A seminested PCR assay was developed in order to amplify the kinetoplast minicircle of Leishmania species from individual sand flies. The kinetoplast minicircle is an ideal target because it is present in 10,000 copies per cell and its sequence is known for most Leishmania species. The two-step PCR is carried out in a single tube using three primers, which were designed within the conserved area of the minicircle and contain conserved sequence blocks. The assay was able to detect as few as 3 parasites per individual sand fly and to amplify minicircle DNA from at least eight Leishmania species. This technique permits the processing of a large number of samples synchronously, as required for epidemiological studies, in order to study infection rates in sand fly populations and to identify potential insect vectors. Comparison of the sequences obtained from sand flies and mammal hosts will be crucial for developing hypotheses about the transmission cycles of Leishmania spp. in areas of endemicity.

Prophage sequences became an integral part of bacterial genomes as a consequence of coevolution, encoding fitness or virulence factors. Such roles have been attributed to phage-derived elements identified in several Gram-negative species: The type VI secretion system (T6SS), the R- and F-type pyocins, and the newly discovered Serratia entomophila antifeeding prophage (Afp), and the Photorhabdus luminescens virulence cassette (PVC). In this study, we provide evidence that remarkably conserved gene clusters, homologous to Afp/PVC, are not restricted to Gram-negative bacteria but are widespread throughout all prokaryotes including the Archaea. Even though they are phylogenetically closer to pyocins, they share key characteristics in common with the T6SS, such as the use of a chaperon-type AAA+ ATPase and the lack of a host cell lysis mechanism. We thus suggest that Afp/PVC-like elements could be classified as phage-like-protein-translocation structures (PLTSs) rather than as pyocins. The reconstruction of phylogeny and the conserved gene content suggest that the diversification of prophage sequences to PLTS occurred in bacteria early in evolution and only once, but PLTS clusters have been horizontally transferred to some of the bacterial lineages and to the Archaea. The adaptation of this element in such a wide host range is suggestive of its versatile use in prokaryotes.

Apoptosis is a normal component of the development and health of multicellular organisms. However, apoptosis is now considered a prerogative of unicellular organisms, including the trypanosomatids of the genera Trypanosoma spp. and Leishmania spp., causative agents of some of the most important neglected human diseases. Trypanosomatids show typical hallmarks of apoptosis, although they lack some of the key molecules contributing to this process in metazoans, like caspase genes, Bcl-2 family genes and the TNF-related family of receptors. Despite the lack of these molecules, trypanosomatids appear to have the basic machinery to commit suicide. The components of the apoptotic execution machinery of these parasites are slowly coming into light, by targeting essential processes and pathways with different apoptogenic agents and inhibitors. This review will be confined to the events known to drive trypanosomatid parasites to apoptosis.

Ten multidrug-resistant Pseudomonas aeruginosa strains producing VIM-1-like acquired metallo-␤-lactamases (MBLs), isolated from four European countries (Greece, Hungary, Italy, and Sweden), were analyzed for genetic relatedness by several methodologies, including fliC sequence analysis, macrorestriction profiling of genomic DNA by pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), and multilocus sequence typing (MLST). The four approaches yielded consistent results overall but showed different resolution powers in establishing relatedness between isolates (PFGE > RAPD > MLST > fliC typing) and could usefully complement each other to address issues in the molecular epidemiology of P. aeruginosa strains producing acquired MBLs. In particular, the recently developed MLST approach was useful in revealing clonal relatedness between isolates when this was not readily apparent using RAPD and PFGE, and it suggested a common ancestry for some of the VIM-1-like MBL-positive P. aeruginosa strains currently spreading in Europe. The MBL producers belonged in three clonal complexes/burst groups (BGs). Of these, one corresponded to the previously described BG4 and included serotype O12 strains from Hungary and Sweden, while the other two were novel and included serotype O11 or nonserotypable strains from Greece, Sweden, and/or Italy. Comparison of the integrons carrying bla VIM-1 -like cassettes of various isolates revealed a remarkable structural heterogeneity, suggesting the possibility that multiple independent events of acquisition of different bla VIM -containing integrons had occurred in members of the same clonal lineage, although a contribution of integrase-mediated cassette shuffling or other recombination mechanisms during the evolution of similar strains could also have played a role in determining this variability.

Relationships among seventy specimens, fifteen species and three genera of phlebotomines were inferred from the phylogenetic analysis of small subunit nuclear rDNA, obtained by the PCR amplification and cloning of almost full-length genes. Outgroups included fifteen dipterans, and single representatives of four other insect orders. The more distant the taxa compared, the larger were the regions of ambiguous sequence alignment that needed to be deleted in order to avoid circularity in performing parsimony analyses. Phlebotomine sequences formed a monophyletic clade within the suborder Nematocera, with the progressively more basal sister groups of Diptera being Culicomorpha, Tipulomorpha and the suborder Brachycera. Within Phlebotominae, subgeneric relationships were resolved and the genus Phlebotomus was shown to be monophyletic, but markers for intraspecific geographical populations were not found and intergeneric relationships were not resolved.