de interés clínico.Ganador al premio Mejor Trabajo Científico de las Cuartas Jornadas de Actualización en Clínica Pediátrica 200

Proteome approaches combined with Fourier transform infrared spectroscopy revealed a distinctive biofilm physiology in Bordetella pertussis

Proteome analysis was combined with whole-cell metabolic fingerprinting to gain insight into the physiology of mature biofilm in Bordetella pertussis, the agent responsible for whooping cough. Recent reports indicate that B. pertussis adopts a sessile biofilm as a strategy to persistently colonize the human host. However, since research in the past mainly focused on the planktonic lifestyle of B. pertussis, knowledge on biofilm formation of this important human pathogen is still limited. Comparative studies were carried out by combining 2-DE and Fourier transform infrared (FT-IR) spectroscopy with multivariate statistical methods. These complementary approaches demonstrated that biofilm development has a distinctive impact on B. pertussis physiology. Results from MALDI-TOF/MS identification of proteins together with results from FT-IR spectroscopy revealed the biosynthesis of a putative acidic-type polysaccharide polymer as the most distinctive trait of B. pertussis life in a biofilm. Additionally, expression of proteins known to be involved in cellular regulatory circuits, cell attachment and virulence was altered in sessile cells, which strongly suggests a significant impact of biofilm development on B. pertussis pathogenesis. In summary, our work showed that the combination of proteomics and FT-IR spectroscopy with multivariate statistical analysis provides a powerful tool to gain further insight into bacterial lifestyles.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Fermentaciones Industriale

Bordetella biofilms: a lifestyle leading to persistent infections

Bordetella bronchiseptica and B. pertussis are Gram-negative bacteria that cause respiratory diseases in animals and humans. The current incidence of whooping cough or pertussis caused by B. pertussis has reached levels not observed since the 1950s. Although pertussis is traditionally known as an acute childhood disease, it has recently resurged in vaccinated adolescents and adults. These individuals often become silent carriers, facilitating bacterial circulation and transmission. Similarly, vaccinated and non-vaccinated animals continue to be carriers of B. bronchiseptica and shed bacteria resulting in disease outbreaks. The persistence mechanisms of these bacteria remain poorly characterized. It has been proposed that adoption of a biofilm lifestyle allows persistent colonization of the mammalian respiratory tract. The history of Bordetella biofilm research is only a decade long and there is no single review article that has exclusively focused on this area. We systematically discuss the role of Bordetella factors in biofilm development in vitro and in the mouse respiratory tract. We further outline the implications of biofilms to bacterial persistence and transmission in humans and for the design of new acellular pertussis vaccines.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Adenylate cyclase influences filamentous haemagglutinin-mediated attachment of Bordetella pertussis to epithelial alveolar cells

Attachment to epithelial cells in the respiratory tract is a key event in Bordetella pertussis colonization. Filamentous haemagglutinin (FHA) is an important virulence factor mediating adhesion to host cells. In this study, the relevance of the interaction between FHA and adenylate cyclase toxin (ACT) during bacterial attachment was investigated. Mutants lacking either FHA or ACT showed significantly decreased adherence to epithelial respiratory cells. The use of several ACT-specific monoclonal antibodies and antiserum showed that the decrease in attachment of strains lacking ACT expression could not be explained by the adhesin-like activity of ACT, or a change of any of the biological activities of ACT. Immunoblot analysis showed that the lack of ACT expression did not interfere with FHA localization. An heparin-inhibitable carbohydrate-binding site is crucial in the process of FHA-mediated bacterial binding to epithelial cells. In the presence of heparin attachment of wild-type B. pertussis, but not of the isogenic ACT defective mutant, to epithelial cells was significantly decreased. These results suggest that ACT enhances the adhesive functions of FHA, and modifies the performance of the FHA heparin-inhibitable carbohydrate binding site. We propose that the presence of ACT in the outer membrane of B. pertussis to play a role in the functionality of FHA.Centro de Investigación y Desarrollo en Fermentaciones Industriale