Molecular and morphological data suggest a new species of big-eared bat (Vespertilionidae: Corynorhinus) endemic to northeastern Mexico.

Corynorhinus mexicanus is an insectivorous bat endemic to Mexico that inhabits the high and humid regions of the Sierra Madre Oriental (SMO), the Trans-Mexican Volcanic Belt (TMVB), and the Sierra Madre Occidental (SMOC). A previous study suggested that C. mexicanus could be a cryptic species complex due to the genetic divergence observed between specimens from the TMVB and SMOC. The present study implemented phylogenetic, population genetics, and morphological analyses to evaluate the hypothesis that C. mexicanus is a species complex. The phylogenetic analysis indicated that C. mexicanus is a polyphyletic species composed of three indirectly related lineages. The estimated divergence times for the lineages suggest that they first originated during the Pliocene, while the second and third shared a common ancestor with C. townsendii 1.55 million years ago, and diverged 600,000 years ago during the Middle Pleistocene. The population genetics analysis reveals the SMO lineage of C. mexicanus is an isolated genetic group and highly diverged from the rest of lineages (SMOC and TMVB). The morphological analyses showed variation in the skull and mandible associated with the lineages and sex of the specimens, highlighting a difference in mandible shape between the specimens of the SMO and the rest of C. mexicanus. The results of this study suggest the presence of an undescribed species of the genus Corynorhinus.

Dysbiosis by Eradication of Helicobacter pylori Infection Associated with Follicular Gastropathy and Pangastropathy.

Dysbiosis plays an important role in the development of bacterial infections in the gastric mucosa, particularly Helicobacter pylori. The international guidelines for the treatment of H. pylori infections suggest standard triple therapy (STT). Nevertheless, because of the increasing resistance rates to clarithromycin, metronidazole has been widely considered in several countries. Unfortunately, the non-justified administration of antibiotics induces dysbiosis in the target organ. We characterized the gastric microbiota of patients diagnosed with follicular gastropathy and pangastropathy attributed to H. pylori infection, before and after the administration of STT with metronidazole. Dominant relative abundances of Cutibacterium were observed in pre-treatment patients, whereas H. pylori was observed at <11%, suggesting the multifactor property of the disease. The correlation of Cutibacterium acnes and H. pylori with gastric infectious diseases was also evaluated using quantitative real-time polymerase chain reaction. The dominance of C. acnes over H. pylori was observed in gastritis, gastropathies, and non-significant histological alterations. None of the microorganisms were detected in the intestinal metaplasia. Post-treatment alterations revealed an increase in the relative abundances of Staphylococcus, Pseudomonas, and Klebsiella. Non-H. pylori gastrointestinal bacteria can be associated with the initiation and development of gastric diseases, such as pathobiont C. acnes.

A polyphasic taxonomy analysis reveals the presence of an ecotype of Rahnella contaminans associated with the gut of Dendroctonus-bark beetles.

Species belonging to the genus Rahnella are dominant members of the core gut bacteriome of Dendroctonus-bark beetles, a group of insects that includes the most destructive agents of pine forest in North and Central America, and Eurasia. From 300 isolates recovered from the gut of these beetles, 10 were selected to describe an ecotype of Rahnella contaminans. The polyphasic approach conducted with these isolates included phenotypic characteristics, fatty acid analysis, 16S rRNA gene, multilocus sequence analyses (gyrB, rpoB, infB, and atpD genes), and complete genome sequencing of two isolates, ChDrAdgB13 and JaDmexAd06, representative of the studied set. Phenotypic characterization, chemotaxonomic analysis, phylogenetic analyses of the 16S rRNA gene, and multilocus sequence analysis showed that these isolates belonged to Rahnella contaminans. The G + C content of the genome of ChDrAdgB13 (52.8%) and JaDmexAd06 (52.9%) was similar to those from other Rahnella species. The ANI between ChdrAdgB13 and JaDmexAd06 and Rahnella species including R. contaminans, varied from 84.02 to 99.18%. The phylogenomic analysis showed that both strains integrated a consistent and well-defined cluster, together with R. contaminans. A noteworthy observation is the presence of peritrichous flagella and fimbriae in the strains ChDrAdgB13 and JaDmexAd06. The in silico analysis of genes encoding the flagellar system of these strains and Rahnella species showed the presence of flag-1 primary system encoding peritrichous flagella, as well as fimbriae genes from the families type 1, α, ß and σ mainly encoding chaperone/usher fimbriae and other uncharacterized families. All this evidence indicates that isolates from the gut of Dendroctonus-bark beetles are an ecotype of R. contaminans, which is dominant and persistent in all developmental stages of these bark beetles and one of the main members of their core gut bacteriome.

A Mouthwash with Cetylpyridinium Chlorideis reducing salivary SARS-CoV-2 viral load in +COVID-19 / Un enjuague con Cloruro de Cetilpiridinio redujo la carga salival del virus SARSCoV-2 en pacientes con Covid-19

Aim: The aim of this randomised, double-blind, placebo-controlled pilot clinical trial is to evaluate the capacity of a mouthwash to reduce SARS-CoV-2 viral load in the saliva of patients with COVID-19. Methods: Twenty-three symptomatic SARS-CoV-2-positive outpatients were selected andrandomised into two groups and registered at NTC 04563689. Both groups rinsed and gargled for one minute with either distilled water (Placebo) or with 0.05% Cetylpyridinium chloride (CPC) plus 0.12% Chlorhexidine (CHX) mouthwash (PERIOAID Intensive Care). Saliva samples were collected before the use of placebo or mouthwash and after 15 minutes and 1 and 2 hours of either of the above treatment. A saliva sample was also taken five days after regular use of placebo or mouthwash twice daily. The virus was detected by qRT-PCR. Results: A great heterogeneity in the viral load values was observed at baseline in both groups for nasopharyngeal and saliva samples. Most of the patients who used the mouthwash (8/12) had a significant decrease in baseline viral load after 15 min (greater than 99% reduction). This inhibitory effect was maintained for up to two hours in 10 of the 12 patients. At five days, SARS-CoV-2 RNA was detectedin only 1 patient from the mouthwash group and in 5 from the placebo group. Conclusions: This study points out that a CPC mouthwash can reduce the viral load in saliva of COVID-positive patients. This finding may be important in transmission control of SARS-CoV-2. Nevertheless, the clinical relevance of CPC mouthwash-reduction on SARS-CoV-2 shedding in saliva requires further study.

Objetivo: El objetivo de este ensayo clínico piloto aleatorizado, doble ciego y controlado con placebo es evaluar la capacidad de un enjuague bucal para reducir la carga viral del SARS-CoV-2 en la saliva de pacientes con COVID-19. Materiales y métodos:Veintitrés pacientes ambulatorios positivos para SARS-CoV-2 sintomáticos fueron seleccionados y aleatorizados en dos grupos y registrados en el NTC 04563689. Ambos grupos se enjuagaron y hicieron gárgaras durante un minuto con agua destilada (placebo) o con cloruro decetilpiridinio al 0 ,05 % (CPC). ) más enjuague bucal con Clorhexidina (CHX) al 0,12% (PERIOAID Intensive Care). Se recolectaron muestras de saliva antes del uso de placebo o enjuague bucal y después de 15 minutos y 1 y 2 horas de cualquiera de los tratamientos anteriores. También se tomó una muestra de saliva cinco días después del uso regular de placebo o enjuague bucal dos veces al día. El virus fue detectado por qRT-PCR. Resultados:Se demostró una gran heterogeneidad en los valores de carga viral al inicio del estudio en grupos ambos para muestras de nasofaringe y saliva. La mayoría de los pacientes que usaron el enjuague bucal (8/12) tuvieron una disminución significativa en la carga viral inicial después de 15 minutos (reducción superior al 99 %). Este efecto inhibidor se mantuvo hasta dos horas en 10 de los 12 pacientes. A los cinco días, se detectó ARN del SARS-CoV-2 en solo 1 paciente del grupo de enjuague bucal y en 5 del grupo de placebo. Conclusiones:Este señala que un enjuague bucal CPC puedereducir la carga viral en saliva de pacientes COVID positivos. Este hallazgo puede ser importante en el control de la transmisión del SARS-CoV-2. Sin embargo, la relevancia clínica de la reducción del enjuague bucal con CPC en la excreción de SARS-CoV-2 en la saliva requiere más estudios.

The Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) Has Digestive Capacity to Degrade Complex Substrates: Functional Characterization and Heterologous Expression of an α-Amylase.

Dendroctonus-bark beetles are natural agents contributing to vital processes in coniferous forests, such as regeneration, succession, and material recycling, as they colonize and kill damaged, stressed, or old pine trees. These beetles spend most of their life cycle under stem and roots bark where they breed, develop, and feed on phloem. This tissue is rich in essential nutrients and complex molecules such as starch, cellulose, hemicellulose, and lignin, which apparently are not available for these beetles. We evaluated the digestive capacity of Dendroctonusrhizophagus to hydrolyze starch. Our aim was to identify α-amylases and characterize them both molecularly and biochemically. The findings showed that D. rhizophagus has an α-amylase gene (AmyDr) with a single isoform, and ORF of 1452 bp encoding a 483-amino acid protein (53.15 kDa) with a predicted signal peptide of 16 amino acids. AmyDr has a mutation in the chlorine-binding site, present in other phytophagous insects and in a marine bacterium. Docking analysis showed that AmyDr presents a higher binding affinity to amylopectin compared to amylose, and an affinity binding equally stable to calcium, chlorine, and nitrate ions. AmyDr native protein showed amylolytic activity in the head-pronotum and gut, and its recombinant protein, a polypeptide of ~53 kDa, showed conformational stability, and its activity is maintained both in the presence and absence of chlorine and nitrate ions. The AmyDr gene showed a differential expression significantly higher in the gut than the head-pronotum, indicating that starch hydrolysis occurs mainly in the midgut. An overview of the AmyDr gene expression suggests that the amylolytic activity is regulated through the developmental stages of this bark beetle and associated with starch availability in the host tree.

Using a Fragment-Based Approach to Identify Alternative Chemical Scaffolds Targeting Dihydrofolate Reductase from Mycobacterium tuberculosis.

Dihydrofolate reductase (DHFR), a key enzyme involved in folate metabolism, is a widely explored target in the treatment of cancer, immune diseases, bacteria, and protozoa infections. Although several antifolates have proved successful in the treatment of infectious diseases, they have been underexplored to combat tuberculosis, despite the essentiality of M. tuberculosis DHFR (MtDHFR). Herein, we describe an integrated fragment-based drug discovery approach to target MtDHFR that has identified hits with scaffolds not yet explored in any previous drug design campaign for this enzyme. The application of a SAR by catalog strategy of an in house library for one of the identified fragments has led to a series of molecules that bind to MtDHFR with low micromolar affinities. Crystal structures of MtDHFR in complex with compounds of this series demonstrated a novel binding mode that considerably differs from other DHFR antifolates, thus opening perspectives for the development of relevant MtDHFR inhibitors.

Feeding ecomorphologies in the fish genus Herichthys (Perciformes: Cichlidae) based on stomach content and lower pharyngeal jaw shape

Speciation is a multifactorial process with factors acting at different scales of space and time. Trophic niche segregation has promoted the diversification of cichlids fishes in lentic (lacustrine) environments, whether this is also the case in lotic (riverine) systems remains unknown. Herichthys is the genus of cichlids with the most boreal distribution in the Americas comprising 12 currently recognized species, most micro-endemic and only two with a wide distribution. In the present work, we analyzed the stomach content and lower pharyngeal jaw morphologies of the species of the genus to evaluate the possible role of feeding ecology in the diversification of the group. Trophic strategies varied widely, including omnivores, piscivores, invertivores, molluskivores, detritivores, herbivores and algivores. Low values of Pianka's index of niche overlap were found in the sympatric micro-endemic species, while in the widely distributed species the indices ranged from low to very high. The analysis of lower pharyngeal jaw morphologies allowed discriminating a shape associated with piscivorous species from other foraging groups. The results of this study suggest that trophic niche segregation is a factor that could promotes diversification within the genus Herichthys although additional studies need to be performed to fully understand the speciation process in this group of Neotropical cichlid fishes.

La especiación es un proceso con múltiples factores que actúan a diferentes escalas de espacio y tiempo. La segregación de nichos tróficos es un proceso que ha promovido la diversificación en cíclidos en entornos lacustres, pero en el caso de los ríos no está claro. Herichthys es un género de cíclidos cuya distribución es la más boreal en América, el cual comprende 12 especies actualmente reconocidas, la mayoría microendémicas y solo dos con una amplia distribución. En el presente trabajo, se analizó el contenido estomacal y las morfologías de la mandíbula faríngea inferior de las especies del género para compararlas y evaluar su posible papel en la diversificación del grupo. La dieta en dichas especies es muy variada e incluyó tanto especies que pueden ser consideradas omnívoras como especialistas. Se encontraron valores bajos del índice de solapamiento alimentario (índice de Pianka) en las especies simpátricas microendémicas, mientras que en las especies ampliamente distribuidas el índice fue muy variable. El análisis de morfometría geométrica de la mandíbula faríngea inferior permite discriminar dos formas principales, una que incluye la especie piscívora y otra que incluye a los otros grupos alimentarios. Los resultados encontrados en este estudio sugieren que la segregación de nicho trófico es un factor que promueve claramente la diversificación dentro del género Herichthys, aunque se deben realizar estudios adicionales para comprender completamente el proceso de especiación en este grupo de peces neotropicales.

Effects of biocide exposure on P. Aeruginosa, E. coli and A. Baumannii complex isolates from hospital and household environments / Efectos de la exposición a biocidas en aislados ambientales de P. Aeruginosa, E. coli y del complejo A. Baumannii hospitalarios y de la comunidad

Background: Bacterial responses to biocide exposure and its effects on survival and persistence remain to be studied in greater detail. Aim: To analyse the viability and survival of environmental isolates from household and hospital settings after biocide exposure. Methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of chlorhexidine (CHxG), benzalkonium chloride (BAC) and triclosan (TC) were determined in isolates of Pseudomonas aeruginosa, Acinetobacter baumannii complex and Escherichia coli collected from hospital and house- holds environments. Viability was monitored after exposure and removal of biocides using agar cultures and flow cytometry. Findings: P. aeruginosa isolates showed greater tolerance for all biocides tested whereas A. baumannii complex and E. coli were less tolerant. When compared with reference strains, biocide tolerance was up to 8 to 13-fold higher for TC and BAC respectively. Flow cytometry showed that biocide exposure may induce viable but non-growing states in P. aeruginosa and E. coli isolates before becoming fully replicative. Changes in the susceptibility profile in one isolate of A. baumannii complex were observed after biocide exposure. Discussion: Bacteria isolates from hospital and households were able to recover after biocide exposure at bactericidal concentrations favouring persistence and spread of biocide-tolerant strains. This study reinforces that cleaning compliance should be monitored by non-culture based tests. Novel formulations in cleaning and disinfection protocols should be revisited in hospitals harbouring P. aeruginosa and A. baumannii multidrug resistant isolates.

Introducción: El efecto de la exposición a biocidas en las poblaciones bacterianas, su viabilidad y persistencia requieren de estudios detallados. Objetivo: analizar la viabilidad y persistencia de bacterias de ambientes hospitalarios y domésticos posterior a la exposición a biocidas. Materiales y Métodos: En un estudio experimental in vitro se determinó la concentración inhibitoria mínima (CIM) y la concentración bactericida (CBM) para chlorhexidina (CHxG), cloruro de benzalconio (BAC) y triclcosan (TC) en aislados de Pseudomonas aeruginosa (10), el complejo Acinetobacter baumannii (5) y Escherichia coli (5) obtenidos de ambientes hospitalarios y domésticos. La viabilidad y susceptibilidad bacteriana después de la exposición y remoción del biocida fue evaluada por citometria de flujo y cultivo. Resultados: Independiente de su procedencia P. aeruginosa presentó mayor tolerancia a todos los biocidas. El complejo A. baumannii y E. coli fueron hasta 8 a 13 veces más tolerantes a BAC y TC que las cepas de referencia. Se observó que la exposición a biocidas altamente efectivos induce formas viables no replicativas en P. aeruginosa y E. coli. Un aislado del complejo A baumannii presentó cambios en el perfil de susceptibilidad posterior a la exposición. Discusión: Aislados tanto de ambiente hospitalario como de la comunidad pueden recuperarse después de la exposición a concentraciones bactericidas de los biocidas favoreciendo la persistencia y diseminación de bacterias no replicativas. Por lo anterior métodos alternativos al cultivo deben utilizarse en el seguimiento de protocolos de limpieza y desinfección. Los tiempos de recuperación de la viabilidad bacteriana deben tenerse en cuenta en la formulación de protocolos para erradicar y/o controlar cepas hospitalarias de P. aeruginosa o A. baumannii multirresistentes.

One-step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme.

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production.

Validación técnica de una PCR: Reacción en cadena de la polimerasa para la detección de Chlamydia trachomatis / Technical validation of a polymerase chain reaction for Chlamydia trachomatis detection

Resumen Objetivo: Estandarizar una técnica de PCR para la detección de Chlamydia trachomatis. Materiales y método: Estudio experimental en el que se optimizaron las condiciones de PCR para la detección in vitro de C. trachomatis. Se utilizó ADN de C. trachomatis VR885D y los cebadores CtP15'-TAGTAACTGCCACTTCATCA-3'y CtP2 5'- TTCCCCTTGTAATTCGTTGC-3, que amplificaron un segmento de 201 pb del plásmido clamidial. Se realizaron diluciones logarítmicas de ADN clamidial y fueron empleados para determinar la sensibilidad analítica expresada como copias de plásmidos y/o de cuerpos elementales. La especificidad analítica de la prueba se evaluó usando los cebadores CtP1 y CtP2 y ADN de microorganismos colonizadores y/o patógenos urogenitales. La variabilidad intraensayo fue evaluada sobre muestras por triplicado, mientras que la variabilidad interensayo se determinó mediante comparación de los resultados obtenidos por tres técnicos en diferentes días. Resultados: Las condiciones de PCR para la amplificación del gen de interés fueron establecidas (94°C/4 min; 40 ciclos de 94°C/1 min, 56°C/1 min. y 72°C/1.5 min; 72°C/4 min); 1.5 mM MgCl2 y 1 U/pL Taq polimerasa. La sensibilidad analítica de la PCR fue de 10-17 g de ADN, equivalentes a una copia del plásmido o menos de un cuerpo elemental de C. trachomatis. Los cebadores CtP1 y CtP2 amplificaron específicamente el ADN de C. trachomatis bajo las condiciones experimentales evaluadas. La repetitibilidad y reproducibilidad de la PCR se determinó con experimentos de variabilidad intra e interensayo respectivamente. Conclusiones: La estandarización de esta PCR es el primer paso para su utilización en el diagnóstico de infecciones por C. trachomatis. Se requieren estudios adicionales de validación clínica de ésta prueba.

Abstract Objective: To standardize a PCR for Chlamydia trachomatis detection. Materials and methods: An experimental study was designed to standardize a C. trachomatis PCR test. Genomic DNA from C. trachomatis serovar D ATCC VR885D was used for the PCR standardization. An amplicon of 201 bp from clamidial plasmid was obtained using primers CtP15'-TAGTAACTGCCACTTCATCA-3' and CtP2 5'- TTCCCCTTGTAATTCGTT-GC-3'. Serial dilutions of clamidial DNA were used to determine the analytical sensitivity. Analytical specificity was tested using DNA from several urogenital microorganisms. Intra assay variability was assessed on triplicate DNA samples, while inter assay variability was assessed comparing the results by three technicians on different days. Results: Established (94°C/4 min; 40 cycles at 94 °C/1 min, 56°C/1 min and 72°C/1.5 min; 72°C/4 min; 1.5 mM of MgCl2 and 1U/pL of Taq polymerase. The analytical sensitivity was 10-17 g of DNA equivalent to one plasmid or less than one elementary body from C. trachomatis. Primers CtP1 and CtP2 amplified specifically C. trachomatis in the experimental conditions evaluated. Intra and inter assay variability demonstrated the repeatability and reproducibility of the PCR respectively. Conclusions: We standardized the experimental conditions for a C. trachomatis PCR that can be used for diagnostic purposes. Other studies are required for further clinical evaluation of this test.

Genetic variation in the Cytb gene of human cerebral Taenia solium cysticerci recovered from clinically and radiologically heterogeneous patients with neurocysticercosis.

Neurocysticercosis (NC) is a clinically and radiologically heterogeneous parasitic disease caused by the establishment of larval Taenia solium in the human central nervous system. Host and/or parasite variations may be related to this observed heterogeneity. Genetic differences between pig and human-derived T. solium cysticerci have been reported previously. In this study, 28 cysticerci were surgically removed from 12 human NC patients, the mitochondrial gene that encodes cytochrome b was amplified from the cysticerci and genetic variations that may be related to NC heterogeneity were characterised. Nine different haplotypes (Ht), which were clustered in four haplogroups (Hg), were identified. Hg 3 and 4 exhibited a tendency to associate with age and gender, respectively. However, no significant associations were found between NC heterogeneity and the different T. solium cysticerci Ht or Hg. Parasite variants obtained from patients with similar NC clinical or radiological features were genetically closer than those found in groups of patients with a different NC profile when using the Mantel test. Overall, this study establishes the presence of genetic differences in the Cytb gene of T. solium isolated from human cysticerci and suggests that parasite variation could contribute to NC heterogeneity.

Genetic variation in the Cytb gene of human cerebral Taenia solium cysticerci recovered from clinically and radiologically heterogeneous patients with neurocysticercosis

Neurocysticercosis (NC) is a clinically and radiologically heterogeneous parasitic disease caused by the establishment of larval Taenia solium in the human central nervous system. Host and/or parasite variations may be related to this observed heterogeneity. Genetic differences between pig and human-derived T. solium cysticerci have been reported previously. In this study, 28 cysticerci were surgically removed from 12 human NC patients, the mitochondrial gene that encodes cytochrome b was amplified from the cysticerci and genetic variations that may be related to NC heterogeneity were characterised. Nine different haplotypes (Ht), which were clustered in four haplogroups (Hg), were identified. Hg 3 and 4 exhibited a tendency to associate with age and gender, respectively. However, no significant associations were found between NC heterogeneity and the different T. solium cysticerci Ht or Hg. Parasite variants obtained from patients with similar NC clinical or radiological features were genetically closer than those found in groups of patients with a different NC profile when using the Mantel test. Overall, this study establishes the presence of genetic differences in the Cytb gene of T. solium isolated from human cysticerci and suggests that parasite variation could contribute to NC heterogeneity. .

[Genotyping of clinical isolates of Aspergillus flavus and its relationship with environmental isolates of an oncohematological center]. / Genotipificación de aislamientos clínicos de Aspergillus flavus y su relación con aislamientos ambientales de un centro oncohematológico.

BACKGROUND: During 4 months, and while conducting an environmental sampling of air, 2 cases of aspergillosis by Aspergillus flavus (A. flavus) were diagnosed at an oncohematological center in Buenos Aires, Argentina. AIMS: The aim of this study was to know the variability and the genetic relationship between the clinical and environmental isolates, obtained in the oncohematological center. METHODS: Two genotyping techniques of different discriminatory power (RAPD and AFLP) were used. A genetic similarity matrix was calculated using Jaccard method and was the basis for the construction of a dendrogram by UPGMA. The level of genetic variability was assessed by measuring the percentage of polymorphic loci, number of effective allele, expected heterocygozity and association index test (I(A)). RESULTS: The dendrogram reveals that the A. flavus isolates recovered from the patients were not genetically related to those gotten from the rooms occupied by the patients. The environmental isolates had higher values of genetic diversity than the clinical isolates. The I(A) estimated for all the isolates suggest that recombination events occurred. CONCLUSIONS: Patients 1 and 2 were not infected with isolates from the nosocomial environment. Clinical and environmental isolates of A. flavus showed high genetic variability among them.

pep27 and lytA in Vancomycin-Tolerant Pneumococci.

Vancomycin therapy failure due to the emergence of tolerance in pneumococci is increasing. The molecular mechanism of tolerance is not clear, but lytA and pep27 are known to be involved. Our aim was to evaluate the expression of both genes in vancomycin-tolerant Streptococcus pneumoniae (VTSP) strains. Eleven VTSP strains from a total of 309 clinical isolates of S. pneumoniae from 1997 to 2006 were classified according to the criteria of Liu and Tomasz. All VTSP strains were evaluated for susceptibility according to CLSI criteria, serotype by the Quellung test, and clonality by PFGE. The expressions of lytA and pep27 were analyzed in different growth phases by RT-PCR with and without vancomycin. Eighty-two percent of VTSP strains showed resistance to penicillin, and 100% were sensitive to vancomycin and cefotaxime. The most frequent serotypes of VTSP strains were 23F (4/11) and 6B (3/11). Clonal relationship was observed in only two strains. No significant changes were observed in pep27 expression in the three phases of growth in VTSP strains with and without vancomycin. Interestingly, pep27 expression in the stationary phase in the non-tolerant reference strain R6 was significantly higher. However, no significant differences in lytA expression were observed between VTSP and R6 strains during the phases of growth analyzed. The absence of changes in pep27 expression in VTSP strains in the stationary phase may be related to their ability to tolerate high antibiotic concentrations, and thus, they survive and remain in the host under the antibiotic selective pressure reflected in therapeutic failure.

Genetic diversity of Taenia solium cysticerci from naturally infected pigs of central Mexico.

This study was designed to explore if each individual case of naturally acquired porcine cysticercosis, living in different geographic rural areas of central Mexico, is caused by one or more different specimens of Taenia solium tapeworm. The genetic variability among cysticerci from the same pig and that from different pigs was assessed by random amplified polymorphic DNA markers (RAPDs), through the percentage of polymorphic loci, the number of effective alleles, the expected heterozygosity and the Shannon index. The parasite population's reproductive structure was estimated through the association index (I(A)), and the degree of genetic differentiation and variation was determined using AMOVA. Using six different random primers, and a total of 181 cysticerci from 14 pigs, 88 different loci were amplified: 85% were polymorphic between pigs and 24% within pigs. The phenogram grouped the cysticerci into eight major clusters, with differences in the genetic distances among all cysticerci from 14 pigs ranging from 0.78 to 1. Most of the cysticerci grouped in accord with their different geographical origin and with their pig of origin. The similarity matrix produced from the phenogram (obtained by UPGMA) and the original similarity matrix yielded a good cophenetic correlation (r=0.82317, P=0.0004), which suggests that the phenogram accurately represents the original genetic similarities between isolates. The combination of I(A) (0.0-0.089) with the genetic diversity index (0.009-0.073) supports the idea that DNA diversity in T. solium cysticerci of naturally infected pigs is within the range expected from a recombination process occurring during sexual reproduction. The small genetic diversity found within the cysticerci of each pig (33.81%), when compared with that between pigs (66.19%), indicates that pigs are rarely infected by different tapeworms. It would then appear that porcine cysticercosis courses with effective concomitant immunity, as occurs in ovine cysticercosis.

Population structure and diversity of the pathogenic fungus Aspergillus fumigatus isolated from different sources and geographic origins

Fifty-five clinical and environmental Aspergillus fumigatus isolates from Mexico, Argentina, France and Peru were analyzed to determine their genetic variability, reproductive system and level of differentiation using amplified fragment length polymorphism markers. The level of genetic variability was assessed by measuring the percentage of polymorphic loci, number of effective alleles, expected heterozygocity and by performing an association index test (I A). The degree of genetic differentiation and variation was determined using analysis of molecular variance at three levels. Using the paired genetic distances, a dendrogram was built to detect the genetic relationship among alleles. Finally, a network of haplotypes was constructed to determine the geographic relationship among them. The results indicate that the clinical isolates have greater genetic variability than the environmental isolates. The I A of the clinical and environmental isolates suggests a recombining population structure. The genetic differentiation among isolates and the dendrogram suggest that the groups of isolates are different. The network of haplotypes demonstrates that the majority of the isolates are grouped according to geographic origin.

Phylogeography of the bark beetle Dendroctonus mexicanus Hopkins (Coleoptera: Curculionidae: Scolytinae).

Dendroctonus mexicanus is polyphagous within the Pinus genus and has a wide geographical distribution in Mexico and Guatemala. We examined the pattern of genetic variation across the range of this species to explore its demographic history and its phylogeographic pattern. Analysis of the mtDNA sequences of 173 individuals from 25 Mexican populations allowed to us identify 53 geographically structured haplotypes. High haplotype and low nucleotide diversities and Tajima's D indicate that D. mexicanus experienced rapid population expansion during its dispersal across mountain systems within its current range. The nested clade phylogeographic analysis indicates that the phylogeographic pattern of D. mexicanus is explained by continuous dispersion among lineages from the Sierra Madre Occidental, the Sierra Madre Oriental and the Trans-Mexican Volcanic Belt. However, we also observed isolation events among haplotypes from the Cofre de Perote/Trans-Mexican Volcanic Belt/Sierra Madre Oriental and the Trans-Mexican Volcanic Belt/Sierra Madre del Sur, which is consistent with the present conformation of mountain systems in Mexico and the emergence of geographical barriers during the Pleistocene.

Genetic polymorphism in Taenia solium cysticerci recovered from experimental infections in pigs.

Taenia solium cysticerci recovered from naturally infected pigs from Mexico, Honduras and Tanzania show a clonal structure and local lineages with probable events of genetic recombination without genetic flow within them, as revealed by RAPD. To evaluate genetic polymorphism from cysticerci recovered from experimental infections, 4 pigs were infected with T. solium eggs obtained from tapeworms released by 3 human carriers, a 10-year-old female, a 25-year-old female, and a 44-year-old male, the 4th pig was infected with a mixture of eggs from the 3 tapeworms. Each pig was orally inoculated with 50,000 eggs. After 16 weeks pigs were humanely euthanized and cysticerci were excised. Parasites recovered from each pig were analyzed by RAPD. The proportion of polymorphic loci and the mean heterozygosity as well as a dendogram and an analysis of principal coordinate and minimum spanning tree were obtained. All four pigs developed viable cysticerci; the percent infection was obtained from the ratio of the number of eggs used for infection and the number of cysticerci counted in each pig after necropsy. Infection varied from 0.2 to 4.2%. The values obtained for the proportion of polymorphic loci (0.14-0.55) and the average of expected heterozygosity (0.06-0.22) in the present experimental infection had a broader range than those reported in the literature from natural infections. The dendogram obtained clustered cysticerci into two main groups; the minimum spanning tree allowed to corroborate the data obtained in the dendogram and gave a better discrimination because in a three-dimensional plot it was easier to see that all cysticerci from each tapeworm were clustered amongst themselves. The results obtained could be hypothetically explained because environmental factors and genetic selection agents present in nature influence natural infections but do not participate in experimental ones.