Alfonso Navas

Incidence of the pinewood nematode Bursaphelenchus xylophlius Steiner & Buhrer, 1934 (Nickle, 1970) in Spain

The pine wood nematode (PWN) Bursaphelenchus xylophilus Steiner & Buhrer, 1934 (Nickle, 1970), is a pathogenic species in the genus Bursaphelenchus Fuchs, 1937. The introduction and establishment of PWN in the Setubal Peninsula, Portugal (Mota et al., 1999), and the evidence that Monochamus galloprovincialis is its insect vector in this country (Sousa et al., 2001), has stimulated studies on its further distribution and its potential vector insects in Europe, including Spain. There is a clear risk of spread of pine wood nematode into Spanish territory from Portugal, both as a result of natural spread and due to the important timber trade between the two countries. The occurrence of Bursaphelenchus spp. in Spain has been previously studied (Abelleira et al., 2002, 2003; Escuer et al., 2003, 2004; Robertson et al., 2008). These studies have shown, in addition, the distribution of insects belonging to the Cerambycidae, Curculionidae and Scolytidae families (Cobos, 1986; Gil Sánchez & Pajares Alonso, 1986; Vives, 2000; Verdugo Paez, 2004), which are known to be vectors of Bursaphelenchus spp. other than B. xylophilus and are often found to be associated with stressed, dead or decaying conifers. Although it is generally accepted that B. xylophilus is the only truly pathogenic species (reviewed by Jones et al., 2008), other species may be involved in pine forest decline in some European regions or, alternatively, may simply be opportunistic infectors of stressed trees (Mamiya, 1999; Caroppo et al., 2000; Skarmoutsos et al., 2000). Pinus pinaster is the most representative

Evolution of Pseudomonas aeruginosa Antimicrobial Resistance and Fitness under Low and High Mutation Rates

ABSTRACT Pseudomonas aeruginosa, a major cause of nosocomial and chronic infections, is considered a paradigm of antimicrobial resistance development. However, the evolutionary trajectories of antimicrobial resistance and the impact of mutator phenotypes remain mostly unexplored. Therefore, whole-genome sequencing (WGS) was performed in lineages of wild-type and mutator (ΔmutS) strains exposed to increasing concentrations of relevant antipseudomonal agents. WGS provided a privileged perspective of the dramatic effect of mutator phenotypes on the accumulation of random mutations, most of which were transitions, as expected. Moreover, a frameshift mutagenic signature, consistent with error-prone DNA polymerase activity as a consequence of SOS system induction, was also seen. This effect was evidenced for all antibiotics tested, but it was higher for fluoroquinolones than for cephalosporins or carbapenems. Analysis of genotype versus phenotype confirmed expected resistance evolution trajectories but also revealed new pathways. Classical mechanisms included multiple mutations leading to AmpC overexpression (ceftazidime), quinolone resistance-determining region (QRDR) mutations (ciprofloxacin), oprD inactivation (meropenem), and efflux pump overexpression (ciprofloxacin and meropenem). Groundbreaking findings included gain-of-function mutations leading to the structural modification of AmpC (ceftazidime), novel DNA gyrase (GyrA) modification (ciprofloxacin), and the alteration of the β-lactam binding site of penicillin-binding protein 3 (PBP3) (meropenem). A further striking finding was seen in the evolution of meropenem resistance, selecting for specific extremely large (>250 kb) genomic deletions providing a growth advantage in the presence of the antibiotic. Finally, fitness and virulence varied within and across evolved antibiotic-resistant populations, but mutator lineages showed a lower biological cost for some antibiotics.

Experimental validation of Haldane's hypothesis on the role of infection as an evolutionary force for Metazoans

A common drawback in evolutionary science is the fact that the evolution of organisms occurs in geological timing, completely out of the time scale of laboratory experimental work. For this reason, some relevant hypotheses on evolution of Metazoans are based on correlations more than on experimental data obtained for testing the robustness of those hypotheses. In the current work, we implement an experimental methodology to analyze the role of infections as a driving force in the evolution of Metazoans (Haldanes hypothesis). To that goal, we have used simple models of virulence with short reproduction times, large populations, and that are easily testable in the laboratory. Using the bacteriovirus nematode Caenorhabditis elegans as a model organism under evolution and their infection by the environmental opportunistic bacterial pathogen Pseudomonas aeruginosa as the selective force, we have demonstrated that bacterial infection selects an evolved nematode lineage resistant to infection, with changes in its respiration and capability of consuming novel food resources. Using an experimental approach, we show that infection is a selective force in the evolution of Metazoans as proposed earlier by Haldane.

Scanning electron microscopy of Anisakis larvae following different treatments

Ingestion of fish parasitized with Anisakis larvae can produce infestation and/or allergy in consumers. Technological and food processing treatments have been applied to parasitized fish in order to kill the larvae and avoid the infestation; however, their influence on allergenicity has not been studied. Four lots of hake (Merluccius merluccius) steaks artificially parasitized with Anisakis larvae were subjected to two storage chilling (5 degrees C +/- 1 degrees C) and freezing (-20 degrees C +/- 1 degrees C) treatments and two food processing treatments of heat (final temperature 86.3 degrees C) and microwave (final temperature 66.9 degrees C) and studied by scanning electron microscopy, environmental scanning electron microscopy (ESEM) (acid [pH = 2] and water preparations), and emission of fluorescence. Anisakis larvae were resistant to acid conditions, remaining alive after treatment. Larvae in the heat- and microwave-treated lots presented coagulated and disrupted zones in the cuticle with release of fluids. The cylindrical shape changed to a dehydrated appearance mainly observed by ESEM. Fluorescence was only noticeable in the frozen larvae. Larvae without apparent changes, together with dehydrated ones, were observed by ESEM in the frozen lot; nevertheless, no disruptions in the cuticle were perceptible. Further studies are needed in order to elucidate if the changes observed in the cuticle reduce the resistance of the parasites to the action of gastric enzymes in the gastrointestinal tract and to determine the release of allergens to the flesh by the live larvae during chilled storage of the fish.

Differential interactions within the Caenorhabditis elegans-Pseudomonas aeruginosa pathogenesis model.

A pathogenesis model based on the interaction between Caenorhabditis elegans and bacterial opportunistic pathogens has recently been developed. In the case of Pseudomonas aeruginosa, the model is based on three different modes of nematode killing (fast killing, slow killing and lethal paralysis) by virulent bacteria that has been incubated in different nutrient media. Using parametric statistics and Probit analysis, we test the reliability of the three different killing systems with respect to bacterial virulence. To accomplish this, we use three P. aeruginosa strains, each with a different level of virulence and one strain of non-virulent Escherichia coli. Probit function proved to be effective in quantifying the virulence of P. aeruginosa. The results of the killing curve analysis using the Probit function demonstrates that the slow-killing test is the most reliable method for quantifying virulence using the C. elegans model of bacterial pathogenesis. Although the greatest virulence differences are observed after long periods of incubation, the Probit analysis clearly shows that the death kinetics of C. elegans depend on the first hours of nematode/bacteria interaction. In contrast, fast killing seems to be non-specific, at least under our experimental conditions, since the killing rates of virulent P. aeruginosa and non-virulent E. coli strains were indistinguishable.

Proteomic profiling and characterization of differential allergens in the nematodes Anisakis simplex sensu stricto and A. pegreffii.

The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius. They were compared by 2D gel electrophoresis and parallel Western blot (2DE gels were hybridized with pools of sera from Anisakis allergenic patients). Unambiguous spot differences were detected and protein assignation was made by MALDI‐TOF/TOF analysis or de novo sequencing. Seventy‐five gel spots were detected and the corresponding proteins were identified. Differentially expressed proteins for A. simplex s.s., A. pegreffii, and their hybrid are described and results are statistically supported. Twenty‐eight different allergenic proteins are classified according to different families belonging to different biological functions. These proteins are described for the first time as antigenic and potentially new allergens in Anisakis. Comparative proteomic analyses of allergenic capacities are useful for diagnosis, epidemiological surveys, and clinical research. All MS data have been deposited in the ProteomeXchange with identifier PXD000662 (http://proteomecentral.proteomexchange.org/dataset/PXD000662).

Isolation of active DNA-binding nuclear proteins from tomato galls induced by root-knot nematodes

We describe 2 methods for extraction of DNA-binding proteins from root-knot nematode feeding sites (ie, galls). DNA-binding activity was assayed by electrophoretic mobility shift assays using fragments from the root-knot nematode-responsiveLEMMI9 and 35S promoters. In noninfected tissue, the method based on nuclei enrichment through a Percoll cushion was superior for isolation of DNA-protein binding activity with both promoters. With infected roots; the method based on crude extracts performed better with theLEMMI9 promoter, whereas nuclei-enriched extracts worked better with the 35S promoter. Therefore, both methods can be used to extract proteins for DNA-binding assays from infected roots, but the method of choice may depend on the promoter under study.

ACI-1 from Acidaminococcus fermentans: Characterization of the First β-Lactamase in Anaerobic Cocci

ABSTRACT Acidaminococcus fermentans belongs to the group of strictly anaerobic gram-negative cocci. All previously describedAcidaminococcus strains are susceptible to β-lactam antibiotics. An A. fermentans strain (RYC-MR95) resistant to penicillin and expanded-spectrum cephalosporin (amoxicillin and cefotaxime MICs, 64 μg/ml) was isolated from a human perianal abscess. A fragment encoding a β-lactamase from genomic DNA was cloned in Escherichia coli K-12 strain HB101, and the recombinant strain expressed resistance to amoxicillin (MIC, 1,024 μg/ml) and cefotaxime (MIC, 4 μg/ml). Clavulanic acid decreased the MICs to 8 and 0.03 μg/ml, respectively. Analysis of the nucleotide sequence revealed a new class A β-lactamase, ACI-1. In accordance with its biochemical properties, we propose to assign ACI-1 to functional group 2be. The ACI-1 enzyme (estimated pI 4.3) had <50% amino acid identity with any other class A β-lactamases, the closest being ROB-1 from Haemophilus influenzae (44%). ACI-1 was closer to class A β-lactamases from some gram-positive organisms (41 to 44% amino acid identity withBacillus β-lactamases) than to most class A enzymes from gram-negative organisms (TEM-1, 24.6%). The aci1 gene had a G+C content of 42.1%, in contrast with 56% G+C content for genomic DNA from A. fermentans, thus suggesting that aci1 may have been obtained by horizontal gene transfer.

Incidence of plant-parasitic nematodes in natural and semi-natural mountain grassland and the host status of some common grass species

A plant-parasitic nematode survey was undertaken in mountainous pastures of southern Spain. Sixty-three species of plant-parasitic nematodes distributed over 25 genera were associated with pastures and grasslands in southern Spain. Paratylenchus species (P. microdorus, P. similis, P. nanus and P. ciccaronei) were the most abundant and prevalent plant-parasitic nematodes found. Pratylenchus neglectus and P.thornei were widely distributed in 55 and 41% of sites, respectively. Amplimerlinius globigerus, Helicotylenchus digonicus, H. dihystera, H. tunisiensis, Merlinius brevidens, M. microdorus, Rotylenchus unisexus and Scutylenchus quadrifer were found in more than 10% of the fields, on occasion at densities of more than 200 per 100 cm³ of soil. A further 49 species of plant-parasitic nematodes were identified and reported. A pot experiment, using a range of grasses and forage legumes adapted to the zone, ascertained the host status of the most abundant plant-parasitic nematodes. Helicotylenchus digonicus, M. microdorus and P.microdorus populations were maintained or increased by all grasses or legumes tested. In general, grasses were better hosts of P.neglectus and P.thornei than the pasture legumes tested. Medicago minima reduced P.thornei densities after 3 months of growing.

Nematodes as bioindicators of dry pasture recovery after temporary rye cultivation

Abstract Soil and plant parasitic nematodes were sampled from the top 10 cm of a luvisol in dry grassland north of Madrid (Central Spain). A natural pasture and two grazed pastures which had been cropped with rye 1 and 6 yr before, respectively, were compared by multivariate statistical analysis of the nematode fauna. Results from stepwise discriminant analysis demonstrated the influence of agricultural management on the abundance of the nematode community of these pasture soils. Specifically, it was found that Paratylenchus and Rhabditidae can have a discriminative value of the recovery of these soil ecosystems. Among the indices used to analyze the abundance, diversity and maturity of the nematode community, only plant parasite index (PPI) detected significant differences among pastures, decreasing its values as recovery time after human intervention increased, with the significantly highest value in the recently cropped (C) pasture. It is concluded that length of time after cropping influences the abundance more than the diversity of the nematodes present in pastures of these areas, and that multivariate statistical analyses are the most useful tests to detect differences. The role of nematodes as biological indicators of the degree of recovery of these soils after human disturbance is noted.