Software for identifying bacteria - PIBWin

The PIBWin programme provides probabilistic identification of unknown bacterial isolates against identification matrices of known strains. Bryant TN. PIBWin - software for probabilistic identification. Journal of Applied Microbiololgy. 2004;97(6):1326-7.

Variación del estado de portador de Staphylococcus aureus en una población de estudiantes de medicina

Para evaluar en estudiantes de medicina la variación del estado de portador de Staphylococcus aureus y su resistencia antimicrobiana, antes y después de la práctica clínica, se realizó un estudio longitudinal en una cohorte de 159 estudiantes de cuarto y noveno semestre universitario. Se tomaron muestras de las zonas periamigdalianas y/o pared posterior de orofaringe, de las fosas nasales y las manos, se cultivaron en agar sangre de cordero al 5% y se incubaron en aerobiosis a 37°C, durante 48 horas. La identificación de Staphylococcus aureus se realizó según las características macroscópicas y pruebas bioquímicas. La susceptibilidad a los antimicrobianos se evaluó mediante el método de difusión de disco, por la técnica de Kirby-Bauer, siguiendo las normas internacionales del Clinical and Laboratory Standards Institute (CLSI), con los siguientes antimicrobianos: ciprofloxacina, vancomicina, oxacilina, cefalotina,clindamicina y rifampicina. La edad promedio de los alumnos de cuarto semestre fue 19,1±1,2 años y el género femenino fue 2/1 más frecuente que el masculino. Se analizaron la presencia de antecedentes como: infecciones, alergias, estado de fumador, otras patologías no infecciosas, uso de antibióticos en los últimos tres meses y procedimientos quirúrgicos u hospitalizaciones seis meses previos a la toma de las muestras. No hubo relación significativa entre la incidencia del estado de portador y los antecedentes estudiados. Se observó un aumento significativo del 15,1%, con respecto al grupo de estudiantes de cuarto semestre, en el estado de portador de S. aureus en el grupo de estudiantes de noveno semestre, después de haber estado expuestos durante tres años al ambiente hospitalario, (p=0,001 Test exacto de Mc Nemar). De los portadores, el 16,4% presentó la bacteria en manos (p<0,001), el 13,8% en fosas nasales (p=0,0015) y el 3,2% en faringe. Por otra parte,el 35,8% de los portadores presentó persistencia, de los cuales el 25,2% fue en fosas nasales; el 4,4%, en faringe y el 3,8% en manos. En cuanto a la resistencia a los antimicrobianos, el 1,9% de las cepas aisladas de los estudiantes de cuarto semestre presentó resistencia: una a ciprofloxacina y dos a clindamicina (tres estudiantes). Por su parte, el 2,5% de las cepas aisladas de estudiantes de noveno semestre fue resistente: una a cefalotina, ciprofloxacina, oxacilina y clindamicina, una a cefalotina y oxacilina y dos a clindamicina (cuatro estudiantes). En el 1,3% del grupo estudiado se aislaron cepas de Staphylococcus aureus Resistentes a la Meticilina (MRSA, por sus siglas en inglés). Estos resultados no muestran diferencias significativas (p=1.000). 

Aerobic en silla de ruedas : propuesta de adaptación en el ámbito escolar.

Se propone una adaptación del aerobic aplicable al entorno escolar habitual, con el objetivo de trabajar a nivel motor y actitudinal. Se comentan aspectos específicos a tener en cuenta en el aeróbic en silla de ruedas, unidades didácticas y tablas de ejercicios.

The role of fungi and bacteria on the organic matter decomposition process in streams: interaction and relevance in biofilms

L'objectiu d'aquest estudi és el d'investigar sobre l'ús de matèria orgànica per part dels fongs i bacteris que colonitzen diferents substrats bentònics en rius Mediterranis i analitzar l'efecte dels factors ambientals i antròpics sobre l'estabilitat estructural i funcional de les comunitats del biofilm. La metodologia emprada en aquest estudi consisteix en: i) anàlisi de la biomassa bacteriana i fúngica, ii) anàlisi de la composició de les comunitats bentòniques (identificació d'hifomicets aquàtics i anàlisi del 16S rDNA bacterià), i iii) anàlisi de l'activitat enzimàtica extracel·lular relacionada amb el reciclatge de matèria orgànica en rius.

Lactic acid bacteria as bioprotective agents against foodborne pathogens and spoilage microorganisms in fresh fruits and vegetables

La present tesi doctoral es centra en l'aplicació dels bacteris de l'àcid lactic (BAL) com a agents bioprotectors davant microorganismes patògens i deteriorants.Es van aïllar i seleccionar BAL de fruites i hortalisses fresques i es van assajar in vitro davant 5 microorganismes fitopatògens i 5 patògens humans.Es van realitzar assajos d'eficàcia en pomes Golden Delicious amb tots els aïllats enfront les infeccions causades pel fong Penicillium expansum. La soca més eficaç era Weissella cibaria TM128, que reduïa el diàmetre de les infeccions en un 50%.Les soques seleccionades es van assajar enfront els patògens Salmonella typhimurium, Escherichia coli i Listeria monocytogenes en enciams Iceberg i pomes Golden Delicious.Els BAL interferien eficientment amb el creixemet de S. typhimurium, and L. monocytogenes, però van mostrar poc efecte enfront E. coli.Finalment, es van realitzar assajos dosi-resposta amb les soques Leuconostoc mesenteroides CM135, CM160 and PM249 enfront L. monocytogenes. De totes les soques assajades, la soca CM160 va ser la més efectiva.

Nitrosospira spp. can produce nitrous oxide via a nitrifier denitrification pathway

Nitrous oxide (N2O) emission from soils is a major contributor to the atmospheric loading of this potent greenhouse gas. It is thought that autotrophic ammonia oxidizing bacteria (AOB) are a significant source of soil-derived N2O and a denitrification pathway (i.e. reduction of NO2- to NO and N2O), so-called nitrifier denitrification, has been demonstrated as a N2O production mechanism in Nitrosomonas europaea. It is thought that Nitrosospira spp. are the dominant AOB in soil, but little information is available on their ability to produce N2O or on the existence of a nitrifier denitrification pathway in this lineage. This study aims to characterize N2O production and nitrifier denitrification in seven strains of AOB representative of clusters 0, 2 and 3 in the cultured Nitrosospira lineage. Nitrosomonas europaea ATCC 19718 and ATCC 25978 were analysed for comparison. The aerobically incubated test strains produced significant (P < 0.001) amounts of N2O and total N2O production rates ranged from 2.0 amol cell(-1) h(-1), in Nitrosospira tenuis strain NV12, to 58.0 amol cell(-1) h(-1), in N. europaea ATCC 19718. Nitrosomonas europaea ATCC 19718 was atypical in that it produced four times more N2O than the next highest producing strain. All AOB tested were able to carry out nitrifier denitrification under aerobic conditions, as determined by production of N-15-N2O from applied N-15-NO2-. Up to 13.5% of the N2O produced was derived from the exogenously applied N-15-NO2-. The results suggest that nitrifier denitrification could be a universal trait in the betaproteobacterial AOB and its potential ecological significance is discussed.

Soft topographic map for clustering and classification of bacteria

In this work a new method for clustering and building a topographic representation of a bacteria taxonomy is presented. The method is based on the analysis of stable parts of the genome, the so-called “housekeeping genes”. The proposed method generates topographic maps of the bacteria taxonomy, where relations among different type strains can be visually inspected and verified. Two well known DNA alignement algorithms are applied to the genomic sequences. Topographic maps are optimized to represent the similarity among the sequences according to their evolutionary distances. The experimental analysis is carried out on 147 type strains of the Gammaprotebacteria class by means of the 16S rRNA housekeeping gene. Complete sequences of the gene have been retrieved from the NCBI public database. In the experimental tests the maps show clusters of homologous type strains and present some singular cases potentially due to incorrect classification or erroneous annotations in the database.

Iron Uptake and Homeostasis in Microorganisms

Preface. Iron is considered to be a minor element employed, in a variety of forms, by nearly all living organisms. In some cases, it is utilised in large quantities, for instance for the formation of magnetosomes within magnetotactic bacteria or during use of iron as a respiratory donor or acceptor by iron oxidising or reducing bacteria. However, in most cases the role of iron is restricted to its use as a cofactor or prosthetic group assisting the biological activity of many different types of protein. The key metabolic processes that are dependent on iron as a cofactor are numerous; they include respiration, light harvesting, nitrogen fixation, the Krebs cycle, redox stress resistance, amino acid synthesis and oxygen transport. Indeed, it is clear that Life in its current form would be impossible in the absence of iron. One of the main reasons for the reliance of Life upon this metal is the ability of iron to exist in multiple redox states, in particular the relatively stable ferrous (Fe2+) and ferric (Fe3+) forms. The availability of these stable oxidation states allows iron to engage in redox reactions over a wide range of midpoint potentials, depending on the coordination environment, making it an extremely adaptable mediator of electron exchange processes. Iron is also one of the most common elements within the Earth’s crust (5% abundance) and thus is considered to have been readily available when Life evolved on our early, anaerobic planet. However, as oxygen accumulated (the ‘Great oxidation event’) within the atmosphere some 2.4 billion years ago, and as the oceans became less acidic, the iron within primordial oceans was converted from its soluble reduced form to its weakly-soluble oxidised ferric form, which precipitated (~1.8 billion years ago) to form the ‘banded iron formations’ (BIFs) observed today in Precambrian sedimentary rocks around the world. These BIFs provide a geological record marking a transition point away from the ancient anaerobic world towards modern aerobic Earth. They also indicate a period over which the bio-availability of iron shifted from abundance to limitation, a condition that extends to the modern day. Thus, it is considered likely that the vast majority of extant organisms face the common problem of securing sufficient iron from their environment – a problem that Life on Earth has had to cope with for some 2 billion years. This struggle for iron is exemplified by the competition for this metal amongst co-habiting microorganisms who resort to stealing (pirating) each others iron supplies! The reliance of micro-organisms upon iron can be disadvantageous to them, and to our innate immune system it represents a chink in the microbial armour, offering an opportunity that can be exploited to ward off pathogenic invaders. In order to infect body tissues and cause disease, pathogens must secure all their iron from the host. To fight such infections, the host specifically withdraws available iron through the action of various iron depleting processes (e.g. the release of lactoferrin and lipocalin-2) – this represents an important strategy in our defence against disease. However, pathogens are frequently able to deploy iron acquisition systems that target host iron sources such as transferrin, lactoferrin and hemoproteins, and thus counteract the iron-withdrawal approaches of the host. Inactivation of such host-targeting iron-uptake systems often attenuates the pathogenicity of the invading microbe, illustrating the importance of ‘the battle for iron’ in the infection process. The role of iron sequestration systems in facilitating microbial infections has been a major driving force in research aimed at unravelling the complexities of microbial iron transport processes. But also, the intricacy of such systems offers a challenge that stimulates the curiosity. One such challenge is to understand how balanced levels of free iron within the cytosol are achieved in a way that avoids toxicity whilst providing sufficient levels for metabolic purposes – this is a requirement that all organisms have to meet. Although the systems involved in achieving this balance can be highly variable amongst different microorganisms, the overall strategy is common. On a coarse level, the homeostatic control of cellular iron is maintained through strict control of the uptake, storage and utilisation of available iron, and is co-ordinated by integrated iron-regulatory networks. However, much yet remains to be discovered concerning the fine details of these different iron regulatory processes. As already indicated, perhaps the most difficult task in maintaining iron homeostasis is simply the procurement of sufficient iron from external sources. The importance of this problem is demonstrated by the plethora of distinct iron transporters often found within a single bacterium, each targeting different forms (complex or redox state) of iron or a different environmental condition. Thus, microbes devote considerable cellular resource to securing iron from their surroundings, reflecting how successful acquisition of iron can be crucial in the competition for survival. The aim of this book is provide the reader with an overview of iron transport processes within a range of microorganisms and to provide an indication of how microbial iron levels are controlled. This aim is promoted through the inclusion of expert reviews on several well studied examples that illustrate the current state of play concerning our comprehension of how iron is translocated into the bacterial (or fungal) cell and how iron homeostasis is controlled within microbes. The first two chapters (1-2) consider the general properties of microbial iron-chelating compounds (known as ‘siderophores’), and the mechanisms used by bacteria to acquire haem and utilise it as an iron source. The following twelve chapters (3-14) focus on specific types of microorganism that are of key interest, covering both an array of pathogens for humans, animals and plants (e.g. species of Bordetella, Shigella, , Erwinia, Vibrio, Aeromonas, Francisella, Campylobacter and Staphylococci, and EHEC) as well as a number of prominent non-pathogens (e.g. the rhizobia, E. coli K-12, Bacteroides spp., cyanobacteria, Bacillus spp. and yeasts). The chapters relay the common themes in microbial iron uptake approaches (e.g. the use of siderophores, TonB-dependent transporters, and ABC transport systems), but also highlight many distinctions (such as use of different types iron regulator and the impact of the presence/absence of a cell wall) in the strategies employed. We hope that those both within and outside the field will find this book useful, stimulating and interesting. We intend that it will provide a source for reference that will assist relevant researchers and provide an entry point for those initiating their studies within this subject. Finally, it is important that we acknowledge and thank wholeheartedly the many contributors who have provided the 14 excellent chapters from which this book is composed. Without their considerable efforts, this book, and the understanding that it relays, would not have been possible. Simon C Andrews and Pierre Cornelis

Optimal protection of stabilised dry live bacteria from bile toxicity in oral dosage forms by bile acid adsorbent resins

We previously found that dried live bacteria of a vaccine strain can be temporarily sensitive to bile acids and suggested that Bile Adsorbing Resins (BAR) can be used in oral vaccine tablets to protect dried bacteria from intestinal bile. Here, we report a quantitative analysis of the ability of BAR to exclude the dye bromophenol blue from penetrating into matrix tablets and also sections of hard capsule shells. Based on this quantitative analysis, we made a fully optimised formulation, comprising 25% w/w of cholestyramine in Vcaps™ HPMC capsules. This gave effectively 100% protection of viability from 4% bile, with 4200-fold more live bacteria recovered from this formulation compared to unprotected dry bacteria. From the image analysis, we found that the filler material or compaction force used had no measurable effect on dye exclusion but did affect the rate of tablet hydration. Increasing the mass fraction of BAR gave more exclusion of dye up to 25% w/w, after which a plateau was reached and no further dye exclusion was seen. More effective dye exclusion was seen with smaller particle sizes (i.e. cholestyramine) and when the BAR was thoroughly dried and disaggregated. Similar results were found when imaging dye penetration into capsule sections or tablets. The predictions of the dye penetration study were tested using capsules filled with dried attenuated Salmonella vaccine plus different BAR types, and the expected protection from bile was found, validating the imaging study. Surprisingly, depending on the capsule shell material, some protection was given by the capsule alone without adding BAR, with Vcaps™ HPMC capsules providing up to 174-fold protection against 1% bile; faster releasing Vcaps Plus™ HPMC capsules and Coni Snap™ gelatin capsules gave less protection.

Protection of live bacteria from bile acid toxicity using bile acid adsorbing resins

We previously demonstrated that a dry, room temperature stable formulation of a live bacterial vaccine was highly susceptible to bile, and suggested that this will lead to significant loss of viability of any live bacterial formulation released into the intestine using an enteric coating or capsule. We found that bile and acid tolerance is very rapidly recovered after rehydration with buffer or water, raising the possibility that rehydration in the absence of bile prior to release into the intestine might solve the problem of bile toxicity to dried cells. We describe here a novel formulation that combines extensively studied bile acid adsorbent resins with the dried bacteria, to temporarily adsorb bile acids and allow rehydration and recovery of bile resistance of bacteria in the intestine before release. Tablets containing the bile acid adsorbent cholestyramine release 250-fold more live bacteria when dissolved in a bile solution, compared to control tablets without cholestyramine or with a control resin that does not bind bile acids. We propose that a simple enteric coated oral dosage form containing bile acid adsorbent resins will allow improved live bacterial delivery to the intestine via the oral route, a major step towards room temperature stable, easily administered and distributed vaccine pills and other bacterial therapeutics