Gino Naclerio

Novel Synthetic, Salt-Resistant Analogs of Human Beta-Defensins 1 and 3 Endowed with Enhanced Antimicrobial Activity

ABSTRACT Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.

Purification and partial characterization of bacillocin 490, a novel bacteriocin produced by a thermophilic strain of Bacillus licheniformis

BackgroundApplications of bacteriocins as food preservatives have been so far limited, principally because of their low antimicrobial activity in foods. Nisin is the only bacteriocin of significant use, but applications are restricted principally because of its very low activity at neutral or alkaline pH. Thus the isolation of new bacteriocins active in foods is desirable.ResultsWe isolated a Bacillus licheniformis thermophilic strain producing a bacteriocin with some novel features, named here bacillocin 490. This bacteriocin was inactivated by pronase E and proteinase K and was active against closely related Bacillus spp. both in aerobic and in anaerobic conditions. Bactericidal activity was kept during storage at 4°C and was remarkably stable in a wide pH range. The bacteriocin was partially purified by elution after adhesion to cells of the food-isolated strain Bacillus smithii and had a rather low mass (2 KDa). Antimicrobial activity against B. smithii was observed also when this organism was grown in water buffalo milk.ConclusionsBacillocin 490 is a novel candidate as a food anti-microbial agent since it displays its activity in milk, is stable to heat treatment and during storage, is active in a wide pH range and has bactericidal activity also at high temperature. These features may allow the use of bacillocin 490 during processes performed at high temperature and as a complementary antimicrobial agent of nisin against some Bacillus spp. in non-acidic foods. The small size suggests its use on solid foods.

Influence of Precipitation and Soil on Transport of Fecal Enterococci in Fractured Limestone Aquifers

ABSTRACT Limestone aquifers provide the main drinking water resources of southern Italy. The groundwater is often contaminated by fecal bacteria because of the interaction between rocks having high permeability and microbial pollutants introduced into the environment by grazing and/or manure spreading. The microbial contamination of springwater in picnic areas located in high mountains can cause gastrointestinal illness. This study was carried out in order to analyze the interaction between Enterococcus faecalis and the soil of a limestone aquifer and to verify the influence of this interaction on the time dependence of groundwater contamination. E. faecalis was chosen because, in the study area involved, it represents a better indicator than Escherichia coli. The research was carried out through field (springwater monitoring) and laboratory experiments (column tests with intact soil blocks). The transport of bacterial cells through soil samples was analyzed by simulating an infiltration event that was monitored in the study area. Comparison of laboratory results with data acquired in the field showed that discontinuous precipitation caused an intermittent migration of microorganisms through the soil and produced, together with dispersion in the fractured medium (unsaturated and saturated zones), an articulated breakthrough at the spring. The short distances of bacterial transport in the study area produced a significant daily variability of bacterial contamination at the field scale.

Expression of the heat shock gene clpL of Streptococcus thermophilus is induced by both heat and cold shock

BackgroundHeat and cold shock response are normally considered as independent phenomena. A small amount of evidence suggests instead that interactions may exist between them in two Lactococcus strains.ResultsWe show the occurrence of molecular relationships between the mechanisms of cold and heat adaptations in Streptococcus thermophilus, a lactic acid bacterium widely used in dairy fermentation, where it undergoes both types of stress. We observed that cryotolerance is increased when cells are pre-incubated at high temperature. In addition, the production of a protein, identified as ClpL, a member of the heat-shock ATPase family Clp A/B, is induced at both high and low temperature. A knock-out clpL mutant is deficient in both heat and cold tolerance. However lack of production of this protein does not abolish the positive effect of heat pre-treatment towards cryotolerance.ConclusionDual induction of ClpL by cold and heat exposure of cells and reduced tolerance to both temperature shocks in a clpL mutant indicates that the two stress responses are correlated in S. thermophilus. However this protein is not responsible by itself for cryotolerance of cells pre-treated at high temperature, indicating that ClpL is necessary for the two phenomena, but does not account by itself for the relationships between them.

Role of organic matter and clay fraction on migration of Escherichia coli cells through pyroclastic soils, southern Italy

A comparative study on the adsorption of Escherichia coli cells to two different pyroclastic soils collected in southern Italy (carbonate Apennines) was performed in laboratory using surfactant-free solutions and solutions with the surfactants sodium dodecyl sulphate (SDS, anionic) and octylphenoxypolyethoxyethanol (Triton X-100, non-ionic). Both soils are rich in organic matter (up to 35%), but only one contains a clay fraction (2-5%). The experiments demonstrated that E. coli cells are significantly adsorbed to the clay fraction of the soil, while the organic matter content does not play a significant role. The pore size exclusion phenomenon is another factor to consider when analyzing the retention of E. coli cells within such soils. However, despite the existence of different factors that enhance bacterial cells retention, a high percent of E. coli cells is transported through soil media. The not absolute protection of such soils against microbial pollution is supported not only by the results of the column experiments at lab scale, but also by the findings of a field monitoring at site scale.

Alteration of cell morphology and viability in a recA mutant of Streptococcus thermophilus upon induction of heat shock and nutrient starvation

We identified the recA gene of the moderately thermophilic bacterium Streptococcus thermophilus and investigated the role of its product in the adaptation to heat shock and nutrient starvation. Expression of recA was required for optimal viability and normal cell morphology upon induction of both stresses. Normal induction of GroEL and ClpL in a recA knock-out mutant suggests that the RecA role in heat shock and nutrient starvation response of S. thermophilus is independent from the intracellular accumulation of these stress-specific chaperones.

Filtration of Bacillus subtilis and Bacillus cereus spores in a pyroclastic topsoil, carbonate Apennines, southern Italy

A comparative study on the filtration of Bacillus subtilis and Bacillus cereus spores in a pyroclastic topsoil was performed in laboratory using surfactant-free solutions and solutions with the surfactant sodium dodecyl sulphate (SDS) (anionic). The results of the column experiments demonstrate that the SDS does not significantly influence the retention of both B. subtilis and B. cereus spores. Since the SDS is adsorbed through hydrophobic interaction with the organic matter of soil media, these results suggest that hydrophobic interaction between spores and organic matter does not play a significant role on filtration processes within the studied topsoil. This statement is of utmost importance taking into consideration the hydrophobic nature of Bacillus spores and the very high organic matter content in the studied topsoil (20-34%). Conversely, the retention of the analyzed spores seems to be influenced by the pore size exclusion phenomenon.

Impaired Temperature Stress Response of a Streptococcus thermophilus deoD Mutant

ABSTRACT An insertional deoD mutant of Streptococcus thermophilus strain SFi39 had a reduced growth rate at 20°C and an enhanced survival capacity to heat shock compared to the wild type, indicating that the deoD product is involved in temperature shock adaptation. We report evidence that ppGpp is implicated in this dual response.

Seawater intrusion in the Guanahacabibes Peninsula (Pinar del Rio Province, western Cuba): effects on karst development and water isotope composition

The water resources in the Guanahacabibes Peninsula are distributed in two areas. The northeastern area is characterized by swamps, wetlands and lagoons, with a low contribution of seawater, whereas the area in the southwestern plain shows a considerable development of the karst structures that limits the existence of superficial waters but permits the ingression of the surrounding seawater. In this latter area, the groundwater showed a marked increase in salinity with the depth. In particular, groundwater with a seawater fraction of 0.1 had the lowest Ca-(Mg)-carbonates saturation indexes calculated by modeling the mixing between freshwater and seawater using different software, thermodynamic databases and equations for activity coefficients. Generally, seawater and groundwaters with an added seawater fraction above 0.60–0.65 showed similar oversaturated indexes in high-Mg calcites and pure Ca-carbonates (calcite and aragonite). Differently, in the groundwater that showed carbonates undersaturation (generally with a seawater fraction between 0.02 and 0.60), the saturation indexes in high-Mg calcites were 0.2 lower than pure Ca-carbonates. Locally, the bacterial reduction of the dissolved sulfate enhanced the dissolution of the limestone, contributing to the increased development of the karst structures and the seawater intrusion. Finally, the presence near the coastline of fresh Ca- and Na-bicarbonate waters was in accordance with the upward flow of the shallow freshwater during the formation of the saline wedge. However, the oxygen and hydrogen stable isotope composition of the waters showed a probable contribution to the area from a deep aquifer that is recharged in the highest reliefs of the province (Cordillera de Guaniguanico).

Winter survival of microbial contaminants in soil：An in situ verification

The aim of the research was to evaluate, at site scale, the influence of freezing and freeze/thaw cycles on the survival of faecal coliforms and faecal enterococci in soil, in a climate change perspective. Before the winter period and during grazing, viable cells of faecal coliforms and faecal enterococci were detected only in the first 10 cm below ground, while, after the winter period and before the new seasonal grazing, a lower number of viable cells of both faecal indicators was detected only in some of the investigated soil profiles, and within the first 5 cm. Taking into consideration the results of specific investigations, we hypothesise that the non-uniform spatial distribution of grass roots within the studied soil can play an important role in influencing this phenomenon, while several abiotic factors do not play any significant role. Taking into account the local trend in the increase of air temperature, a different distribution of microbial pollution over time is expected in spring waters, in future climate scenarios. The progressive increase in air temperature will cause a progressive decrease in freeze/thaw cycles at higher altitudes, minimising cold shocks on microbial cells, and causing spring water pollution also during winter.