Azole-Resistant Invasive Aspergillosis: Relationship to Agriculture

Azole resistance in Aspergillus fumigatus has been increasingly reported particularly over the last decade. Two routes of acquisition are described: selection of resistance during long term azole therapy in the clinical setting, and primary acquisition of resistant isolates from the environment due to the considerable use of azole fungicides in agriculture and for material preservation. Three specific resistance genotypes have been found in azole naïve patients. Two of these have also been found in the environment and are characterized by a tandem repeat in the promoter region of the target gene coupled with point mutation(s) in CYP51A (TR₃₄/L98H and TR₄₆/Y121F/T289A). In the third a single target enzyme alteration (G432S) is found. These resistant “environmental” strains have been detected in many West-European countries as well as in the Asia-Pacifics. Noticeably, these two continents account for the highest fungicide use in the global perspective (37 % and 24 %, respectively). Among the 25 azole fungicides, five have been associated with the potential to select for the TR₃₄/L98H genotype; three of these are among those most frequently used. Although the number of antifungal fungicide compounds and classes available is impressive compared to the armamentarium in human medicine, azoles will remain the most important group in agriculture due to superior field performance and significant resistance in fungal pathogens to other compounds. Hence, further spread of environmental resistant Aspergillus genotypes may occur and will depend on the fitness of each resistant phenotype and the pattern of azole fungicide use.     

Filling the gap - COI barcode resolution in eastern Palearctic birds

Background

Many fish species experience long periods of fasting in nature often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as myofibrillar proteins act as a major energy source. To investigate the mechanisms of metabolic reorganisation with fasting and refeeding in a saltwater stage of Atlantic salmon (Salmo salar L.) we analysed the expression of genes involved in myogenesis, growth signalling, lipid biosynthesis and myofibrillar protein degradation and synthesis pathways using qPCR.

Results

Hierarchical clustering of gene expression data revealed three clusters. The first cluster comprised genes involved in lipid metabolism and triacylglycerol synthesis (ALDOB, DGAT1 and LPL) which had peak expression 3-14d after refeeding. The second cluster comprised ADIPOQ, MLC2, IGF-I and TALDO1, with peak expression 14-32d after refeeding. Cluster III contained genes strongly down regulated as an initial response to feeding and included the ubiquitin ligases MuRF1 and MAFbx, myogenic regulatory factors and some metabolic genes.

Conclusion

Early responses to refeeding in fasted salmon included the synthesis of triacylglycerols and activation of the adipogenic differentiation program. Inhibition of MuRF1 and MAFbx respectively may result in decreased degradation and concomitant increased production of myofibrillar proteins. Both of these processes preceded any increase in expression of myogenic regulatory factors and IGF-I. These responses could be a necessary strategy for an animal adapted to long periods of food deprivation whereby energy reserves are replenished prior to the resumption of myogenesis.     

Genetic Diversity in the Worldwide <Emphasis Type="Italic">Botrychium lunaria</Emphasis> (Ophioglossaceae) Complex,with New Species and New Combinations

The Botrychium lunaria (Ophioglossaceae) complex worldwide includes the named species B. lunaria, B. crenulatum, B. tunux, and B. yaaxudakeit. These species have been distinguished from each other morphologically and genetically. This study further investigates the genetic diversity and geographic distribution of this complex, examining a large number of plants worldwide. Enzyme electrophoresis was used to examine allelic variation of 22 loci for 1574 plants of putative B. lunaria, B. crenulatum and B. tunux from North America, Eurasia, and New Zealand, and B. dusenii from the Falkland Islands. Variation in allelic composition assessed by genetic identity and cluster analysis using the programs PopGene and STRUCTURE as well as morphology and geography indicated that the complex is composed of six distinct entities; two of which warrant recognition as new species, B. neolunaria, endemic to North America, and B. nordicum, sister to the B. lunaria complex, from Iceland and Norway; and a new combination, B. lunaria var. melzeri , endemic to Greenland, Iceland, and Norway. The new taxa are described in this paper. Three entities within B. tunux are discussed but not proposed for recognition at this time. Botrychium lanceolatum, included in this study, is composed of three morphologically and genetically distinct entities warranting taxonomic recognition.     

Blastocystis: subtyping isolates using pyrosequencing technology

Blastocystis is a prevalent single-celled enteric parasite of unresolved clinical significance. Efforts based on molecular methodologies to establish whether pathogenicity is linked to specific isolates of the genetically diverse genus of Blastocystis have been scarce and so far yielded ambiguous results which can be difficult to interpret. To alleviate some of the problems related to unravelling the molecular epidemiology of Blastocystis infections we developed and evaluated a simple and high-throughput sequence analysis (SQA) pyrosequencing technique based on the detection of genotype-specific nucleotide polymorphisms in the 18S small subunit rRNA gene for a rapid and cost-effective post-PCR screening of Blastocystis genotypes. The method was effectively capable of genotyping 48/48 isolates positive by nested PCR in approximately one hour, and in 94% of the cases the isolate detected by PCR and pyrosequencing was also detected by one of two different PCR assays with subsequent dideoxy sequencing.     

Development and Application of a Blastocystis Subtype-Specific PCR Assay Reveals that Mixed-Subtype Infections Are Common in a Healthy Human Population

The human gut is host to a diversity of microorganisms, including the single-celled microbial eukaryote Blastocystis. Research has shown that most carriers host a single Blastocystis subtype (ST), which is unusual given the considerable within-host species diversity observed for other microbial genera in this ecosystem. However, our limited knowledge of both the incidence and biological significance of Blastocystis diversity within hosts (i.e., so-called mixed infections) is likely due to problems with existing methodologies. Here, we developed and applied Blastocystis ST-specific PCRs for the investigation of the most common subtypes of Blastocystis (ST1 to ST4) to a healthy human cohort (n = 50). We detected mixed infections in 22% of the cases, all of which had been identified as single-ST infections in a previous study using state-of-the-art methods. Our results show that certain STs occur predominantly as either single (ST3 and 4) or mixed (ST1) infections, which may reflect inter alia transient colonization patterns and/or cooperative or competitive interactions between different STs. Comparative analyses with other primers that have been used extensively for ST-specific analysis found them unsuitable for detection of mixed- and, in some cases, single-ST infections. Collectively, our data shed new light on the diversity of Blastocystis within and between human hosts. Moreover, the development of these PCR assays will facilitate future work on the molecular epidemiology and significance of mixed infections in groups of interest, including health and disease cohorts, and also help identify sources of Blastocystis transmission to humans, including identifying potential animal and environmental reservoirs.     

The impact of genetic diversity in protozoa on molecular diagnostics

Detection of intestinal parasitic protists, commonly referred to as 'intestinal protozoa,' by PCR is increasingly used not only for identification or confirmation but also as a first-line diagnostic tool. Apart from the ability to sample correctly and extract parasite DNA directly from faeces, primer and probe specificity and sensitivity affect predictive values and hence the utility of diagnostic assays. Molecular characterization of intestinal protists is necessary to design primers and probes because this is the basic material for current and future improved diagnostic PCRs for either detecting all genetic variants or specifically differentiating among such variants. As an example, this paper highlights the existence of interspecific and intraspecific genetic diversity among intestinal, unicellular parasites and its implications for nucleic acid-based diagnostic assays.     

Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reaction

Blastocystis hominis is a common enteric parasite of worldwide distribution. Its pathogenetic potential has not yet been established, although numerous case reports suggest that B. hominis may cause the development of various gastrointestinal symptoms and disorders. The detection of the parasite in stool specimens is conventionally done by microscopy of direct smears, fecal concentrates, or permanently stained smears; however, morphology-based diagnosis is problematic. The aim of this study was to develop and evaluate a polymerase chain reaction (PCR) technique for the direct detection of B. hominis in human stool samples. Primers were based on small subunit ribosomal DNA and able to detect > or =32 parasites/200 mg stool artificially spiked with cultured B. hominis. In the evaluation of 43 clinical specimens, the PCR was tested against the formol ethyl acetate concentration technique (FECT) and a culture technique, proving 100% test specificity and a significantly higher sensitivity than the FECT. The PCR method is recommended for screening clinical specimens for B. hominis infection and for use in prevalence studies.     

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background  

Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.