Osvaldo Yantorno

Yeast growth and β-galactosidase production during aerobic batch cultures in lactose-limited synthetic medium

Abstract A lactose-limited synthetic medium was used to analyse the dynamics of batch growth and β-galactosidase production in the yeasts Kluyveromyces and Candida , under fully aerobic conditions. In shake flasks cultures, no significant differences were observed in the growth yields among 18 tested strains and most of them showed comparable enzyme formation. Five of these strains, K. lactis CBS 2360, CBS 683, NRRL 1118, K. marxianus NRRL 1109 and C. pseudotropicalis NCYC 744 were further studied in fermenter cultures. Metabolic behaviour was characteristic of the Crabtree-negative yeasts. Ethanol formation was absent or very low (less than 5% of the lactose metabolized). During the exponential growth phase typical fluxes for lactose and oxygen uptake were 3·0 and 13 15·5 mmol g −1 H −1 respectively. Enzyme levels were constant during the exponential phase and incresed 1·3–1·8-fold during the brief deceleration phase.

A spectroscopy approach for the study of the interactions of bioactive vanadium species with bovine serum albumin

The interest in biological functions (benefits or toxics effects) of vanadium species has grown enormously in recent years. In this work, different spectroscopic methods were applied to study the effects of the interaction of vanadyl and vanadate species with bovine serum albumin (BSA), considered as the most abundant plasma protein. UV-Vis, Fourier transform infrared (FT-IR), and FT-Raman spectroscopies were used to investigate changes in secondary and tertiary structures of BSA induced by the binding of oxovanadium(IV) and vanadate(V) species (VO(2+) and VO3(-), respectively). Correlations between the metal ion binding mode, protein conformational transitions, and structural variations were established.

FHA-mediated cell-substrate and cell-cell adhesions are critical for Bordetella pertussis biofilm formation on abiotic surfaces and in the mouse nose and the trachea

Bordetella spp. form biofilms in the mouse nasopharynx, thereby providing a potential mechanism for establishing chronic infections in humans and animals. Filamentous hemagglutinin (FHA) is a major virulence factor of B. pertussis, the causative agent of the highly transmissible and infectious disease, pertussis. In this study, we dissected the role of FHA in the distinct biofilm developmental stages of B. pertussis on abiotic substrates and in the respiratory tract by employing a murine model of respiratory biofilms. Our results show that the lack of FHA reduced attachment and decreased accumulation of biofilm biomass on artificial surfaces. FHA contributes to biofilm development by promoting the formation of microcolonies. Absence of FHA from B. pertussis or antibody-mediated blockade of surface-associated FHA impaired the attachment of bacteria to the biofilm community. Exogenous addition of FHA resulted in a dose-dependent inhibitory effect on bacterial association with the biofilms. Furthermore, we show that FHA is important for the structural integrity of biofilms formed on the mouse nose and trachea. Together, these results strongly support the hypothesis that FHA promotes the formation and maintenance of biofilms by mediating cell-substrate and inter-bacterial adhesions. These discoveries highlight FHA as a key factor in establishing structured biofilm communities in the respiratory tract.

Release of Outer Membrane Vesicles from Bordetella pertussis

Abstract. The aim of the study reported here was to investigate the production of Bordetella pertussis outer membrane vesicles (OMVs). Numerous vesicles released from cells grown in Stainer-Scholte liquid medium were observed. The formation of similar vesicle-like structures could also be artificially induced by sonication of concentrated bacterial suspensions. Immunoblot analysis showed that OMVs contain adenylate cyclase-hemolysin (AC-Hly), among other polypeptides, as well as the lipopolysaccharide (LPS). Experiments carried out employing purified AC-Hly and OMVs isolated from B. pertussis AC-Hly− showed that AC-Hly is an integral component of the vesicles. OMVs reported here contain several protective immunogens and might be considered a possible basic material for the development of acellular pertussis vaccines.

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.

Genetic diversity of Burkholderia contaminans isolates from cystic fibrosis patients in Argentina

ABSTRACT 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.

Analyses of lipopolysaccharides, outer membrane proteins and DNA fingerprints reveal intraspecies diversity in Moraxella bovis isolated in Argentina.

Intra-specific diversity within Moraxella bovis was investigated analysing DNA fingerprints, outer membrane proteins (OMP) and lipopolysaccharides (LPS) profiles. Three collection strains and 57 isolates of M. bovis, collected during 3 years from cattle with infectious bovine keratoconjunctivitis (IBK) symptoms, from diverse geographical locations of Argentina, were examined. The LPS and OMP profiles were studied through SDS-PAGE analysis and genotype was determined by PCR-DNA fingerprinting. Genotyping identified five DNA types while analysis of LPS and OMP profiles identified three rough LPS types and three OMP types among the 60 isolates of M. bovis including the three collection strains. None of the three methods employed to assess diversity was discriminating when used alone because the degree of heterogeneity in each group of surface structures was limited, but when data of each typing method were combined, 15 distinct subgroups were determined. This subgrouping was clearly able to differentiate isolates of the same genotype. These typing methods appear to be useful to assess different aspects of the disease such as the diversity within a population of M. bovis associated to epidemic conditions, track the causal agent in an outbreak of the disease, monitoring vaccination programs and studies on virulence.

Whole-bacterial cell enzyme-linked immunosorbent assay for cell-bound Moraxella bovis pili

Infectious bovine keratoconjunctivitis (IBK), caused by Moraxella bovis, is a disease of major importance in cattle industry. M. bovis has several virulence factors among which pili are crucial antigen for the protective capacity of vaccines against this disease. The production of vaccines against IBK therefore requires a reliable technique for cellular piliation level assessment on cells to be included as vaccine components. In this study we describe a specific whole-bacterial cell enzyme-linked immunosorbent assay (bact-ELISA) capable of detecting pili antigen on M. bovis cell surface. A sequential competitive bact-ELISA was developed using highly piliated M. bovis cells as antigen. Samples to be analyzed were allowed to react with anti-pilus serum prior to incubation in wells coated with piliated cells of M. bovis. This assay proved useful for the rapid, sensitive and reproducible evaluation of piliation on M. bovis cells, and represents an important tool for cellular piliation monitoring daburing M. bovis cells production in stirred bioreactors.

Microcycle conidiation in the entomopathogenic fungus Beauveria bassiana bals. (vuill.)

Abstract The present work documents the experimental conditions under which the three types of two local strains of Beauveria bassiana conidia enter into a microcycle conidiation process (conidiation without vegetative phase). Different levels of different C (glucose and starch) and N sources (KNO 3 , NH 4 Cl, glutamate and peptone) were added to a basal medium containing buffer phosphate and microelements. No microcycle conidiation was observed in media containing more than 0.00139 g N l −1 for every N source and all C source concentrations assayed (except when C level was 0 g l −1 ). The absence of the C source, at every level of the N source, produced daughter conidia at the tip of very short germ tubes. The percentage of germinating conidia which underwent microcycle conidiation in 48 h of incubation was not modified by the C source level. Nevertheless, the higher the concentration of the C source added, the longer and the stronger the vacuolated germinating tubes produced. The secondary conidia produced by microcycle conidiation were viable and capable of repeating the cycle for at least two generations. Finally, taking into account the morphological changes during microcycle conidiation three distinct phases which characterized the process were established. The development of reproducible experimental systems where microcycle conidiation can be expressed is important in a complete study of B. bassiana conidiogenesis.

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.