A chromatographic approach to distinguish Gram-positive from Gram-negative bacteria using exogenous volatile organic compound metabolites.

This paper utilized L-alanine aminopeptidase activity as a useful approach to distinguish between Gram-negative and Gram-positive bacteria. This was done using two enzyme substrates, specifically 2-amino-N-phenylpropanamide and 2-amino-N-(4-methylphenyl)propanamide which liberated the volatile compounds aniline and p-toluidine, respectively. Two complementary analytical techniques have been used to identify and quantify the VOCs, specifically static headspace multicapillary column gas chromatography ion mobility spectrometry (SHS-MCC-GC-IMS) and headspace solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS). Superior limits of detection were obtained using HS-SPME-GC-MS, typically by a factor of x6 such that the LOD for aniline was 0.02µg/mL and 0.01µg/mL for p-toluidine. In addition, it was also possible to determine indole interference-free by HS-SPME-GC-MS at an LOD of 0.01µg/mL. The approach was applied to a range of selected bacteria: 15 Gram-negative and 7 Gram-positive bacteria. Use of pattern recognition, in the form of Principal Component Analysis, confirmed that it is possible to differentiate between Gram-positive and Gram-negative bacteria using the enzyme generated VOCs, aniline and p-toluidine. The exception was Stenotrophomonas maltophilia which showed negligible VOC concentrations for both aniline and p-toluidine, irrespective of the analytical techniques used and hence was not characteristic of the other Gram-negative bacteria investigated. The developed methodology has the potential to be applied for clinical and food applications.

MALDI-TOF: a useful tool for laboratory identification of uncommon glucose non-fermenting Gram-negative bacteria associated with cystic fibrosis.

The predisposition of patients with cystic fibrosis (CF) for recurrent pulmonary infections can result in poor prognosis of the disease. Although the clinical significance in CF of micro-organisms, such as Staphylococcus aureus, Haemophilus influenzae and Pseudomonas aeruginosa, is well established, the implication of uncommon glucose non-fermenting Gram-negative bacilli (UGNF-GNB) in respiratory samples from CF patients is still unclear. Because of limitations of traditional methods used in most clinical laboratories, the accurate identification of these microbes is a challenge. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) is an alternative tool for efficient identification of bacteria. This was a retrospective study to evaluate different identification methods in a collection of UGNF-GNB isolated from children with CF during a period of three years. The performance of MALDI-TOF was compared to that of 16S rDNA gene sequencing and to a conventional and automated phenotypic identification. The discriminatory power of MALDI-TOF (75.0 % agreement) was superior to automated techniques (67.1 % agreement) and to conventional phenotypical identification (50.0 % agreement). MALDI-TOF also demonstrated high accuracy in identifying Stenotrophomonas maltophilia, Achromobacter xylosoxidans and Chryseobacterium indologenes, but had limited utility in identifying Pandoraea spp. and some species of Acinetobacter and Chryseobacterium (other than C. indologenes). Although MALDI-TOF identified only 75 % of the isolates in comparison with 16S rDNA gene sequencing, the prompt identification and high discriminatory power exhibited by MALDI-TOF make it a useful tool for the characterization of micro-organisms that are difficult to identify using routine methods.

Hydration, ionic valence and cross-linking propensities of cations determine the stability of lipopolysaccharide (LPS) membranes.

The supra-molecular structure of LPS aggregates governs outer membrane permeability and activation of the host immune response during Gram-negative bacterial infections. Molecular dynamics simulations unveil at atomic resolution the subtle balance between cation hydration and cross-linking ability in modulating phase transitions of LPS membranes.

Pesquisa de genes de resistência a quinolonas em bacilos Gram negativos de origem clínica e ambiental / Research for genes of quinolone resistance in Gram negative bacilli from clinical and environmental origin

Introdução. Quinolonas são antimicrobianos sintéticos que inibem as enzimas DNA-girase e topoisomerase IV resultando na morte bacteriana. São altamente eficazes no tratamento de infecções bacterianas, especialmente causadas por bactérias Gram negativas, e portanto amplamente utilizados na medicina humana e veterinária, na qual também são empregados como profiláticos. Porém, o uso indiscriminado e inadequado levou ao aumento de bactérias resistentes a estes compostos. Esta resistência pode ocorrer devido a mutações nas enzimas DNA-girase e topoisomerase IV, e também por genes contidos em plasmídeos. Estes últimos são os principais responsáveis pela disseminação e circulação da resistência entre o meio ambiente e o ambiente hospitalar. Objetivos. Pesquisar genes de resistência a antimicrobianos do grupo das quinolonas em bactérias Gram negativas de origem clínica e ambiental que apresentam resistência fenotípica a este grupo. Material e Métodos. 73 cepas de Enterobacteriaceae e Aeromonas sp. de origem clínica e ambiental foram selecionadas para o estudo, e avaliadas quanto à sensibilidade aos antimicrobianos do grupo das quinolonas e à pesquisa de genes de resistência a este mesmo grupo e mutações no gene que codifica a enzima DNA-girase por meio de PCR e sequenciamento. Resultados. Das 73 cepas previamente selecionadas para compor o estudo, 65 foram utilizadas, devido à exclusão de perfis clonais similares. Nestas, foram observados os genes, qnrS1 (1,5 por cento ), qnrS2 (26,2 por cento ), qnrB1 (3,1 por cento ), qnrB19 (12,3 por cento ), qnrD1 (1,5 por cento ), aac(6)-Ib-cr (10,8 por cento ), oqxA (43,1 por cento ) e oqxB (41,5 por cento ), e duas variantes determinadas qnrB-like (3,1 por cento ) e qnrB69-like (1,5 por cento ). Os genes qnrA, qnrC e qepA não foram identificados. Mutações na enzima DNA-girase foram observadas em 97,9 por cento das cepas positivas para algum dos genes pesquisados...

Introduction. Quinolones are synthetic antimicrobial agents that inhibit DNA gyrase and topoisomerase IV enzymes resulting in bacterial death. They are highly effective in the treatment of bacterial infections, especially the ones caused by Gram negative bacteria, as well as for prophylaxy. Therefore they are widely used in human and veterinary medicine. However, indiscriminate and improper use led to an increase of bacteria resistance to these compounds. This resistance can be due to mutations in DNA gyrase and topoisomerase IV enzymes and also by genes contained in plasmids, which are mainly responsible for the spread and transmission of resistance between the environment and the hospital set. Objectives. To search for genes of resistance to quinolone antimicrobial agents in Gram-negative bacteria from clinical and environmental strains that present phenotypic resistance to this group. Material and Methods. 73 strains of Enterobacteriaceae and Aeromonas spp., from clinical and environmental origin, were selected for this study, and evaluated for antimicrobial susceptibility of quinolone and search of resistance genes in this same group and also for mutations in the gene encoding the enzyme DNA gyrase by PCR and sequencing. Results. Of the 73 strains previously selected to compose this study, 65 were used, due to the exclusion of similar clonal profiles. In these, genes qnrS1 (1.5 per cent ), qnrS2 (26.2 per cent ) qnrB1 (3.1 per cent ), qnrB19 (12.3 per cent ) qnrD1 (1.5 per cent ) aac(6')-Ib-cr (10.8 per cent ) oqxA (43.1 per cent ) and oqxB (41.5 per cent ) were observed, and two variants were named as qnrB-like (3.1 per cent ) and qnrB69-like (1.5 per cent ). The qnrA, qnrC and qepA genes were not identified. Mutations in DNA gyrase enzyme were observed in 97.9 per cent of the positive strains for at least one of the genes studied. It was possible to establish the association of aac(6')-Ib-cr with class 1 integron gene in four strains...

Ultrastructure and cytochemistry of lipid granules in the many-celled magnetotactic prokaryote, 'Candidatus Magnetoglobus multicellularis'.

Conspicuous cytoplasmic granules are reported in a magnetotactic multicellular prokaryote named 'Candidatus Magnetoglobus multicellularis'. Unfortunately, this microorganism, which consists of an assembly of gram-negative bacterial cells, cannot yet be cultivated, limiting the biochemical analysis of the granules and preventing in vitro studies with starvation/excess of nutrients. In this scenario, light and electron microscopy techniques were used to partially address the nature of the granules. Besides magnetosomes, three types of inclusions were observed: small (mean diameter=124 nm) polyhydroxyalkanoate-like (PHA) granules, large (diameters ranging from 0.11 to 2.5 microm) non-PHA lipid granules, and rare phosphorus-rich granules, which probably correspond to polyphosphate bodies. The PHA granules were rounded in projection, non-reactive with OsO(4), and suffered the typical plastic deformation of PHAs after freeze fracturing. The nature of the large granules, consisting of round globular structures (mean diameter=0.76 microm), was classified as non-PHA based on the following data: (a) multilayered structure in freeze-fracture electron microscopy, typical of non-PHA lipids; (b) Nile blue fluorescence imaging detected non-PHA lipids; (c) imidazole buffered osmium tetroxide and ruthenium red cytochemistry stained the globules, which appeared as electron-dense granules instead of electron lucent as PHAs do. Most likely, 'Candidatus Magnetoglobus multicellularis' stores carbon mainly as unusual lipid granules, together with smaller amounts of PHAs.

Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization.

The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.

[Integrons and their relationship with resistance phenotype in Gram negative bacilli isolated in the Hospital Torres Galdames, Iquique, Chile]. / Integrones y su relación con el fenotipo de resistencia en bacilos gramnegativos aislados en el Hospital Torres Galdames de Iquique, Chile.

UNLABELLED: The antimicrobial resistance is coded in genetic elements which generate a horizontal flow of information, particularly in conditions that are under strong selective pressure like the nosocomial environment. In that sense, in the last decades, gram negative bacilli have become important agents of nosocomial infection. In order to investigate the presence of integrons among clinical isolates of gram negative bacilli and their relationship with their resistance profile, we studied 88 strains isolated from clinical specimens of different wards of the Hospital Torres Galdames, during the June-December period, 2004. They were identified according to biochemical tests. The antimicrobial susceptibility was evaluated by agar diffusion method. The integron presence was investigated by polymerase chain reaction (PCR). A cluster analysis was carried out to study the relationship between the presence of integrons and the resistance profile. The genotyping of the isolates was carried out by ERIC-PCR technique. RESULTS: Of the isolated strains, 18% corresponded to Proteus mirabilis, 17% to Escherichia coli, and 32% to Non Fermentative Gram Negative bacilli. Most isolates presented high resistance to the antibiotics studied: 83% to ampicillin, 85% to cephalotin, 82% to ceftriaxone, 82% to ciprofloxacin, 81% to gentamycin and 82% to cotrimoxazole. Seventy-five % of the 88 strains presented integrons. Class 2 integrons were found to be the most common. The results of the cluster analysis did not show a clear relationship among the presence of the integrons and the resistance profile. With the available information it is not possible to relate the integron presence with a certain resistance pattern. The patterns of bands obtained with the technique ERIC-PCR revealed a great genetic variety among the analyzed isolations, defining diverse genotypes, distributed in the different services of the hospital where they were isolated.

Integrones y su relación con el fenotipo de resistencia en bacilos gramnegativos aislados en el Hospital Torres Galdames de Iquique, Chile / Integrons and their relationship with resistance phenotype in Gram negative bacilli isolated in the Hospital Torres Galdames, Iquique, Chile

La resistencia antimicrobiana es codificada por algunos elementos genéticos que generan un flujo horizontal, particularmente, en ambientes que están sometidos a una fuerte presión selectiva, como ocurre en el ambiente hospitalario. En tal sentido, los bacilos gramnegativos, en el último tiempo, han cobrado importancia como agentes de infección nosocomial. Objetivo. Investigar la presencia de integrones en aislados clínicos de bacilos gramnegativos y su relación con el fenotipo de resistencia, Material y Métodos. Se analizaron 88 aislados clínicos de distintos servicios del Hospital Torres Galdames, durante el período: junio a diciembre de 2004. Fueron identificadas de acuerdo con su perfil bioquímico y se determinó la susceptibilidad a antimicrobianos mediante el método de difusión en agar. La presencia de integrones se detectó mediante RPC. Se realizó un análisis de cluster para estudiar la relación entre el fenotipo de resistencia y la presencia de integrones. Las cepas fueron genotipificadas mediante ERIC-PCR. Resultados. Dieciocho por ciento de las cepas aisladas correspondió a Proteus mirabilis, 17 por ciento a Escherichia coli y 32 por ciento a bacilos gramnegativos no fermentadores. La mayoría de los aislados presentó una elevada resistencia a los antimicrobianos evaluados: ampicilina 83 por ciento, cefalotina 82 por ciento, ceftriaxona 82 por ciento, ciprofloxacina 81 por ciento, gentamicina 81 por ciento y cotrimoxazol 82 por ciento. De las 88 cepas, 75 por ciento presentó integrones, siendo más común la clase 2. Los resultados del análisis de cluster no revelaron una clara relación entre la presencia de éstos y el perfil de resistencia para los antimicrobianos ensayados. Con la información disponible no fue posible relacionar la presencia de integrones con un determinado patrón de resistencia. Los patrones de bandas obtenidos con la técnica de ERIC-PCR revelaron una gran variedad genética entre las cepas analizadas, definiendo...

The antimicrobial resistance is coded in genetic elements which generate a horizontal flow of information, particularly in conditions that are under strong selective pressure like the nosocomial environment. In that sense, in the last decades, gram negative bacilli have become important agents of nosocomial infection. In order to investigate the presence of integrons among clinical isolates of gram negative bacilli and their relationship with their resistance profile, we studied 88 strains isolated from clinical specimens of different wards of the Hospital Torres Galdames, during the June-December period, 2004. They were identified according to biochemical tests. The antimicrobial susceptibility was evaluated by agar diffusion method. The integron presence was investigated by polymerase chain reaction (PCR). A cluster analysis was carried out to study the relationship between the presence of integrons and the resistance profile. The genotyping of the isolates was carried out by ERIC-PCR technique. Results: Of the isolated strains, 18 percent corresponded to Proteus mirabilis, 17 percent to Escherichia coli, and 32 percent to Non Fermentative Gram Negative bacilli. Most isolates presented high resistance to the antibiotics studied: 83 percent to ampicillin, 85 percent to cephalotin, 82 percent to ceftriaxone, 82 percent to ciprofloxacin, 81 percent to gentamycin and 82 percent to cotrimoxazole. Seventy-five percent of the 88 strains presented integrons. Class 2 integrons were found to be the most common. The results of the cluster analysis did not show a clear relationship among the presence of the integrons and the resistance profile. With the available information it is not possible to relate the integron presence with a certain resistance pattern. The patterns of bands obtained with the technique ERIC-PCR revealed a great genetic variety among the analyzed isolations, defining diverse genotypes, distributed in the different services...

Adsorption of protein GlnB of Herbaspirillum seropedicae on Si(111) investigated by AFM and XPS.

The protein GlnB-Hs (GlnB of Herbaspirillum seropedicae) in diazotroph micro-organisms signalizes levels of nitrogen, carbon, and energy for a series of proteins involved in the regulation of expression and control of the activity of nitrogenase complex that converts atmospheric nitrogen in ammonia, resulting in biological nitrogen fixation. Its structure has already been determined by X-ray diffraction, revealing a trimer of (36 kDa) with lateral cavities having hydrophilic boundaries. The interactions of GlnB-Hs with the well-known Si(111) surface were investigated for different incubation times, protein concentrations in initial solution, deposition conditions, and substrate initial state. The protein solution was deposited on Si(111) and dried under controlled conditions. An atomic force microscope operating in dynamic mode shows images of circular, linear, and more complex donut-shaped protein arrangement, and also filament types of organization, which vary from a few nanometers to micrometers. Apparently, the filament formation was favored because of protein surface polarity when in contact with the silicon surface, following some specific orientation. The spin-coating technique was successfully used to obtain more uniform surface covering.