Aleš Lapanje

Selective antimicrobial activity of maggots against pathogenic bacteria

Maggot therapy, also known as biosurgery, is an ancient method for the healing of chronic infected wounds. Although clinicians have reported on the beneficial activities of the Lucilia sericata larvae that have been used for healing chronic wounds, the selectivity of this therapy against the different pathogenic micro-organisms that are found in chronic wounds has never been analysed. In the present study, we have investigated the in vitro activities of larval excreta/secreta both against selected bacterial strains that frequently occur in chronically infected wounds, and against bacteria isolated directly from the larvae and their excreta/secreta. Additionally, the antibacterial activities were investigated in in vivo studies, by comparing bacterial diversity in wounds before and after the application of L. sericata larvae. In conclusion, larval therapy is highly recommended, particularly for the treatment of wounds infected with Gram-positive bacteria, like Staphylococcus aureus, but less so for wounds infected with Gram-negative bacteria, especially Proteus spp. and Pseudomonas spp. strains. Bacteria from the genus Vagococcus were resistant to the maggot excreta/secreta.

Isolation of bacterial endophytes from germinated maize kernels

The germination of surface-sterilized maize kernels under aseptic conditions proved to be a suitable method for isolation of kernel-associated bacterial endophytes. Bacterial strains identified by partial 16S rRNA gene sequencing as Pantoea sp., Microbacterium sp., Frigoribacterium sp., Bacillus sp., Paenibacillus sp., and Sphingomonas sp. were isolated from kernels of 4 different maize cultivars. Genus Pantoea was associated with a specific maize cultivar. The kernels of this cultivar were often overgrown with the fungus Lecanicillium aphanocladii; however, those exhibiting Pantoea growth were never colonized with it. Furthermore, the isolated bacterium strain inhibited fungal growth in vitro.

Long-term Hg pollution-induced structural shifts of bacterial community in the terrestrial isopod (Porcellio scaber) gut

In previous studies we detected lower species richness and lower Hg sensitivity of the bacteria present in egested guts of Porcellio scaber (Crustacea, Isopoda) from chronically Hg polluted than from unpolluted environment. Basis for such results were further investigated by sequencing of 16S rRNA genes of mercury-resistant (Hgr) isolates and clone libraries. We observed up to 385 times higher numbers of Hgr bacteria in guts of animals from polluted than from unpolluted environment. The majority of Hgr strains contained merA genes. Sequencing of 16S rRNA clones from egested guts of animals from Hg-polluted environments showed elevated number of bacteria from Pseudomonas, Listeria and Bacteroidetes relatives groups. In animals from pristine environment number of bacteria from Achromobacter relatives, Alcaligenes, Paracoccus, Ochrobactrum relatives, Rhizobium/Agrobacterium, Bacillus and Microbacterium groups were elevated. Such bacterial community shifts in guts of animals from Hg-polluted environment could significantly contribute to P. scaber Hg tolerance.

Gut bacterial community structure (Porcellio Scaber, Isopoda, Crustacea) as a measure of community level response to long‐term and short‐term metal pollution

Prokaryotes are of high importance in the assessment of environmental pollution effects. Due to fast responsiveness of bacterial communities to environmental physicochemical factors, it is difficult to compare results of bacterial community investigations on the temporal and spatial scale. To reduce the effects of variable physicochemical environmental conditions on bacterial microbiota when investigating the specific impact of contaminants on bacterial communities, we investigated the bacterial community in the gut of terrestrial isopods (Porcellio scaber, Isopoda, Crustacea) from clean and metal-polluted environments. Animals were collected from a chronically mercury-polluted site, a chronically multiple metal-(Cd, Pb, Zn) polluted site, and two reference sites. In addition, animals from an unpolluted site were laboratory exposed to 5 microg Hg/g food in order to compare the effect of acute and chronic Hg exposure. The bacterial gut microbiota was investigated by temporal temperature gradient gel electrophoresis (TTGE) and clone library construction based on polymerase chain reaction amplified 16S rRNA genes. The major bacterial representatives of the emptied gut microbiota in the animals from the chronically polluted environments seemed not affected when analyzed by TTGE. The detailed bacterial community structure investigated by 16S rRNA clone library construction, however, showed that the community from the Hg-polluted site also was affected severely (242.4 operational taxonomic units [OTU] in the polluted and 650.6 OTU in the unpolluted environment). When animals were acutely exposed to mercury, changes of bacterial community structures already were seen on TTGE profiles and no additional analysis was needed. We suggest the use of P. scaber gut bacterial community structure as a measure of effects caused by both long- and short-term exposure to pollution.

Anaerobic bacteria in the gut of terrestrial isopod Crustacean Porcellio scaber.

Anaerobic bacteria fromPorcellio scaber hindgut were identified and, subsequently, isolated using molecular approach. Phylogenetic affiliation of bacteria associated with the hindgut wall was determined by analysis of bacterial 16S rRNA gene sequences which were retrieved directly from washed hindguts ofP. scaber. Sequences from bacteria related to obligate anaerobic bacteria from generaBacteroides andEnterococcus were retrieved, as well as sequences from ‘A1 subcluster’ of the wall-less mollicutes. Bacteria from the genusDesulfotomaculum were isolated from gut wall and cultivated under anaerobic conditions. In contrast to previous reports which suggested the absence of anaerobic bacteria in the isopod digestive system due to short retention time of the food in the tube-like hindgut, frequent renewal of the gut cuticle during the moulting process, and unsuccessful attempts to isolate anaerobic bacteria from this environment our results indicate the presence of resident anaerobic bacteria in the gut ofP. scaber, in spite of apparently unsuitable,i.e. predominately oxic, conditions.

Intestinal Microbiota of Terrestrial Isopods

Isopods are a cosmopolitan group of crustaceans, which inhabit marine environments ranging from deep-sea to intertidal areas, surface and underground freshwaters and terrestrial environments fromhigh humidity to dry habitats, including deserts. More than one third of the described isopodan species (approximately 9000) belong to the terrestrial Oniscidea or woodlice (slaters, sowbugs and pillbugs; Schamlfuss 2003). The phylogeny and systematics of Oniscidea were analysed and presented by several authors (Schmalfuss 1989; Erhard 1998; Tabacaru and Danielopol 1999). In general, Oniscidea are assigned to five sections, namely Diplocheta, Tylida, Microcheta, Synocheta andCrinocheta, with 33 families altogether (Erhard 1998). Amphibious species of the family Ligiidae (slaters) and members of Mesoniscidae represent only approximately 50 of the described species, the rest belonging to the higher oniscideans,mostly to troglobiontic Synocheta and “truly terrestrial” Crinocheta. Terrestrial isopods are effective herbivorous scavengers feedingpredominantly on decayed plant material, fungi and algae, thus participating in decomposition and cycling of energy and organic matter in the terrestrial environments (Hopkin 1991; Zimmer and Topp 1997). Due to their significant ecological role and their ability to survive in polluted environments, a substantial amount of research was focused on these organisms, and as a result a comprehensive knowledge accumulated on their biology. Species like Porcellio scaber and Oniscus asellus, for example, are among the most studied organisms in terrestrial ecophysiology and ecotoxicology (Hopkin 1989; Drobne 1997). Terrestrial isopods have diverse feeding strategies including coprophagy and occasional cannibalism. The nutritional importance and significance of coprophagy in the field was not demonstrated

Fast Prediction of DNA Melting Bubbles Using DNA Thermodynamic Stability

DNA melting bubbles are the basis of many DNA-protein interactions, such as those in regulatory DNA regions driving gene expression, DNA replication and bacterial horizontal gene transfer. Bubble formation is affected by DNA duplex stability and thermally induced duplex destabilization (TIDD). Although prediction of duplex stability with the nearest neighbor (NN) method is much faster than prediction of TIDD with the Peyrard-Bishop-Dauxois (PBD) model, PBD predicted TIDD defines regulatory DNA regions with higher accuracy and detail. Here, we considered that PBD predicted TIDD is inherently related to the intrinsic duplex stabilities of destabilization regions. We show by regression modeling that NN duplex stabilities can be used to predict TIDD almost as accurately as is predicted with PBD. Predicted TIDD is in fact ascribed to non-linear transformation of NN duplex stabilities in destabilization regions as well as effects of neighboring regions relative to destabilization size. Since the prediction time of our models is over six orders of magnitude shorter than that of PBD, the models present an accessible tool for researchers. TIDD can be predicted on our webserver at http://tidd.immt.eu.

Band smearing of PCR amplified bacterial 16S rRNA genes: Dependence on initial PCR target diversity

Band smearing in agarose gels of PCR amplified bacterial 16S rRNA genes is understood to comprise amplicons of varying sizes arising from PCR errors, and requires elimination. We consider that with amplified heterogeneous DNA, delayed electro-migration is caused not by PCR errors but by dsDNA structures that arise from imperfect strand pairing. The extent of band smearing was found to be proportional to the sequence heterogeneity in 16S rRNA variable regions. Denaturing alkaline gels showed that all amplified DNA was of the correct size. A novel bioinformatic approach was used to reveal that band smearing occurred due to imperfectly paired strands of the amplified DNA. Since the smear is a structural fraction of the correct size PCR product, it carries important information on richness and diversity of the target DNA. For accurate analysis, the origin of the smear must first be identified before it is eliminated by examining the amplified DNA in denaturing alkaline gels.

DNA structure at the plasmid origin-of-transfer indicates its potential transfer range

Horizontal gene transfer via plasmid conjugation enables antimicrobial resistance (AMR) to spread among bacteria and is a major health concern. The range of potential transfer hosts of a particular conjugative plasmid is characterised by its mobility (MOB) group, which is currently determined based on the amino acid sequence of the plasmid-encoded relaxase. To facilitate prediction of plasmid MOB groups, we have developed a bioinformatic procedure based on analysis of the origin-of-transfer (oriT), a merely 230 bp long non-coding plasmid DNA region that is the enzymatic substrate for the relaxase. By computationally interpreting conformational and physicochemical properties of the oriT region, which facilitate relaxase-oriT recognition and initiation of nicking, MOB groups can be resolved with over 99% accuracy. We have shown that oriT structural properties are highly conserved and can be used to discriminate among MOB groups more efficiently than the oriT nucleotide sequence. The procedure for prediction of MOB groups and potential transfer range of plasmids was implemented using published data and is available at http://dnatools.eu/MOB/plasmid.html.

Cyanogenic Pseudomonas spp. strains are concentrated in the rhizosphere of alpine pioneer plants

HCN producing bacteria have previously been isolated from alpine mineral soil and their ecophysiology was presumed to be associated with mineral weathering. Nevertheless, the high ecological patchiness of the alpine environment calls for an extensive and detailed analysis of the spatial distribution of HCN producing bacterial populations and their associated weathering traits. Our results of such an analysis showed that primarily the rhizosphere of pioneer plants was rich in HPPs, harbouring the most potent HCN producers. HCN production incidence and intensity were dependent on the plant-associated microhabitat and type of bedrock/mineral soil, however the HCN+ phenotype was not associated with one of the particular genotypes which we determined by BOX-PCR. In HPP isolates, HCN production most commonly co-occurred with the production of hydroxamate-type siderophores, but was less often associated with inorganic phosphate solubilization activity and the production of catechol-type siderophores. These observations indicate that a plants physiotype, not species, provide physicochemical conditions that determine selective pressure, which enables the growth of Pseudomonas spp. with a random genotype, but phenotypically predetermined to increase mineral weathering via a particular combination of phosphate solubilization and iron complexation with siderophores and HCN.