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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.

The cell envelope of the Gram-negativeBurkholderia cepaciacomplex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals, such as those living with cystic fibrosis. To systematically identify cell envelope-associated resistance and susceptibility determinants at the genome level, we constructed a high-density, randomly-barcoded transposon mutant library in the clinical isolateB. cenocepaciaK56-2 and exposed it to a panel of more than twenty cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profiled over a hundred new functional associations and identified novel mediators of antibiotic susceptibility in the Bcc cell envelope. We revealed new connections between β-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism, which highlight a vulnerability in sharing this lipid intermediate. We then show that the clinically relevant synergy of the β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. Importantly, we found that avibactam more strongly potentiates the activity of aztreonam and meropenem than ceftazidime in a panel of Bcc clinical isolates. Finally, we characterize for first time in the Bcc the iron and receptor-dependent activity of the novel siderophore-cephalosporin antibiotic, cefiderocol. Overall, our work has implications for antibiotic target prioritization, and for using additional combinations of β-lactam/β-lactamase inhibitors that can extend the utility of our current clinical arsenal of antibacterial therapies.Author SummaryWhile the Gram-negative cell envelope is a major barrier to antibiotic action, we have an incomplete picture of how each component contributes to antibiotic resistance. To answer this question from a genome-wide perspective in a model of antibiotic-resistant bacteria, we constructed a library of randomly-barcoded transposon mutants inBurkholderia cenocepaciaand exposed it to a panel of diverse cell envelope-targeting antibiotics. We identified individual genes and whole pathways associated with antibiotic resistance, including several novel players. Focusing on clinically relevant antibiotics, our strategy dissected how the synergy of the important β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam primarily depends upon inhibition of a single β-lactamase. Additionally, we are the first to characterise the molecular basis of the antibiotic mechanism of cefiderocol inBurkholderia, a new and very potent siderophore-cephalosporin conjugate drug. Encouragingly, we found that cefiderocol susceptibility was greatest at physiological iron concentrations. Overall, we highlight several avenues that can be taken to enhance or develop new therapeutic strategies againstBurkholderiainfection.

The identification of the most competent embryos for transfer to the uterus constitutes the main challenge of in vitro fertilization (IVF). We established a metabolomic-based approach by applying Fourier transform infrared (FTIR) spectroscopy on 130 samples of 3-day embryo culture supernatants from 26 embryos that implanted and 104 embryos that failed. On examining the internal structure of the data by unsupervised multivariate analysis, we found that the supernatant spectra of nonimplanted embryos constituted a highly heterogeneous group. Whereas ∼40% of these supernatants were spectroscopically indistinguishable from those of successfully implanted embryos, ∼60% exhibited diverse, heterogeneous metabolic fingerprints. This observation proved to be the direct result of pregnancy's multifactorial nature, involving both intrinsic embryonic traits and external characteristics. Our data analysis strategy thus involved one-class modelling techniques employing soft independent modelling of class analogy that identified deviant fingerprints as unsuitable for implantation. From these findings, we could develop a noninvasive Fourier-transform-infrared-spectroscopy–based approach that represents a shift in the fundamental paradigm for data modelling applied in assisted-fertilization technologies.

La enfermedad por coronavirus 2019 (COVID-19) se informó por primera vez en Wuhan, China, el 31 de diciembre de 2019, declarándose emergencia de salud pública de importancia internacional en primera instancia y posteriormente elevada a la categoría de pandemia. Ante la grave situación sanitaria producto de la propagación del SARS-CoV-2, el Ministerio de Salud de la Provincia de Buenos Aires y la Universidad Nacional de La Plata establecieron un convenio de cooperación con la finalidad de que la Universidad Nacional de La Plata provea capacidad diagnóstica al sistema sanitario. Luego de un período de adecuación de los laboratorios e implementación de protocolos se inició tempranamente con el diagnóstico del SARS-CoV-2, habiendo procesado a fines de septiembre de 2020 más de 32.000 muestras provenientes de diversos Municipios de las Regiones Sanitarias I, II, IV, VI, X y XI de la Provincia de Buenos Aires. Los laboratorios participantes de las Facultades de Ciencias Exactas, Ciencias Médicas y Ciencias Veterinarias forman parte de los Laboratorios de referencia de COVID-19 de la Provincia de Buenos Aires y articulan dicha labor con diversas actividades de investigación, docencia y desarrollo con la finalidad de proveer soluciones al sistema de salud en el contexto de la pandemia. de salud en el contexto de la pandemia.

The identification of the most competent embryos for transfer to the uterus constitutes the main challenge of

The identification of the most competent embryos for transfer to the uterus constitutes the main challenge of in-vitro fertilization (IVF). We established a metabolomic-based approach applying Fourier Transform Infrared spectroscopy (FTIR) on 130 samples of 3-days embryo culture supernatants from 26 embryos that implanted and 104 that failed. Examining the internal structure of the data by unsupervised multivariate analysis, it was observed that the supernatants of nonimplanted embryos contained highly heterogeneous spectral features. These features were overlapping with metabolic-implantation fingerprints, thus demonstrating that in establishing embryo-assessment models a one-class modelling involving only the samples with positive-implantation outcomes should be applied. Analysis of variance confirmed that the women´s age (>40 years) undermined the implantation of the embryos exhibiting implantation metabolomics, and also that constituted a condition triggering embryos to express nonimplantation metabolomics. We conclude that IVF-success rates can be significantly improved if FTIR spectroscopy is used as an embryo-selection criterion.

The concern regarding toxicity from the presence of deoxynivalenol (DON) in wheat that affects both economy and public health leads to the need to find appropriate detection methods for determining the degree of DON contamination in terms of the equipment available and the speed required for obtaining the incidence. The objective of this study was to compare the performance of two alternative analytical techniques for DON quantification for use in the food industry with a reference technique. Samples of wheat and the commercial by-products were analysed by high-performance liquid chromatography (HPLC) with an ultraviolet detector as the reference method and the results compared with those obtained from a rapid lateral-flow immunochromatographic device (Reveal Q+) and of a Fourier-transform-infrared (FTIR) spectroscopy technique. Pearson’s correlation coefficient between the HPLC and Reveal-Q+ data (0.45), although significant (P, Facultad de Ciencias Exactas, Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI)

The effect of casein (CAS) and inulin (INUL), at ratios commonly found during infant formula processing, on the conformational changes of whey proteins (WP) was investigated. Model systems were prepared as aqueous dispersions of WP isolate alone or with calcium caseinate and/or INUL, and heated for 30 min at66, 70 or 75 C. Heat treatment of WP induced denaturation and aggregation. Solubility of WP system decreased by 25% when heated at 75 C. Upon heating, the presence of CAS did not prevent WP denaturation, but increased protein solubility up to 100% in WPeCAS system and prevented aggregation. INULaffected the secondary structure of WP and increased the denaturation temperature, although it did not prevent aggregation nor solubility loss at 75 C. In conclusion, presence of CAS prevented aggregationand solubility loss of WP during heating while INUL induced WP conformational changes impacting the denaturation stage. Fil: Rodríguez Arzuaga, Mariana. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina. Laboratorio Tecnológico del Uruguay; Uruguay Fil: Bosch, María Alejandra. 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; Argentina Fil: Añon, Maria Cristina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina Fil: Abraham, Analia Graciela. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina

Bordetella pertussisis the causative agent of whooping cough or pertussis a highly contagious disease that affects respiratory tract Although vaccination has reduced mortality due to pertussis infection the efficacy of both current cellular and a cellular formulations produced from bacteria grown in stirred bioreactors is limited Thus the interest in the development of new vaccines increased In this scenario the hypothesis that B pertussiscolonizes and persists in their host through biofilm development started to be analyzed The biofilm formation starts with the adhesion of free floating bacteria to surfaces Then the production of extracellular polymeric substance is induced and this contributes to increase the anchor to the surface The resultant mature biofilm is more resistant to the external physical chemical or biological attack We here reviewed the main results showing the fitness of B pertussisreference strains and clinical isolates to adopt the biofilm lifestyle We outline the implications of biofilm development in the proteomic of cells growing in liquid media planktonic cells and in biofilm sessile cells In particular an over expression of the Bordetella intermediate protein A BipA a typical marker of intermediate phase in the surface of biofilm cells is analyzed Considering that bacterial biofilm are often immersed in liquid fluid and are subjected to hydrodynamic forces we here highlight the importance of shear stress in initial adhesion of B pertussison surfaces Finally the differences in the capacity of biofilm formation between reference strains and clinical isolates recovered in Argentina and USA are reviewed We conclude that elucidating the molecular mechanisms involved in the association of biofilm and pathogenesis of Bordetella pertussis will be important for the development of new and more effective vaccines to control the resurgence of whooping cough

Identification of microorganisms by Fourier transform infrared (FT-IR) spectroscopy is known as a promising alternative to conventional identification techniques in clinical, food, and environmental microbiology. In this study we demonstrate the application of FT-IR hyperspectral imaging for rapid, objective, and cost-effective diagnosis of pathogenic bacteria. The proposed method involves a relatively short cultivation step under standardized conditions, transfer of the microbial material onto suitable IR windows by a replica method, FT-IR hyperspectral imaging measurements, and image segmentation by machine learning classifiers, a hierarchy of specifically optimized artificial neural networks (ANN). For cultivation, aliquots of the initial microbial cell suspension were diluted to guarantee single-colony growth on solid agar plates. After a short incubation period when microbial microcolonies achieved diameters between 50 and 300 μm, microcolony imprints were produced by using a specifically developed stamping device which allowed spatially accurate transfer of the microcolonies' upper cell layers onto IR-transparent CaF

Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). These opportunistic pathogens are also widely distributed in natural and man-made environments. After a 12-year epidemiological surveillance involving Bcc bacteria from respiratory secretions of Argentinean patients with CF and from hospital settings, we found six isolates of the Bcc with a concatenated species-specific allele sequence that differed by more than 3% from those of the Bcc with validly published names. According to the multilocus sequence analysis (MLSA), these isolates clustered with the agricultural soil strain, Burkholderia sp. PBP 78, which was already deposited in the PubMLST database. The isolates were examined using a polyphasic approach, which included 16S rRNA, recA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), DNA base composition, average nucleotide identities (ANIs), fatty acid profiles, and biochemical characterizations. The results of the present study demonstrate that the seven isolates represent a single novel species within the Bcc, for which the name Burkholderia puraquae sp. nov. is proposed. Burkholderia puraquae sp. nov. CAMPA 1040T (=LMG 29660T=DSM 103137T) was designated the type strain of the novel species, which can be differentiated from other species of the Bcc mainly from recA gene sequence analysis, MLSA, ANIb, MALDI-TOF MS analysis, and some biochemical tests, including the ability to grow at 42 ºC, aesculin hydrolysis, and lysine decarboxylase and β-galactosidase activities., Facultad de Ciencias Exactas, Centro de Investigación y Desarrollo en Fermentaciones Industriales, Instituto de Biotecnologia y Biologia Molecular

We report here the draft genome sequence of Burkholderia puraquae type strain CAMPA 1040, a member of the Burkholderia cepacia complex. This strain, isolated from a hemodialysis water reservoir, harbors several stress tolerance genes, such as the systems for low oxygen survival, for copper tolerance, and for osmotic stress resistance., Facultad de Ciencias Exactas

Fusarium graminearum is the primary causal agent of Fusarium head blight of wheat in Argentina. This disease affects crop yields and grain quality also reducing the wheat end-use, and causing mycotoxin contamination. The aim of this work was to analyze the phenotypic characteristics associated with phenotypic diversity and aggressiveness of 34 F. graminearum sensu stricto isolates recovered from Argentinean fields in the 2008 growing season using the Fourier Transform Infrared (FTIR) dried film technology. We applied this technique also to search for spectral specific markers associatedwith aggressiveness. The combination of FTIR technologywith hierarchical cluster analysis allowed us to determine that this population constitutes a highly diverse and heterogeneous group of fungi with significant phenotypic variance. Still, when the spectral features of a set of these isolates were compared against their aggressiveness, as measured by disease severity, thousand grains weight, and relative yield reduction, we found that the more aggressive isolates were richer in lipid content. Therefore, we could define several spectroscopic markers (> C — H stretching modes in the 3000–2800 window, > C=O and — C — O vibrational modes of esters at 1765–1707 cm−1 and 1474–900 cm−1, respectively),, mostly assigned to lipid content that could be associated with F. graminearum aggressiveness. All together, by the application of FTIR techniques and simple multivariate analyses, it was possible to gain significant insights into the phenotypic characterization of F. graminearum local isolates, and to establish the existence of a direct relationship between lipid content and fungal aggressiveness. Considering that lipids have a major role as mediators in the interaction between plants and fungi our results could represent an attractive outcome in the study of Fusarium pathogenesis., Centro de Investigación y Desarrollo en Fermentaciones Industriales

Background: Pandoraea species are considered emerging pathogens in the context of cystic fibrosis (CF) and are difficult to identify by conventional biochemical methods. These multidrug resistant bacteria remain poorly understood particularly in terms of natural resistance, mechanisms of acquired resistance and impact on the prognosis of the disease and the lung function. Among them, Pandoraea sputorum has been previously described in few cases of CF patients from Spain, Australia, France and United States, underlining the need of more clinical data for a better knowledge of its pathogenicity. This is the first report relating to P. sputorum in a CF patient in Argentina. Case presentation: Pandoraea sputorum was identified in a nine-year-old cystic fibrosis patient from Argentina, after treatment failure during an exacerbation. The isolates were successfully identified by combining molecular techniques based on 16S rRNA sequencing and mass spectrometry (MS) methods, after reassessing previous misidentified isolates by conventional methods. After first isolation of P. sputorum, patient's clinical condition worsened but later improved after a change in the treatment. Although isolates showed susceptibility to trimethoprim-sulfamethoxazole and imipenem, in our case, the antibiotic treatment failed in the eradication of P. sputorum. Conclusions: All combined data showed a chronic colonization with P. sputorum associated to a deterioration of lung function. We noted that the presence of P. sputorum can be underestimated in CF patients and MALDI-TOF MS appears to be a promising means of accurate identification of Pandoraea species., Centro de Investigación y Desarrollo en Fermentaciones Industriales, Instituto de Biotecnologia y Biologia Molecular, Facultad de Ciencias Exactas

espanolNo todos los dias se abre un concurso para redisenar el principal eje de una metropolis. El proyecto ganador del concurso Nueva Alameda Providencia �organizado por el Gobierno Regional Metropolitano de Santiago� no solo busca renovar la avenida mas emblematica de la capital de Chile, sino tambien transformarla en una experiencia urbana excepcional donde el espacio publico, en vez de competir con los distintos sistemas de transporte, sea el que los define y reordena. EnglishNot every day a competition to redesign a metropolis�s main axis is opened. The awarded entry for the New Alameda Providencia contest, organized by the Regional Metropolitan Government of Santiago, not only seeks to renew the most emblematic avenue in the Chilean capital, but also to transform it into an exceptional urban experience in which public space, rather than competing with the various transport systems, is the one that defines and reorders them.

Kefiran, an exopolysaccharide produced by microorganisms present in kefir grains, is a glucogalactan that has several health promoting properties. In the present work the effect of different sugars (glucose, galactose, sucrose) and polyols (glycerol and sorbitol) as plasticizers on kefiran film properties was evaluated. Kefiran films were brittle and rigid and all the tested plasticizers improved film properties. They were all transparent with low opacity. Unplasticized kefiran films X-ray diffraction patterns were similar to those of plasticized ones, showing an amorphous-crystalline structure with low crystallinity degree. All films presented low aw values (below 0.5) conferring protection properties against microorganism growth. The lowest permeability value was obtained with glucose as plasticizer whereas the best mechanical properties were obtained with glycerol addition. The relative high intensities of the bands assigned to water molecules stretching and bending modes in ATR/FT-IR average spectra of glycerol–kefiran films were correlated with their relative high water content compared to the other plasticized films. The analysis of the bands due to C–H stretching modes showed that glucose and glycerol could be reducing the interaction between the adjacent chains in the kefiran polymer. It would demonstrate the importance of the analysis of kefiran, plasticizers and water molecules interactions when films properties are studied.

Two styrene–butadiene–styrene block (SBS) copolymers which differ in weight average molecular weight and two base asphalts (from local crude source, Argentina) had been blended under different experimental conditions. Rotational viscosity, fluorescence microscopy, size exclusion chromatography (SEC) and FT-IR spectrometry were used in order to study the rheological and morphological changes of the blends during their manufacturing. Our results show that the high shear rate and temperature used during this procedure induce the SBS copolymer degradation and the degradation fragments distribution into maltenic asphalt phase. These events are responsible for the observed changes. The optimal time which produces the best rheological properties of modified asphalt depends on the blend composition and on the shear rate used.

The epidemiological and clinical management of cystic fibrosis (CF) patients suffering from acute pulmonary exacerbations or chronic lung infections demands continuous updating of medical and microbiological processes associated with the constant evolution of pathogens during host colonization. In order to monitor the dynamics of these processes, it is essential to have expert systems capable of storing and subsequently extracting the information generated from different studies of the patients and microorganisms isolated from them. In this work we have designed and developed an on-line database based on an information system that allows to store, manage and visualize data from clinical studies and microbiological analysis of bacteria obtained from the respiratory tract of patients suffering from cystic fibrosis. The information system, named Cystic Fibrosis Cloud database is available on the http://servoy.infocomsa.com/cfc_database site and is composed of a main database and a web-based interface, which uses Servoy's product architecture based on Java technology. Although the CFC database system can be implemented as a local program for private use in CF centers, it can also be used, updated and shared by different users who can access the stored information in a systematic, practical and safe manner. The implementation of the CFC database could have a significant impact on the monitoring of respiratory infections, the prevention of exacerbations, the detection of emerging organisms, and the adequacy of control strategies for lung infections in CF patients.

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. Fil: Martina, Pablo F. 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; Argentina Fil: Feliziani, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Juan, Carlos. Instituto de Investigación Sanitaria de Palma. Hospital Universitario Son Espases. Servicio de Microbiología y Unidad de Investigación; España Fil: Bettiol, Marisa Paula. Provincia de Buenos Aires. Ministerio de Salud. Hospital de Niños "Sor María Ludovica" de la Plata; Argentina Fil: Gatti, Blanca. Provincia de Buenos Aires. Ministerio de Salud. Hospital de Niños "Sor María Ludovica" de la Plata; Argentina Fil: 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; Argentina Fil: Smania, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Oliver, Anntonio. Instituto de Investigación Sanitaria de Palma. Hospital Universitario Son Espases. Servicio de Microbiología y Unidad de Investigación; España Fil: Bosch, María Alejandra. 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; Argentina

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 (KNO3, NH4Cl, 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.

The difficulties in preventing and treating infectious bovine keratoconjunctivitis (IBK) and the consequent impact on the cattle industry worldwide emphasize the need to better understand this infectious process along with the biology of Moraxella bovis, its primary causative agent. Although there is increasing evidence that bacterial biofilms participate in a variety of ocular infections by direct biofilm formation on the surfaces of the eye, IBK has not been considered as a biofilm-based disease so far, and even more, no information is currently available regarding the ability of M. bovis to adopt a biofilm lifestyle. In the present research, we demonstrated the capacity of M. bovis clinical isolates and reference strains to form biofilms on different abiotic surfaces and culture conditions, and provided qualitative and quantitative information on the biofilm growth and architecture of mature biofilms. In addition, our data indicated that the type IV pili play a critical role in the biofilm formation in vitro. Most significantly, we proved that through exposure to MgCl2 type IV pili are removed from the cell surface, not only preventing M. bovis biofilm formation but also disassembling preformed biofilms. These results could constitute a new approach in the understanding of M. bovis colonization process in cattle eye and/or nasal cavity, and may aid in the development of future antimicrobial strategies for the control of IBK. © 2013 Elsevier B.V. Fil: Prieto, Claudia Inés. Universidad Nacional de La Plata; Argentina Fil: Serra, Diego Omar. 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; Argentina Fil: Martina, Pablo F.. 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; Argentina Fil: Jacobs, Melisa. 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; Argentina Fil: Bosch, María Alejandra. Universidad Nacional de La Plata; Argentina Fil: 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; Argentina

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 BOXPCR 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., Centro de Investigación y Desarrollo en Fermentaciones Industriales, Instituto de Biotecnologia y Biologia Molecular

A simple statistical model is presented to describe the germination kinetics of filamentous fungal spore populations grown either in soluble or on solid substrates. The model attempts to reduce large amounts of data to three parameters of relevant physiological significance: one related to the maximum proportion of germination in a spore population, another related to the kinetics of the process, and another — the synchronization factor — that measures the extent of synchronization in the population of germinating spores. The latter is a very important parameter, which is introduced for the first time in a model of spore germination, and which, together with the other two, would be considered in relation to filamentous fungal fermentations in physiological and technological studies. These parameters were estimated by the Maximum Likelihood method. The fit of our experimental data, together with associated data reported in the literature indicated the validity of the model for the description of the time course of germination for a large variety of strains grown under different physical and chemical environmental conditions.

Pathogenic bacteria delay wound healing through several different mechanisms such as persistent production of inflammatory mediators or maintenance of necrotic neutrophils, which release cytolytic enzymes and free oxygen radicals. One of the most frequent pathogens isolated from infections in chronic wounds is Pseudomonas aeruginosa. This bacterium is extremely refractory to therapy and to host immune attack when it forms biofilms. Therefore, antibiotics and antiseptics are becoming useless in the treatment of these infections. In previous works, we demonstrated that Lactobacillus plantarum has an important antipathogenic capacity on P. aeruginosa. The aim of the present work was to elucidate the mechanism involved in the control of growth of P. aeruginosa on different surfaces by L. plantarum. For this purpose, we investigated the effects of L. plantarum supernatants on pathogenic properties of P. aeruginosa, such as adhesion, viability, virulence factors, biofilm formation, and quorum sensing signal expression. L. plantarum supernatants were able to inhibit pathogenic properties of P. aeruginosa by a quorum quenching mechanism. The antipathogenic properties mentioned above, together with the immunomodulatory, tissue repair, and angiogenesis properties in the supernatants of L. plantarum, make them an attractive option in infected chronic wound treatment.

Water balances, based on the evaluation of water released from the fermentor, were estimated in a solid-state fermentation process performed without forced aeration. It was proved that the net gain or loss of water during the course of the process depends on the dry weight of the solid substrate at the beginning of the process and also on the initial water activity. A linear relationship was found between the wet weight losses of cultures recorded after the end of the growth period and the initial dry weight of the substrate which enables the evaluation of the water released from the vessels. These values were included into water balances from which it was possible to calculate the water produced by metabolism of carbohydrates, which was estimated at 0.40 mol of H 2 O per C-mol of glucose consumed.

Pathogenic bacteria delay wound healing through several different mechanisms such as persistent production of inflammatory mediators or maintenance of necrotic neutrophils, which release cytolytic enzymes and free oxygen radicals. One of the most frequent pathogens isolated from infections in chronic wounds is Pseudomonas aeruginosa. This bacterium is extremely refractory to therapy and to host immune attack when it forms biofilms. Therefore, antibiotics and antiseptics are becoming useless in the treatment of these infections. In previous works, we demonstrated that Lactobacillus plantarum has an important antipathogenic capacity on P. aeruginosa. The aim of the present work was to elucidate the mechanism involved in the control of growth of P. aeruginosa on different surfaces by L. plantarum. For this purpose, we investigated the effects of L. plantarum supernatants on pathogenic properties of P. aeruginosa, such as adhesion, viability, virulence factors, biofilm formation, and quorum sensing signal expression. L. plantarum supernatants were able to inhibit pathogenic properties of P. aeruginosa by a quorum quenching mechanism. The antipathogenic properties mentioned above, together with the immunomodulatory, tissue repair, and angiogenesis properties in the supernatants of L. plantarum, make them an attractive option in infected chronic wound treatment.

Species belonging to the Burkholderia cepacia complex (BCC) are capable of causing chronic respiratory tract infections in patients suffering from cystic fibrosis as well 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 work, 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.

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., Centro de Investigación y Desarrollo en Fermentaciones Industriales, Facultad de Ciencias Exactas

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 Fil: Serra, Diego. 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; Argentina Fil: Bosch, María Alejandra. 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; Argentina Fil: Russo, Daniela Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Rodriguez, Maria Elena. 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; Argentina Fil: Zorreguieta, Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Schmitt, Juergen. Synthon GmbH; Alemania Fil: Naumann, Dieter. Robert Koch Institut; Alemania Fil: 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; Argentina

Fourier transform infrared (FT-IR) spectroscopy was used in combination with multivariate statistical analysis for differentiation of lactic bacteria isolated from kefir grains. Twelve reference strains and 42 lactobacilli isolates from four local kefir grains, previously identified by biochemical traditional techniques at species level were included in this study. The spectra were analysed by hierarchical clustering analysis (HCA) using Pearson's product-moment correlation coefficient and Ward's algorithm. The differentiation between homo- and heterofermentative lactobacilli, proposed as a first level in the classification scheme, was performed with vector normalized first derivatives spectra in the windows 1789-1700, 1059-935, 3000-2927 and 896-833 cm(-1). For heterofermentative lactobacilli the windows 1780-1750, 1500-1200, 2950-2930 and 900-700 cm(-1) were found to contribute to the maximal separation among L. kefir, L. parakefir and Lactobacillus brevis. It was also demonstrated that although this model was robust against small variations in growth temperature (+/-5 degrees C) and growth time (+/-5 h), the make of culture medium used (Biokar or Difco) affected the separation of heterofermentative lactobacilli at species level. For homofermentative lactobacilli the spectral regions 1230-900, 1777-1690, 1357-1240 and 2960-2870 cm(-1), were selected for discrimination among 5 different species that are normally present in kefir grains: L. plantarum, L. acidophilus, L. kefirgranum, L. kefiranofaciens and L. cassei. The classification and discrimination schemes proposed in this work completely matched with the identification obtained by classical biochemical techniques at species level.

Although Bordetella pertussis, the etiologic agent of whooping cough, adheres and grows on the ciliated epithelium of the respiratory tract, it has been extensively studied only in liquid cultures. In this work, the phenotypic expression of B. pertussis in biofilm growth is described as a first approximation of events that may occur in the colonization of the host. The biofilm developed on polypropylene beads was monitored by chemical methods and Fourier transform infrared (FT-IR) spectroscopy. Analysis of cell envelopes revealed minimal differences in outer membrane protein (OMP) pattern and no variation of lipopolysaccharide (LPS) expression in biofilm compared with planktonically grown cells. Sessile cells exhibited a 2.4- to 3.0-fold higher carbohydrate/protein ratio compared with different types of planktonic cells. A 1.8-fold increased polysaccharide content with significantly increased hydrophilic characteristics was observed. FT-IR spectra of the biofilm cells showed higher intensity in the absorption bands assigned to polysaccharides (1,200-900 cm(-1) region) and vibrational modes of carboxylate groups (1,627, 1,405, and 1,373 cm(-1)) compared with the spectra of planktonic cells. In the biofilm matrix, uronic-acid-containing polysaccharides, proteins, and LPS were detected. The production of extracellular carbohydrates during biofilm growth was not associated with changes in the specific growth rate, growth phase, or oxygen limitation. It could represent an additional virulence factor that may help B. pertussis to evade host defenses.

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.

89 Hypermutation in Burkholderia cepacia complex is highly prevalent in chronic respiratory infections of cystic fibrosis patients A. Bosch1, P. Martina2, S. Feliziani3, C. Juan4, M. Bettiol5, B. Gatti6, B. Valeiras7, O. Yantorno7, A. Smania8, A. Oliver4. 1CINDEFI-CONICET-CCT La Plata, Universidad Nacional de La Plata, Chemistry, La Plata, Argentina; 2CINDEFIUNLP-CONICET-CCT La Plata, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Chemistry, La Plata, Argentina; 3CIQUIBIC-CONICET Centro de Investigaciones en Quimica Biologica de Cordoba, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba, Argentina; 4Servicio de Microbiologia y Unidad de Investigacion, Hospital Universitario Son Espases, Palma de Mallorca, Spain, Palma de Mallorca, Spain; 5Sala de Microbiologia, Hospital de Ninos de La Plata ‘Sor Maria Ludovica’, Microbiologia, La Plata, Argentina; 6Sala de Microbiologia, Hospital de Ninos de La Plata ‘Sor Maria Ludovica’, La Plata, Argentina; 7CINDEFI-UNLP-CONICET-CCT La Plata, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina; 8CIQUIBIC-CONICET Centro de Investigaciones en Quimica Biologica de Cordoba, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba, Argentina

89 Hypermutation in Burkholderia cepacia complex is highly prevalent in chronic respiratory infections of cystic fibrosis patients A. Bosch1, P. Martina2, S. Feliziani3, C. Juan4, M. Bettiol5, B. Gatti6, B. Valeiras7, O. Yantorno7, A. Smania8, A. Oliver4. 1CINDEFI-CONICET-CCT La Plata, Universidad Nacional de La Plata, Chemistry, La Plata, Argentina; 2CINDEFIUNLP-CONICET-CCT La Plata, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Chemistry, La Plata, Argentina; 3CIQUIBIC-CONICET Centro de Investigaciones en Quimica Biologica de Cordoba, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba, Argentina; 4Servicio de Microbiologia y Unidad de Investigacion, Hospital Universitario Son Espases, Palma de Mallorca, Spain, Palma de Mallorca, Spain; 5Sala de Microbiologia, Hospital de Ninos de La Plata ‘Sor Maria Ludovica’, Microbiologia, La Plata, Argentina; 6Sala de Microbiologia, Hospital de Ninos de La Plata ‘Sor Maria Ludovica’, La Plata, Argentina; 7CINDEFI-UNLP-CONICET-CCT La Plata, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina; 8CIQUIBIC-CONICET Centro de Investigaciones en Quimica Biologica de Cordoba, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba, Argentina

Solid-state fermentation of rice bran or rice-bran-husk mixtures byMetarhizium anisopliae proved to be highly successful in spore yield. Optimum sporulation response on bran medium occurred when the initial water activity values ranged from 0.982 to 0.999. When bran was supplemented with 50% rice husk, the spore yield almost doubled, but a narrower initial water activity range (0.997–0.999) was optimum. Under these conditions, spore yields were 5–15 times higher (depending on the strain) than those currently obtained on the basis of rice grain fermentations., Centro de Investigación y Desarrollo en Fermentaciones Industriales