Relationships between Legionella and Aeromonas spp. and associated lake bacterial communities across seasonal changes in an anthropogenic eutrophication gradient.

Anthropogenic eutrophication of lakes threatens their homeostasis and carries an increased risk of development of potentially pathogenic microorganisms. In this paper we show how eutrophication affects seasonal changes in the taxonomic structure of bacterioplankton and whether these changes are associated with the relative abundance of pathogenic bacteria of the genera Legionella and Aeromonas. The subject of the study was a unique system of interconnected lakes in northern Poland (Great Masurian Lakes system), characterized by the presence of eutrophic gradient. We found that the taxonomic structure of the bacterial community in eutrophic lakes was significantly season dependent. No such significant seasonal changes were observed in meso-eutrophic lakes. We found that there is a specific taxonomic composition of bacteria associated with the occurrence of Legionella spp. The highest positive significant correlations were found for families Pirellulaceae, Mycobacteriaceae and Gemmataceae. The highest negative correlations were found for the families Sporichthyaceae, Flavobacteriaceae, the uncultured families of class Verrucomicrobia and Chitinophagaceae. We used also an Automatic Neural Network model to estimate the relative abundance of Legionella spp. based on the relative abundance of dominant bacterial families. In the case of Aeromonas spp. we did not find a clear relationship with bacterial communities inhabiting lakes of different trophic state. Our research has shown that anthropogenic eutrophication causes significant changes in the taxonomic composition of lake bacteria and contributes to an increase in the proportion of potentially pathogenic Legionella spp.

Comparison of protease and aminopeptidase activities in meconium: A pilot study.

The successive accumulation of proteases and aminopeptidases in meconium are important physiological components of the intrauterine environment in which a fetus develops. The aim of the present study was to assess the changes in the activities of these enzymes in meconium of healthy infants, and to investigate whether there were any statistically significant associations between activity of the enzymes of interest and the mode of delivery. The activities of proteases and aminopeptidases were determined in meconium portions (n=110) using the substrates BODIPY FL casein and L-leucine-7-amido-4-methylcoumarin hydrochloride, respectively. Serial meconium samples (2-5 per neonate) were collected from healthy infants born vaginally (n=14), and by a cesarean section (n=16). Protease activity (104 RFU/h) was lower in the first meconium sample compared with the final sample from the same infant (3.99±2.03 vs. 5.76±2.24, respectively, mean ± standard deviation; P=0.004). Conversely, there was no significant difference in aminopeptidase activity (103 nM/l/h) between consecutive meconium samples (P=0.702). The ratios of the first-meconium sample enzyme activity to the last-meconium sample enzyme activity were lower for proteases compared with aminopeptidases (0.76±0.48 vs. 1.35±1.04, respectively mean ± standard deviation; P=0.014), and sustained in the infants born by a cesarean section (P=0.008). Spearman's correlation coefficient analysis between the first and last meconium samples showed the correlation increased in the infants born vaginally compared with the rest of the infants (proteases, R=0.618 vs. R=0.314; aminopeptidases, R=0.688 vs. R=0.566). Aminopeptidase activity did not exhibit any notable dynamic changes during meconium accumulation in the fetal intestine. In infants born vaginally compared with those born by a cesarean section, the activity of both proteases and aminopeptidases in the first meconium sample showed an improved correlation with the activity of the final meconium sample. This may suggest that in the intrauterine environment, during accumulation of meconium in the digestive tract of the fetus, the activity and/or levels of these enzymes and the substrates they catalyze were more stable in newborns born vaginally compared with infants born by caesarean section.

Structural and functional microbial diversity along a eutrophication gradient of interconnected lakes undergoing anthropopressure.

We present the results of an analysis of the 16S rRNA-based taxonomical structure of bacteria together with an analysis of carbon source utilization ability using EcoPlate (Biolog, USA) metabolic fingerprinting assessment against the backdrop of physicochemical parameters in fifteen interconnected lakes. The lakes exhibit a wide spectrum of trophic gradients and undergo different intensities of anthropopressure. Sequences of V3-V4 16S rRNA genes binned by taxonomic assignment to family indicated that bacterial communities in the highly eutrophicated lakes were distinctly different from the bacterial communities in the meso-eutrophic lakes (ANOSIM r = 0.99, p = 0.0002) and were characterized by higher richness and more diverse taxonomical structure. Representatives of the Actinobacteria, Proteobacteria, Cyanobacteria, Planctomycetes, Verrucomicrobia, Bacteroides phyla predominated. In most cases their relative abundance was significantly correlated with lake trophic state. We found no similar clear relationship of community-level physiological profiling with lake trophic state. However, we found some significant links between the taxonomic and metabolic structure of the microbes in the studied lakes (Mantel's correlation r = 0.22, p = 0.006). The carbon source utilization ability of the studied microorganisms was affected not only by the taxonomic groups present in the lakes but also by various characteristics like a high PO43- concentration inhibiting the utilization of phosphorylated carbon.

Quantitative description of respiration processes in meso-eutrophic and eutrophic freshwater environments.

We propose a modification of measurement methodology allowing the overall respiration rate (VResp) close to the in situ conditions; size of the labile, respirable organic matter pool (OMResp); and its turnover time (Tt) to be calculated. In addition to the respiration of dissolved substrates by free-living bacteria, the respiration of attached bacteria and other planktonic organisms is also taken into account. In case study we evaluated the modified, quantitative description of respiration processes in surface waters of lakes of different trophic status: mezzo-eutrophic and eutrophic. In both types of studied environments, VResp oscillated between 1.0 µmol C l-1 h-1 and 3.0 µmol C l-1 h-1, and the size of the OMResp pool varied from 39.3 µM C to 828.7 µM C. Despite of higher OMResp concentrations in eutrophic lakes, we found a lower susceptibility of OM to respiration processes in eutrophic than in meso-eutrophic lakes but similar VResp in both types of lakes. We conclude that the proposed method allows a fast quantitative description of labile organic matter utilization by aerobic aquatic microorganisms.

Total proteolytic activity and concentration of alpha-1 antitrypsin in meconium for assessment of the protease/antiprotease balance.

BACKGROUND: During intrauterine life, various proteolytic enzymes and their main inhibitor, alpha-1 antitrypsin, accumulate naturally in meconium. A protease/antiprotease balance is required to maintain the biological stability of the environment in which the fetus develops. METHODS: The pool of active proteases was determined using the EnzChek Protease Assay Kit. The concentration of alpha-1 antitrypsin in meconium was measured by enzyme-linked immunosorbent assay. Serial portions of meconium (n=80) were collected from healthy full-term neonates (n=19). RESULTS: Mean concentrations of active proteases and alpha-1 antitrypsin were 1.55 [standard deviation (SD) 1.3]mgg-1 (range 0.15-6.17) and 3.72 (SD 1.78)mgg-1 (range 0.76-8.55), respectively, with significant correlation (Rs=0.32, p=0.004). A significant increase in the concentration of active proteases was found between the first and last meconium portions (p<0.05). The proteases in the last meconium portions had a higher reaction velocity and affinity for the substrate than the proteases in the first meconium portions. The active protease:alpha-1 antitrypsin ratio was <0.5 in all first meconium portions, but was higher in the last meconium portions. CONCLUSIONS: Strong correlation between the concentrations of active proteases and alpha-1 antitrypsin in meconium may indicate their mutual interaction in the intrauterine environment. Alpha-1 antitrypsin maintains the protease/antiprotease balance during fetal development.

Microbial Biomass and Enzymatic Activity of the Surface Microlayer and Subsurface Water in Two Dystrophic Lakes.

Nutrient and organic matter concentration, microbial biomass and activities were studied at the surface microlayers (SML) and subsurface waters (SSW) in two small forest lakes of different water colour. The SML in polyhumic lake is more enriched with dissolved inorganic nitrogen (0.141 mg l-1) than that of oligohumic lake (0.124 mg l-1), the former also contains higher levels of total nitrogen (2.66 mg l-1). Higher activities of lipase (Vmax 2290 nmol l-1 h-1 in oligo- and 6098 in polyhumic) and glucosidase (Vmax 41 nmol l-1 h-1 in oligo- and 49 in polyhumic) were in the SMLs in both lakes. Phosphatase activity was higher in the oligohumic SML than in SSW (Vmax 632 vs. 339 nmol l-1 h-1) while in polyhumic lake was higher in SSW (Vmax 2258 nmol l-1 h-1 vs. 1908 nmol l-1 h-1). Aminopeptidase activity in the SSW in both lakes was higher than in SMLs (Vmax 2117 in oligo- and 1213 nmol l-1 h-1 in polyhumic). It seems that solar radiation does inhibit neuston microbial community as a whole because secondary production and the share of active bacteria in total bacteria number were higher in SSW. However, in the oligohumic lake the abundance of bacteria in the SML was always higher than in the SSW (4.07 vs. 2.69 × 106 cells ml-1) while in the polyhumic lake was roughly equal (4.48 vs. 4.33 × 106 cells ml-1) in both layers. Results may also suggest that surface communities are not supplemented by immigration from bulk communities. The SML of humic lakes may act as important sinks for allochthonous nutrient resources and may then generate considerable energy pools for microbial food webs.

Coomassie Blue G250 for Visualization of Active Bacteria from Lake Environment and Culture.

Bacteria play a fundamental role in the cycling of nutrients in aquatic environments. A precise distinction between active and inactive bacteria is crucial for the description of this process. We have evaluated the usefulness of Coomassie Blue G250 for fluorescent staining of protein containing potentially highly active bacteria. We found that the G250 solution has excitation and emission properties appropriate for direct epifluorescence microscopy observations. It enables fast and effective fluorescent visualization of living, protein-rich bacteria, both in freshwater environment and culture. Our results revealed that the number of G250-stained bacteria from eutrophic lake was positively correlated with other standard bacterial activity markers, like number of bacteria containing 16S rRNA, bacterial secondary production or maximal potential leucine-aminopeptidase activity. In case of the E. coli culture, the percentage of bacteria visualized with G250 was similar to that of bacteria which accumulated tetracycline. Compared to other common methods utilizing fluorogenic substances for bacteria staining, the approach we evaluated is inexpensive and less hazardous (for example mutagenic) to the environment and researchers. It can be regarded as an additional or alternative method for protein rich, active bacteria staining.

Preliminary studies on the evolution of carbon assimilation abilities within Mucorales.

Representatives of Mucorales belong to one of the oldest lineages of terrestrial fungi. Although carbon is of fundamental importance for fungal growth and functioning, relatively little is known about enzymatic capacities of Mucorales. The evolutionary history and the variability of the capacity to metabolize different carbon sources among representatives of the order Mucorales was studied using Phenotypic Microarray Plates. The ability of 26 strains belonging to 23 nonpathogenic species of Mucorales to use 95 different carbon sources was tested. Intraspecies variability of carbon assimilation profiles was lower than interspecies variation for some selected strains. Although similarities between the phylogenetic tree and the dendrogram created from carbon source utilization data were observed, the ability of the various strains to use the analyzed substrates did not show a clear correlation with the evolutionary history of the group. Instead, carbon assimilation profiles are probably shaped by environmental conditions.

Factors controlling bacteria and protists in selected Mazurian eutrophic lakes (North-Eastern Poland) during spring.

BACKGROUND: The bottom-up (food resources) and top-down (grazing pressure) controls, with other environmental parameters (water temperature, pH) are the main factors regulating the abundance and structure of microbial communities in aquatic ecosystems. It is still not definitively decided which of the two control mechanisms is more important. The significance of bottom-up versus top-down controls may alter with lake productivity and season. In oligo- and/or mesotrophic environments, the bottom-up control is mostly important in regulating bacterial abundances, while in eutrophic systems, the top-down control may be more significant. RESULTS: The abundance of bacteria, heterotrophic (HNF) and autotrophic (ANF) nanoflagellates and ciliates, as well as bacterial production (BP) and metabolically active cells of bacteria (CTC, NuCC, EST) were studied in eutrophic lakes (Mazurian Lake District, Poland) during spring. The studied lakes were characterized by high nanoflagellate (mean 17.36 ± 8.57 × 103 cells ml-1) and ciliate abundances (mean 59.9 ± 22.4 ind. ml-1) that were higher in the euphotic zone than in the bottom waters, with relatively low bacterial densities (4.76 ± 2.08 × 106 cells ml-1) that were lower in the euphotic zone compared to the profundal zone. Oligotrichida (Rimostrombidium spp.), Prostomatida (Urotricha spp.) and Scuticociliatida (Histiobalantium bodamicum) dominated in the euphotic zone, whereas oligotrichs Tintinnidium sp. and prostomatids Urotricha spp. were most numerous in the bottom waters. Among the staining methods used to examine bacterial cellular metabolic activity, the lowest percentage of active cells was recorded with the CTC (1.5-15.4%) and EST (2.7-14.2%) assay in contrast to the NuCC (28.8-97.3%) method. CONCLUSIONS: In the euphotic zone, the bottom-up factors (TP and DOC concentrations) played a more important role than top-down control (grazing by protists) in regulating bacterial numbers and activity. None of the single analyzed factors controlled bacterial abundance in the bottom waters. The results of this study suggest that both control mechanisms, bottom-up and top-down, simultaneously regulated bacterial community and their activity in the profundal zone of the studied lakes during spring. In both lake water layers, food availability (algae, nanoflagellates) was probably the major factor determining ciliate abundance and their composition. In the bottom waters, both groups of protists appeared to be also influenced by oxygen, temperature, and total phosphorus.

Persistence of bacterial proteolytic enzymes in lake ecosystems.

This study analyzes proteolytic enzyme persistence and the role of dead (or metabolically inactive) aquatic bacteria in organic matter cycling. Samples from four lakes of different trophic status were used. Irrespective of the trophic status of the examined lakes, bacterial aminopeptidases remained active even 72 h after the death of the bacteria that produced them. The total pool of proteolytic enzymes in natural lake water samples was also stable. We found that the rates of amino acid enzymatic release from proteinaceous matter added to preserved lake water sample were constant for at least 96 h (r(2)  = 0.99, n = 17, P ≤ 0.0001, V(max)  = 84.6 nM h(-1) ). We also observed that proteases built into bacterial cell debris fragments remained active for a long time, even after the total destruction of cells. Moreover, during 24 h of incubation time, about 20% of these enzymatically active fragments adsorbed onto natural seston particles, becoming a part of the 'attached enzymes system' that is regarded as the 'hot-spot' of protein degradation in aquatic ecosystems.

The dynamics of protein decomposition in lakes of different trophic status--reflections on the assessment of the real proteolytic activity in situ.

Abstract The aim of this paper is to discuss the methodology of our investigation of the dynamics of protein degradation and the total in situ proteolytic activity in meso/eutrophic, eutrophic, and hypereutrophic freshwater environments. Analysis of the kinetics and rates of enzymatic release of amino acids in water samples preserved with sodium azide allows determination of the concentrations of labile proteins (C(LAB)), and their half-life time (T(1/2)). Moreover, it gives more realistic information on resultant activity in situ (V(T(1/2))) of ecto- and extracellular proteases that are responsible for the biological degradation of these compounds. Although the results provided by the proposed method are generally well correlated with those obtained by classical procedures, they better characterize the dynamics of protein degradation processes, especially in eutrophic or hypereutrophic lakes. In these environments, processes of protein decomposition occur mainly on the particles and depend primarily on a metabolic activity of seston-attached bacteria. The method was tested in three lakes. The different degree of eutrophication of these lakes was clearly demonstrated by the measured real proteolytic pattern and confirmed by conventional trophic state determinants.