Nickel-organo compounds as potential enzyme precursors under simulated early Earth conditions.

The transition from inorganic catalysis through minerals to organic catalysis by enzymes is a necessary step in the emergence of life. Our work is elucidating likely reactions at the earliest moments of Life, prior to the existence of enzymatic catalysis, by exploring essential intersections between nickel bioinorganic chemistry and pterin biochemistry. We used a prebiotically-inspired acetylene-containing volcanic hydrothermal experimental environment to shed light on the efficient formation of nickel-organo complexes. The simplest bis(dithiolene)nickel complex (C2H2S2)2Ni was identified by UV/Vis spectroscopy, mass spectrometry, nuclear magnetic resonance. Its temporal progression and possible function in this simulated early Earth atmosphere were investigated by isolating the main bis(dithiolene)nickel species from the primordial experimental setup. Using this approach, we uncovered a significant diversity of nickel-organo compositions by identifying 156 elemental annotations. The formation of acetaldehyde through the subsequent degradation of these organo-metal complexes is intriguing, as it is reminiscent of the ability of Pelobacter acetylenicus to hydrate acetylene to acetaldehyde via its bis(dithiolene)-containing enzyme acetylene hydratase. As our findings mechanistically characterize the role of nickel sulfide in catalyzing the formation of acetaldehyde, this fundamental pre-metabolic reaction could play the role of a primitive enzyme precursor of the enzymatic acetylene metabolism and further strengthen the role of acetylene in the molecular origin of life.

C2-addition patterns emerging from acetylene and nickel sulfide in simulated prebiotic hydrothermal conditions.

Chemical complexity is vital not only for the origin of life but also for biological evolution. The chemical evolution of a complex prebiotic mixture containing acetylene, carbon monoxide (CO), and nickel sulfide (NiS) has been analyzed with mass spectrometry as an untargeted approach to reaction monitoring. Here we show through isotopic 13C-labelling, multiple reaction products, encompassing diverse CHO and CHOS compounds within the complex reaction mixture. Molecules within the same chemical spaces displayed varying degrees of 13C-labelling, enabling more robust functional group characterization based on targeted investigations and differences in saturation levels among the described classes. A characteristic C2-addition pattern was detected in all compound classes in conjunction with a high diversity of thio acids, reminiscent of extant microbial C2-metabolism. The analysis involved a time-resolved molecular network, which unveiled the behavior of sulfur in the system. At the onset of the reaction, early formed compounds contain more sulfur atoms compared to later emerging compounds. These results give an essential insight into the still elusive role of sulfur dynamics in the origin of life. Moreover, our results provide temporally resolved evidence of the progressively increasing molecular complexity arising from a limited number of compounds.

Changes in adenoviral chromatin organization precede early gene activation upon infection.

Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome packaging changes during infection and subsequent transcriptional activation is currently unclear. Here, we combined RNA-seq, MNase-seq, ChIP-seq, and single genome imaging during early adenovirus infection to unveil the structure- and time-resolved dynamics of viral chromatin changes as well as their correlation with gene transcription. Our MNase mapping data indicates that the adenoviral genome is arranged in precisely positioned nucleoprotein particles with nucleosome-like characteristics, that we term adenosomes. We identified 238 adenosomes that are positioned by a DNA sequence code and protect about 60-70 bp of DNA. The incoming adenoviral genome is more accessible at early gene loci that undergo additional chromatin de-condensation upon infection. Histone H3.3 containing nucleosomes specifically replaces pVII at distinct genomic sites and at the transcription start sites of early genes. Acetylation of H3.3 is predominant at the transcription start sites and precedes transcriptional activation. Based on our results, we propose a central role for the viral pVII nucleoprotein architecture, which is required for the dynamic structural changes during early infection, including the regulation of nucleosome assembly prior to transcription initiation. Our study thus may aid the rational development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy.

Biotechnological potential and initial characterization of two novel sesquiterpene synthases from Basidiomycota Coniophora puteana for heterologous production of δ-cadinol.

BACKGROUND: Terpene synthases are versatile catalysts in all domains of life, catalyzing the formation of an enormous variety of different terpenoid secondary metabolites. Due to their diverse bioactive properties, terpenoids are of great interest as innovative ingredients in pharmaceutical and cosmetic applications. Recent advances in genome sequencing have led to the discovery of numerous terpene synthases, in particular in Basidiomycota like the wood rotting fungus Coniophora puteana, which further enhances the scope for the manufacture of terpenes for industrial purposes. RESULTS: In this study we describe the identification of two novel (+)-δ-cadinol synthases from C. puteana, Copu5 and Copu9. The sesquiterpene (+)-δ-cadinol was previously shown to exhibit cytotoxic activity therefore having an application as possible, new, and sustainably sourced anti-tumor agent. In an Escherichia coli strain, optimized for sesquiterpene production, titers of 225 mg l-1 and 395 mg l-1, respectively, could be achieved. Remarkably, both enzymes share the same product profile thereby representing the first two terpene synthases from Basidiomycota with identical product profiles. We solved the crystal structure of Copu9 in its closed conformation, for the first time providing molecular details of sesquiterpene synthase from Basidiomycota. Based on the Copu9 structure, we conducted structure-based mutagenesis of amino acid residues lining the active site, thereby altering the product profile. Interestingly, the mutagenesis study also revealed that despite the conserved product profiles of Copu5 and Copu9 different conformational changes may accompany the catalytic cycle of the two enzymes. This observation suggests that the involvement of tertiary structure elements in the reaction mechanism(s) employed by terpene synthases may be more complex than commonly expected. CONCLUSION: The presented product selectivity and titers of Copu5 and Copu9 may pave the way towards a sustainable, biotechnological production of the potentially new bioactive (+)-δ-cadinol. Furthermore, Copu5 and Copu9 may serve as model systems for further mechanistic studies of terpenoid catalysis.

Characterization of Sunflower Oil Extracts from the Lichen Usnea barbata.

The increasing global emergence of multidrug resistant (MDR) pathogens is categorized as one of the most important health problems. Therefore, the discovery of novel antimicrobials is of the utmost importance. Lichens provide a rich source of natural products including unique polyketides and polyphenols. Many of them display pharmaceutical benefits. The aim of this study was directed towards the characterization of sunflower oil extracts from the fruticose lichen, Usnea barbata. The concentration of the major polyketide, usnic acid, was 1.6 mg/mL extract as determined by NMR analysis of the crude mixture corresponding to 80 mg per g of the dried lichen. The total phenolics and flavonoids were determined by photometric assays as 4.4 mg/mL (gallic acid equivalent) and 0.27 mg/mL (rutin equivalent) corresponding to 220 mg/g and 13.7 mg/g lichen, respectively. Gram-positive (e.g., Enterococcus faecalis) and Gram-negative bacteria, as well as clinical isolates of infected chickens were sensitive against these extracts as determined by agar diffusion tests. Most of these activities increased in the presence of zinc salts. The data suggest the potential usage of U. barbata extracts as natural additives and mild antibiotics in animal husbandry, especially against enterococcosis in poultry.

Reprogramming of host glutamine metabolism during Chlamydia trachomatis infection and its key role in peptidoglycan synthesis.

Obligate intracellular bacteria such as Chlamydia trachomatis undergo a complex developmental cycle between infectious, non-replicative elementary-body and non-infectious, replicative reticulate-body forms. Elementary bodies transform to reticulate bodies shortly after entering a host cell, a crucial process in infection, initiating chlamydial replication. As Chlamydia fail to replicate outside the host cell, it is unknown how the replicative part of the developmental cycle is initiated. Here we show, using a cell-free approach in axenic media, that the uptake of glutamine by the bacteria is crucial for peptidoglycan synthesis, which has a role in Chlamydia replication. The increased requirement for glutamine in infected cells is satisfied by reprogramming the glutamine metabolism in a c-Myc-dependent manner. Glutamine is effectively taken up by the glutamine transporter SLC1A5 and metabolized via glutaminase. Interference with this metabolic reprogramming limits the growth of Chlamydia. Intriguingly, Chlamydia failed to produce progeny in SLC1A5-knockout organoids and mice. Thus, we report on the central role of glutamine for the development of an obligate intracellular pathogenic bacterium and the reprogramming of host glutamine metabolism, which may provide a basis for innovative anti-infection strategies.

Prevalencia de patógenos causantes de enfermedad diarreica aguda en el área Metropolitana de Asunción y Central

Introducción: Las enfermedades diarreicas agudas (EDA) constituyen un problema de salud pública y son una de las causas más importantes de mortalidad y morbilidad en niños a nivel mundial. Objetivo: Determinar la prevalencia de enteropatógenos causantes de EDA en el área metropolitana de Asunción y Central. Materiales y métodos: Se realizó un estudio observacional, descriptivo de corte transverso. Se analizaron 743 muestras de heces diarreicas, en las cuales se investigó la presencia de Salmonella spp., Shigella spp., Campylobacter spp., Escherichia coli diarreigénicas y Rotavirus, utilizando técnicas de referencia. Resultados: En el 31,2% (232/743) de las muestras fue posible identificar al menos uno de los patógenos entéricos investigados, siendo las E. coli diarreigénicas fueron las bacterias identificadas con mayor frecuencia, seguido por Rotavirus, Campylobacter spp., Shigella spp. y en último lugar, Salmonella spp. Conclusión: La población más afectada corresponde a niños menores de 5 años. El principal patógeno identificado como agente causal de diarreas fueron las E. coli diarreigénicas en primer lugar, seguido por Rotavirus, Campylobacter spp., Shigella spp. y Salmonella spp. En algunas muestras se detectaron más de un patógeno entérico, encontrando incluso casos de coinfección con hasta cuatro patógenos diferentes.

Introduction: Acute diarrheal diseases (ADD) constitute a public health problem and are one of the most important causes of mortality and morbidity in children worldwide. Objective: To determine the prevalence of enteropathogens causing ADD in the metropolitan area of ​​Asunción and Central. Materials and methods: An observational, descriptive cross-sectional study was conducted. 743 samples of diarrheic feces were analyzed, in which the presence of Salmonella spp., Shigella spp., Campylobacter spp., Escherichia coli diarreigenic and Rotavirus was investigated, using reference techniques. Results: In 31.2% (232/743) of the samples it was possible to identify at least one of the enteric pathogens investigated, being the diarrhenetic E. coli were the most frequently identified bacteria, followed by Rotavirus, Campylobacter spp., Shigella spp. and lastly, Salmonella spp. Conclusion: The most affected population corresponds to children under 5 years of age. The main pathogen identified as the causative agent of diarrhea was diarrigenic E. coli, followed by Rotavirus, Campylobacter spp., Shigella spp. and Salmonella spp. In some samples more than one enteric pathogen was detected, even finding cases of coinfection with up to four different pathogens.

The enzymes OSC1 and CYP716A263 produce a high variety of triterpenoids in the latex of Taraxacum koksaghyz.

Only very little is known about the resin composition of natural rubber from the dandelion species Taraxacum koksaghyz, thus its full characterization could provide new insights into how the isoprenoid end-products influence the physical properties of natural rubber, and this resin might be a good source of highly diverse triterpenoids. Here, we present a comprehensive analysis of the triterpenoid composition in an acetone extract and identified 13 triterpenes and triterpenoids also including the so far unknown pentacyclic compounds lup-19(21)-en-3-ol (1) and its ketone lup-19(21)-en-3-one (2). We purified single triterpenes from the acetone extract by developing a two-step HPLC system that is adapted to the structural differences of the described triterpenoids. Furthermore, we isolated six different oxidosqualene cyclases (OSCs) and two P450 enzymes, and we functionally characterized TkOSC1 and CYP716A263 in Nicotiana benthamiana and Saccharomyces cerevisiae in detail. TkOSC1 is a multifunctional OSC that was capable of synthesizing at least four of the latex-predominant pentacyclic triterpenes (taraxasterol, α-, ß-amyrin and lup-19(21)-en-3-ol) while CYP716A263 oxidized pentacyclic triterpenes at the C-3 position. The identified enzymes responsible for biosynthesis and modification of pentacyclic triterpenes in T. koksaghyz latex may represent excellent tools for bioengineering approaches to produce pentacyclic triterpenes heterologously.

Perfiles genéticos bacterianos y análisis de brotes de las Enfermedades Transmitidas por Alimentos empleando Electroforesis de Campo Pulsado como herramienta para la vigilancia epidemiológica molecular / Bacterial genetic profiles and analysis of outbreaks of foodborne diseases using pulsed field gel electrophoresis as a tool for molecular epidemiological surveillance

Las Enfermedades de Transmisión Alimentaria (ETA) son un problema de salud pública con altos índices de morbilidad y mortalidad a nivel global. La vigilancia y estudio de brotes de las ETA a través de Electroforesis de Campo Pulsado (PFGE) constituye un soporte fundamental para la investigación epidemiológica. El objetivo del estudio es presentar la base de datos de perfiles genéticos bacterianos y analizar brotes de enfermedades transmitidas por alimentos empleando Electroforesis de Campo Pulsado. Estudio descriptivo observacional de carácter retrospectivo, muestreo por conveniencia en el que fueron estudiados 778 aislamientos bacterianos causantes de ETA. La Base de Datos Nacional (BDN) quedó conformada por los siguientes patógenos entéricos; Salmonella spp., Shigella sonnei, Vibrio cholerae, Campylobacter spp., Escherichia coli O157:H7 y Escherichia coli no O157 caracterizados por una diversidad de patrones únicos, clusters y brotes. La BDN de Salmonella spp., quedó representada por un total de 558 cepas con 248 PUN, de las cuales 22,6% (126 cepas) corresponden a Salmonella enterica ser. Typhimurium, 20,6% (115 cepas) a Salmonella enterica ser. Enteritidis, 9,1% (51 cepas) a Salmonella enterica ser. Newport, 1,6% (9 cepas) a Salmonella enterica ser. Muenchen, que al mismo tiempo son los serotipos que están asociados a brotes. Fueron confirmados un total de 13 brotes causados por Salmonella spp.; Shigella sonnei con 113 cepas estudiadas, 57 patrones únicos y 19 clusters detectados. Se identificaron 3 patrones PYJ16X01.0012, PYJ16X01.0034 y PYJ16X01.0014 como los predominantes. Vibrio cholerae con 18 cepas estudiadas, 9 patrones únicos y 4 clusters detectados. Se pudo establecer una relación genética del 100% entre cepas de Vibrio cholerae O1 biotipo El Tor serotipo Ogawa productora de toxinas ctxA y tcpA aislada del caso índice del brote de cólera. Campylobacter spp., con 62 cepas estudiadas, 42 patrones únicos y 10 clusters detectados. La BDN de E. coli productor de toxina shiga O157 y no O157, con 9 y 20 cepas de origen humano respectivamente, caracterizadas según sus factores de virulencia y subtipos. Se reconocieron 8 patrones electroforéticos PUN y 1 cluster para E. coli productor de toxina shiga O157, y 18 PUN y 1 clúster para E. coli productor de toxina shiga no O157.La disponibilidad de una Base de Datos Nacional de patógenos bacterianos transmitidos por alimentos constituye un importante avance para la salud pública, con un gran aporte en la vigilancia y epidemiología del país permitiendo la confirmación y detección de brotes discriminando aislamientos relacionados genéticamente y por consiguiente el estudio de relaciones clonales y probable origen(AU)

Foodborne diseases (FBD) are a problem of public health with high indexes of morbidity and mortality at global level. The surveillance and study of outbreaks of the FBD through pulsed field gel electrophoresis (PFGE) is a fundamental support for epidemiological research. The aim of the study is to present the database of bacterial genetic profiles and analyze outbreaks of FBD using PFGE. This was an observational descriptive retrospective study with convenience sampling in which 778 bacterial isolates causing FBD were studied. The National Database (NDB) was made up of the following enteric pathogens causing FBD: Salmonella spp., Shigella sonnei, Vibrio cholerae, Campylobacter spp., Escherichia coli O157: H7 and Escherichia coli no O157. Each of them was characterized by a diversity of unique patterns, clusters and outbreaks. The NDB of Salmonella spp. was represented by a total of 558 strains with 248 PUN, of which 22.6% (126 strains) correspond to Salmonella enterica ser. Typhimurium, 20.6% (115 strains) to Salmonella enterica ser. Enteritidis, 9.1% (51 strains) to Salmonella enterica ser. Newport, 1.6% (9 strains) to Salmonella enterica ser. Muenchen, which at the same time are the serotypes associated with outbreaks. A total of thirteen outbreaks caused by Salmonella spp., Shigella sonnei with 113 strains studied, 57 unique patterns and 19 clusters detected were confirmed. Three patterns PYJ16X01.0012, PYJ16X01.0034 and PYJ16X01.0014 were identified as the predominant. Vibrio cholerae with 18 strains studied, 9 unique patterns and 4 clusters were detected. A genetic relationship of 100% was established between strains of Vibrio cholerae O1 biotype El Tor serotype Ogawa toxin producer ctxA and tcpA isolated from the index case of the cholera outbreak. Campylobacter spp., with 62 strains studied, 42 unique patterns and 10 clusters were detected. The NDB of O157 and non-O157 Shiga toxin producing E. coli O157, with 9 and 20 strains of human origin respectively, were characterized according to their virulence factors and subtypes. We recognized 8 PUN electrophoretic patterns and 1 cluster for O157 Shiga toxin producing E. coli, and 18 PUN and 1 cluster for non-O157 Shiga toxin producing E. coli. The availability of a National Database of bacterial pathogens transmitted by food constitutes an important advance for public health, with a great contribution to the surveillance and epidemiology of the country allowing the confirmation and detection of outbreaks discriminating genetically related isolates and therefore, the study of clonal relationships and probable origin(AU)

Reversibility of citrate synthase allows autotrophic growth of a thermophilic bacterium.

Biological inorganic carbon fixation proceeds through a number of fundamentally different autotrophic pathways that are defined by specific key enzymatic reactions. Detection of the enzymatic genes in (meta)genomes is widely used to estimate the contribution of individual organisms or communities to primary production. Here we show that the sulfur-reducing anaerobic deltaproteobacterium Desulfurella acetivorans is capable of both acetate oxidation and autotrophic carbon fixation, with the tricarboxylic acid cycle operating either in the oxidative or reductive direction, respectively. Under autotrophic conditions, the enzyme citrate synthase cleaves citrate adenosine triphosphate independently into acetyl coenzyme A and oxaloacetate, a reaction that has been regarded as impossible under physiological conditions. Because this overlooked, energetically efficient carbon fixation pathway lacks key enzymes, it may function unnoticed in many organisms, making bioinformatical predictions difficult, if not impossible.

CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality.

CHD3 and CHD4 (Chromodomain Helicase DNA binding protein), two highly similar representatives of the Mi-2 subfamily of SF2 helicases, are coexpressed in many cell lines and tissues and have been reported to act as the motor subunit of the NuRD complex (nucleosome remodeling and deacetylase activities). Besides CHD proteins, NuRD contains several repressors like HDAC1/2, MTA2/3 and MBD2/3, arguing for a role as a transcriptional repressor. However, the subunit composition varies among cell- and tissue types and physiological conditions. In particular, it is unclear if CHD3 and CHD4 coexist in the same NuRD complex or whether they form distinct NuRD complexes with specific functions. We mapped the CHD composition of NuRD complexes in mammalian cells and discovered that they are isoform-specific, containing either the monomeric CHD3 or CHD4 ATPase. Both types of complexes exhibit similar intranuclear mobility, interact with HP1 and rapidly accumulate at UV-induced DNA repair sites. But, CHD3 and CHD4 exhibit distinct nuclear localization patterns in unperturbed cells, revealing a subset of specific target genes. Furthermore, CHD3 and CHD4 differ in their nucleosome remodeling and positioning behaviour in vitro. The proteins form distinct CHD3- and CHD4-NuRD complexes that do not only repress, but can just as well activate gene transcription of overlapping and specific target genes.

Primeros resultados de la vigilancia integrada de la resistencia antimicrobiana de patógenos transmitidos por alimentos, campylobacter spp y salmonella spp en tres poblaciones distintas. Paraguay. 2011-2012 / First results of the comprehensive surveillance of the antimicrobial resistance of foodborne pathogens, campylobacter spp and salmonella spp in three different populations. Paraguay, 2011-2012

La infección causada por Salmonella spp. y por Campylobacter spp. son las enfermedades transmitidas por alimentos (ETA) reportadas más frecuentemente en el mundo, siendo la carne de pollo uno de los vehículos alimentarios más importantes para ambas. Se presenta los primeros resultados de la vigilancia antimicrobiana integrada de las ETA de Salmonella spp. y Campylobacter spp. en tres poblaciones. En este estudio descriptivo de corte transverso, de casos consecutivos, se recolectaron muestras de diversos orígenes de carne de pollo y distintas poblaciones para su aislamiento, caracterización y perfil de resistencia. Se observó una prevalencia de Campylobacter spp. del 13% en alimentos, 20% en muestras clínicas y 55% en heces cloacales de aves, con alta prevalencia de Campylobacter jejuni en las tres poblaciones; de Salmonella spp fue 6% en alimentos, 13% en muestras clínicas y 3% en heces cloacales de aves, con predominio del serotipo Salmonella ser. Enteritidis en las muestras clínicas y heces cloacales de aves. La resistencia a ciprofloxacina de Campylobacter spp., entre 59-81% se destacó en las tres poblaciones estudiadas. Para Salmonella spp. se observó una resistencia a nitrofurantoina del 73% en heces cloacales de aves, 55% en alimentos y 19,4% en humanos; a tetraciclina, 42% en alimentos, 5% en muestras clínicas y 9% en heces cloacales; para el ácido nalidíxico la resistencia fue del 72% en animales y 53% en muestras clínicas. Es importante fortalecer la vigilancia integrada de la resistencia antimicrobiana en estas tres poblaciones de manera a detectar en forma oportuna mecanismos de resistencia que pudieran afectar al ser humano a través de la cadena alimentaria.

Infection caused by Salmonella ssp. and Campylobacter spp. are the foodborne diseasesreported most frequently throughout the world, and chicken meat is considered one of themost important food vehicles for both. The objective was to present the first resultsobtained from the integrated antimicrobial surveillance of foodborne diseases of Salmonellaspp. and Campylobacter spp in three populations. In this descriptive cross - sectional ofconsecutive sampling, samples were collected from different sources of chicken meat and different populations for isolation, characterization and resistance profile. A prevalence of13% in food, 20% in clinical samples and 55% in cloacal feces was observed in the isolationof Campylobacter spp. with high prevalence of Campylobacter jejuni in all three populationsfollowed by 6% in food, 13% in clinical samples and 3% in birds cloacal feces of Salmonellaspp. with predominance in the isolation of the serotype Salmonella ser. Enteritidis in clinicalsample populations and birds cloacal feces. The resistance of Campylobacter spp. tociprofloxacin of 59-81%, stood out in the three populations under study, in contrast toSalmonella spp. A high resistance to nitrofurantoin of 73% was observed in poultry feces,55% in foods and 19.4% in humans. Resistance to tetracycline was found in foods (42%),5% in clinical samples and 9% in cloacal feces. A resistance of 72% was observed inanimals and 53% in clinical samples for nalidixic acid. It is important to strengthen theintegrated surveillance of antimicrobial resistance in these three populations in order totimely detect mechanisms of resistance that can affect the human being through the foodchain.

Lactate oxidation facilitates growth of Mycobacterium tuberculosis in human macrophages.

Mycobacterium tuberculosis (Mtb) uses alveolar macrophages as primary host cells during infection. In response to an infection, macrophages switch from pyruvate oxidation to reduction of pyruvate into lactate. Lactate might present an additional carbon substrate for Mtb. Here, we demonstrate that Mtb can utilize L-lactate as sole carbon source for in vitro growth. Lactate conversion is strictly dependent on one of two potential L-lactate dehydrogenases. A knock-out mutant lacking lldD2 (Rv1872c) was unable to utilize L-lactate. In contrast, the lldD1 (Rv0694) knock-out strain was not affected in growth on lactate and retained full enzymatic activity. On the basis of labelling experiments using [U-13C3]-L-lactate as a tracer the efficient uptake of lactate by Mtb and its conversion into pyruvate could be demonstrated. Moreover, carbon flux from lactate into the TCA cycle, and through gluconeogenesis was observed. Gluconeogenesis during lactate consumption depended on the phosphoenolpyruvate carboxykinase, a key enzyme for intracellular survival, showing that lactate utilization requires essential metabolic pathways. We observed that the ΔlldD2 mutant was impaired in replication in human macrophages, indicating a critical role for lactate oxidation during intracellular growth.

Detección molecular de Escherichia coli diarreogénica en pacientes pediátricos con síndrome diarreico agudo en Paraguay / Molecular detection of diarrheogenic Escherichia coli in pediatric patients with acute diarrheal syndrome in Paraguay

La Escherichia coli diarreogénica (ECD) se ha clasificado con base en criterios clínicos, epidemiológicos y moleculares en cinco grupos, cada uno con factores de virulencia específicos. El objetivo fue determinar la prevalencia de ECD en pacientes pediátricos con enfermedad diarreica aguda del Laboratorio Central de Salud Publica en el periodo 2012- 2015. Se procesaron muestras de heces con síndrome diarreico agudo, provenientes de pacientes pediátricos, en los cuales se buscó algún gen de virulencia ECD utilizando métodos convencionales de siembra y screening molecular, mediante PCR múltiple con cebadores diseñados específicamente para amplificar los genes de virulencia elt, est, eae, stx, ipaH y aggR. Del total de muestras analizadas, 13% (180/1379) de las muestras presentó algún factor de virulencia compatible con algún patotipo ECD con mayor predominio en niños de 1 a 3 años. La frecuencia de los distintos patotipos fue la siguiente: 61 (34%) ETEC, 40 (22%) EAEC, 41 (23%) EPEC, 27 (15%) EIEC, 7 (4%) STEC y 3 (2%) ETEC/EAEC, 1 (0.5%) ETEC/EAEC/EIEC. El porcentaje de E. coli diarreogénicas detectado tiene similitud con lo reportado en otros países de la región, lo que nos indica que estos patógenos son parte importante de la etiología de la enfermedad diarreica aguda infecciosa en la población infantil en nuestro país. Se debe destacar que para el diagnóstico de las diferentes categorías ECD, es necesario disponer de un procedimiento diagnóstico específico dirigido a la detección de los factores de virulencia utilizando métodos moleculares o métodos inmunológicos.

Diarrheagenic Escherichia coli (DEC) has been classified based on clinical, epidemiological and molecular criteria in five groups, each with specific virulence factors. The objective was to determine the prevalence of DEC in pediatric patients with acute diarrheal disease of the Central Laboratory of Public Health in the 2012-2015 period. A total of 1447 fecal samples of acute diarrheal syndrome from pediatric patients were processed in which a DEC virulence gene was searched using conventional screening and molecular screening methods with multiple PCR primers specifically designed to amplify virulence genes, st, lt, eae, stx, ipaH and aggR. From the total of analyzed samples, 13% (180/1379) of the samples presented some virulence factor compatible with a DEC pathogen type with greater predominance in children from 1 to 3 years. The frequency of the different pathogen types was as follows: 61 (34%) ETEC, 40 (22%) EAEC, 41 (23%) EPEC, 27 (15%) EIEC, 7 (4%) STEC and 3 (2% ETEC/EAEC, 1 (0.5%) ETEC/EAEC/EIEC. The percentage of DEC detected is similar to that reported in other countries of the region, which indicates that these pathogens are an important part of the etiology of acute infectious diarrheal disease in children in our country. It should be noted that for the diagnosis of different DEC categories, it is necessary to have a specific diagnostic procedure aimed at the detection of virulence factors using molecular methods or immunodiagnostic methods.

Metabolic adaptation of Chlamydia trachomatis to mammalian host cells.

Metabolic adaptation is a key feature for the virulence of pathogenic intracellular bacteria. Nevertheless, little is known about the pathways in adapting the bacterial metabolism to multiple carbon sources available from the host cell. To analyze the metabolic adaptation of the obligate intracellular human pathogen Chlamydia trachomatis, we labeled infected HeLa or Caco-2 cells with 13 C-marked glucose, glutamine, malate or a mix of amino acids as tracers. Comparative GC-MS-based isotopologue analysis of protein-derived amino acids from the host cell and the bacterial fraction showed that C. trachomatis efficiently imported amino acids from the host cell for protein biosynthesis. FT-ICR-MS analyses also demonstrated that label from exogenous 13 C-glucose was efficiently shuffled into chlamydial lipopolysaccharide probably via glucose 6-phosphate of the host cell. Minor fractions of bacterial Ala, Asp, and Glu were made de novo probably using dicarboxylates from the citrate cycle of the host cell. Indeed, exogenous 13 C-malate was efficiently taken up by C. trachomatis and metabolized into fumarate and succinate when the bacteria were kept in axenic medium containing the malate tracer. Together, the data indicate co-substrate usage of intracellular C. trachomatis in a stream-lined bipartite metabolism with host cell-supplied amino acids for protein biosynthesis, host cell-provided glucose 6-phosphate for cell wall biosynthesis, and, to some extent, one or more host cell-derived dicarboxylates, e.g. malate, feeding the partial TCA cycle of the bacterium. The latter flux could also support the biosynthesis of meso-2,6-diaminopimelate required for the formation of chlamydial peptidoglycan.

5-methylcytosine-sensitive variants of Thermococcus kodakaraensis DNA polymerase.

DNA methylation of cytosine in eukaryotic cells is a common epigenetic modification, which plays an important role in gene expression and thus affects various cellular processes like development and carcinogenesis. The occurrence of 5-methyl-2'-deoxycytosine (5mC) as well as the distribution pattern of this epigenetic marker were shown to be crucial for gene regulation and can serve as important biomarkers for diagnostics. DNA polymerases distinguish little, if any, between incorporation opposite C and 5mC, which is not surprising since the site of methylation is not involved in Watson-Crick recognition. Here, we describe the development of a DNA polymerase variant that incorporates the canonical 2'-deoxyguanosine 5'-monophosphate (dGMP) opposite C with higher efficiency compared to 5mC. The variant of Thermococcus kodakaraensis (KOD) exo- DNA polymerase was discovered by screening mutant libraries that were built by rational design. We discovered that an amino acid substitution at a single site that does not directly interact with the templating nucleobase, may alter the ability of the DNA polymerase in processing C in comparison to 5mC. Employing these findings in combination with a nucleotide, which is fluorescently labeled at the terminal phosphate, indicates the potential use of the mutant DNA polymerase in the detection of 5mC.

Serorreactividad al antígeno de superficie del virus de la hepatitis B en mujeres que acudieron al laboratorio central de salud pública. Asunción, Paraguay / Seroreactivity against the hepatitis B virus surface antigen in women who attended the central public health laboratory. Asunción, Paraguay

La hepatitis B es un grave problema de salud pública a nivel mundial, aproximadamente cerca de 2 billones de personas tienen evidencia serológica de infección por el virus de la hepatitis B. El objetivo de este trabajo fue describir la frecuencia de hepatitis B e identificar los factores de riesgo asociados en mujeres en edad fértil que acudieron al Laboratorio Central de Salud Pública entre diciembre de 2013 y junio de 2014. Fue un estudio observacional analítico de corte transverso que, previo consentimiento informado, analizó suero de mujeres entre 15 y 44 años con una edad promedio de 26,6 (±6,8) años. Mediante la detección del antígeno de superficie de la hepatitis B por ELISA se identificaron seis casos positivos (0,4%), indicando una endemicidad baja; cifra que ha variado según perfil socio demográfico: según edad, las de 20 y más años presentaron una frecuencia mayor en comparación a las demás (p>0,05). No se observaron diferencias significativas al evaluar la seropositividad según el estado civil, el nivel de escolaridad, la condición de gravidez, los antecedentes de transfusiones, sin embargo, la seropositividad era mayor en las portadoras de tatuajes/piercing que entre las no portadoras, lo que representaba un riesgo 6,2 veces mayor (OR:6,2 IC95%:1,3-31,3). En conclusión, la frecuencia del HBsAg en nuestra población es baja, y el factor de riesgo asociado a su detección fue la presencia de tatuajes y/o piercing.

Hepatitis B is a serious public health problem worldwide; approximately about 2 billionpeople have serologic evidence of infection with hepatitis B virus. The aim of this analyticcross-sectional study was to describe the frequency of hepatitis B and identify risk factorsin women of child bearing age who attended the Central Public Health Laboratory in theperiod 2013 to 2014. Prior informed consent, antigen detection of hepatitis B surface wasperformed by ELISA in women between 15 and 44 years with a mean age of 26.6 (±6.8)years. The identification of six serologic positive cases (0.4%) indicates low endemicity.This figure varied according to socio-demographic profile: according to age, those whowere 20 years old or older had an increased frequency compared to the others (p> 0.05). No significant differences were observed in seropositivity by marital status, level ofeducation, pregnancy, history of transfusion, while seropositivity was higher amongcarriers of tattoos/piercing than among non-carriers, which represented a 6.2 times higherrisk (OR 6.2 95% CI 1.3 to 31.3). In conclusion, the frequency of HBsAg in our populationwas low. The risk factor associated with its detection was the presence of tattoos and / or piercings.

Síndrome diarreico agudo causado por Campylobacter spp. en pacientes menores de 11 años y su resistencia antimicrobiana a las drogas de elección para tratamiento 2010-2012, Paraguay / Acute Diarrhea Syndrome from Campylobacter spp. in Patients under Age 11 Years and Resistance to Drugs of Choice in Paraguay, 2010-2012

Objetivos: Determinar la prevalencia de Campylobacter spp. en pacientes menores de 11 años con síndrome diarreico agudo e indagar la resistencia antimicrobiana con respecto a las drogas de elección para el tratamiento clínico con Ciprofloxacina, Eritromicina y Tetraciclina. Materiales y Métodos: Se realizó un estudio descriptivo, retrospectivo de corte transverso, muestreo no probabilístico de casos consecutivos, cuya muestra tiene un tamaño de 1110 con un nivel de confianza de 95%. La población estudiada fue de pacientes pediátricos menores de 11 años cuyas muestras de heces fueron remitidas por las instituciones integrantes de la Red de Enteropatógenos al Laboratorio Central de Salud Pública en el periodo 2010-2012. Resultados: De 1110 muestras de heces estudiadas se aislaron Campylobacter spp. en 176 de ellas y corresponde al 16% de prevalencia. Se observó resistencia a las quinolonas en un 49 % de las muestras estudiadas, así como una resistencia a Tetraciclina del 28% y 1% de resistencia a los macrólidos (Eritromicina). Conclusión: Se observó una prevalencia importante de Campylobacter spp. como agente etiológico de síndrome diarreico agudo y es relevante la incorporación del aislamiento de este patógeno como agente etiológico rutinario en los análisis de coprocultivos realizados. La resistencia observada a las drogas de elección utilizadas como tratamiento nos obliga a tener en cuenta el control del uso indiscriminado de antimicrobianos

Utilization of host-derived cysteine-containing peptides overcomes the restricted sulphur metabolism of Campylobacter jejuni.

The non-glycolytic food-borne pathogen Campylobacter jejuni successfully colonizes the intestine of various hosts in spite of its restricted metabolic properties. While several amino acids are known to be used by C. jejuni as energy sources, none of these have been found to be essential for growth. Here we demonstrated through phenotype microarray analysis that cysteine utilization increases the metabolic activity of C. jejuni. Furthermore, cysteine was crucial for its growth as C. jejuni was unable to synthesize it from sulphate or methionine. Our study showed that C. jejuni compensates this limited anabolic capacity by utilizing sulphide, thiosulphate, glutathione and the dipeptides γGlu-Cys, Cys-Gly and Gly-Cys as sulphur sources and cysteine precursors. A panel of C. jejuni mutants in putative peptidases and peptide transporters were generated and tested for their participation in the catabolism of the cysteine-containing peptides, and the predicted transporter protein CJJ81176_0236 was discovered to facilitate the growth with the dipeptide Cys-Gly, Ile-Arg and Ile-Trp. It was named Campylobacter peptide transporter A (CptA) and is the first representative of the oligopeptide transporter OPT family demonstrated to participate in the glutathione-derivative Cys-Gly catabolism in prokaryotes. Our study provides new insights into how host- and microbiota-derived substrates like sulphide, thiosulphate and short peptides are used by C. jejuni to compensate its restricted metabolic capacities.