Listeria monocytogenes Attachment to and Detachment from Stainless Steel Surfaces in a Simulated Dairy Processing Environment

The presence of pathogens in dairy products is often associated with contamination via bacteria attached to food-processing equipment, especially from areas where cleaning/sanitation is difficult. In this study, the attachment of Listeria monocytogenes on stainless steel (SS), followed by detachment and growth in foods, was evaluated under conditions simulating a dairy processing environment. Initially, SS coupons were immersed in milk, vanilla custard, and yogurt inoculated with the pathogen (10⁷ CFU/ml or CFU/g) and incubated at two temperatures (5 and 20°C) for 7 days. By the end of incubation, cells were mechanically detached from coupons and used to inoculate freshly pasteurized milk which was subsequently stored at 5°C for 20 days. The suspended cells in all three products in which SS coupons were immersed were also used to inoculate freshly pasteurized milk (5°C for 20 days). When SS coupons were immersed in milk, shorter lag phases were obtained for detached than for planktonically grown cells, regardless of the preincubation temperature (5 or 20°C). The opposite was observed when custard incubated at 20°C was used to prepare the two types of inocula. However, in this case, a significant increase in growth rate was also evident when the inoculum was derived from detached cells. In another parallel study, while L. monocytogenes was not detectable on SS coupons after 7 days of incubation (at 5°C) in inoculated yogurt, marked detachment and growth were observed when these coupons were subsequently transferred and incubated at 5°C in fresh milk or/and custard. Overall, the results obtained extend our knowledge on the risk related to contamination of dairy products with detached L. monocytogenes cells.Listeria monocytogenes is ubiquitous in nature due to its inherent ability to survive and grow under a wide range of adverse environmental conditions, such as refrigeration temperatures, high acidity and salinity, and reduced water activity (16). This microorganism is a major concern for the food industry, since it is the causal agent of listeriosis, a severe disease with high hospitalization and case-fatality rates (approximately 91% and 30%, respectively) (25). According to the European Centre for Disease Control and Prevention, listeriosis was the fifth most common zoonotic infection in Europe in 2006 (14), while it accounts for approximately 28% of the deaths resulting from food-borne illnesses in the United States (34).In the food industry, inadequately cleaned food-processing equipment (e.g., stainless steel [SS] surfaces) constitutes a potential source for L. monocytogenes, resulting in contamination of foods which come in contact with such equipment (36). Even though adherence to strict sanitation practices should minimize the risk of survivors on surfaces, existing evidence suggests that a considerable risk may occur in sites of processing plants which are not easily cleaned or sanitized, such as those that do not allow direct access of sanitation equipment for abrasion (e.g., edges, convex surfaces, etc.) (43, 45). Attachment to surfaces is believed to be important for the survival and persistence of this pathogen in such environments, with some strains able to remain on equipment surfaces for several years (32, 37). Thus, L. monocytogenes has been shown to adhere to and form biofilms on various food contact surfaces under laboratory conditions (3, 42, 44). Furthermore, attached L. monocytogenes cells are more difficult to mechanically remove from surfaces and are more resistant to sanitizers than their free-living counterparts (15, 40).Dairy products have been implicated in outbreaks of listeriosis (10, 31). However, most of the in vitro studies of the growth and survival of L. monocytogenes in such products have used strains previously cultivated planktonically (41). Although the results obtained in these studies are of great value, such studies have not taken into consideration that cells contaminating a product in a food-processing environment are usually attached to surfaces enclosed in biofilms. Limited information is available on the kinetic behavior of L. monocytogenes in dairy products inoculated with detached cells, although preincubation conditions have been shown to influence subsequent growth and survival of L. monocytogenes in foods (7, 13, 17, 18). Given the major physiological differences between attached and planktonic cells (15, 27, 48), an effect on subsequent growth might be possible.Considering the above, the main objective of the present study was to assess the influence of L. monocytogenes preincubation conditions with respect to mode of growth (either attached to SS or grown suspended in dairy products) on the subsequent growth of this pathogen in milk (at 5°C for 20 days). To prepare the two types of inocula, two different growth media (milk and vanilla custard) and temperatures (5 and 20°C) were studied. The unforced detachment of L. monocytogenes cells from SS coupons and growth in two dairy products (milk and custard) at 5°C for 20 days was also evaluated. In the latter case, previous attachment of cells to the coupons was done under especially adverse preincubation conditions (in yogurt at 5°C for 7 days).     

Microbial Decomposer Communities Are Mainly Structured by Trophic Status in Circumneutral and Alkaline Streams

In streams, the release of nitrogen and phosphorus is reported to affect microbial communities and the ecological processes they govern. Moreover, the type of inorganic nitrogen (NO₃, NO₂, or NH₄) may differently impact microbial communities. We aimed to identify the environmental factors that structure aquatic microbial communities and drive leaf litter decomposition along a gradient of eutrophication. We selected five circumneutral (Portuguese) and five alkaline (French) streams differing in nutrient concentrations to monitor mass loss of alder leaves, bacterial and fungal diversity by PCR-denaturing gradient gel electrophoresis, fungal biomass and reproduction, and bacterial biomass during 11 weeks of leaf immersion. The concentrations of inorganic nutrients in the stream water ranged from 5 to 300 μg liter⁻¹ soluble reactive phosphorus, 0.30 to 5.50 mg liter⁻¹ NO₃-N, 2 to 103 μg liter⁻¹ NO₂-N, and <4 to 7,100 μg liter⁻¹ NH₄-N. Species richness was maximum in moderately anthropized (eutrophic) streams but decreased in the most anthropized (hypertrophic) streams. Different species assemblages were found in subsets of streams with different trophic statuses. In both geographic areas, the limiting nutrient, either nitrate or phosphate, stimulated the microbial activity in streams of intermediate trophic status. In the hypertrophic streams, fungal biomass and reproduction were significantly lower, and bacterial biomass dramatically decreased at the site with the highest ammonium concentration. The limiting nutrients that defined the trophic status were the main factor structuring fungal and bacterial communities, whatever the geographic area. A very high ammonium concentration in stream water most probably has negative impacts on microbial decomposer communities.There is evidence that increases in nitrate and phosphate concentrations stimulate microbial respiration and fungal and bacterial activity (biomass buildup, sporulation, and/or productivity) on plant litter, leading to faster leaf decomposition in freshwaters (16, 17, 26, 34). However, fungal demands of nitrate and phosphate are reported to be fulfilled at relatively low levels (1, 12), and further increases in these nutrients in the stream water do not necessarily result in enhanced fungal activity. Besides, the form in which inorganic nutrients are present in streams, their biological availability, and even their toxicity have different ecological consequences. In densely anthropized hypertrophic streams, high levels of nitrate and phosphate were associated with decreased fungal biomass and leaf breakdown, most probably because of the high concentrations of ammonium and ammonia (2). On the other hand, the positive effects of nutrients on biomass and productivity of leaf-associated fungi can be offset by other factors, such as low oxygen concentration and sedimentation, leading to retarded decomposition (26, 33, 34).Changes in inorganic nutrient concentrations in the stream water were reported to alter the structure of fungal communities on plant litter (16, 36). Nutrient additions to moderate levels increased the diversity of fungal communities in circumneutral soft-water Appalachian mountain streams (18) but not in a Mediterranean alkaline stream (1). Moreover, fungal diversity was lower in circumneutral eutrophic streams than in reference streams (10, 35). Fungal diversity has been assessed mostly through the morphological analysis of produced conidia, not taking into account nonsporulating fungi. This raises the question of whether the differential impacts of eutrophication on fungal diversity could be due partly to difficulties in measuring actual diversity. Besides, the study of bacterial diversity on decomposing leaves has been strongly restricted to a few cultivable bacteria (<1%). Molecular typing, such as denaturing gradient gel electrophoresis (DGGE) of a specific rRNA gene region, has proved useful for assessing diversity in both leaf-associated fungi and bacteria (7, 8, 9, 11, 30).We aimed to identify the environmental factors that drive the ecological processes in freshwaters impacted by eutrophication through examination of leaf litter decomposition and associated microbial communities along a gradient of nutrient enrichment. Specifically, we addressed the following two questions: (i) which are the environmental factors that mainly structure the fungal and bacterial communities and (ii) what are the relationships between concentrations of inorganic nutrients in the stream water, leaf litter decomposition, and the activity of associated microbes? We selected 10 stream sites spanning wide concentration ranges of dissolved inorganic nitrogen (NO₃-N, NO₂-N, NH₄-N, and NH₃-N) and soluble reactive phosphorus (SRP), including 5 in northwestern Portugal with circumneutral pH and 5 in southwestern France with an alkaline pH. With these two groups of stream sites, we assessed the structure of and diversity in both sporulating and nonsporulating fungal communities, using asexual spore morphology and DGGE fingerprints of the ITS2 region, and in bacterial communities, using DGGE fingerprints of the 16S rRNA gene region. Additionally, we examined leaf mass loss and microbial activity on decomposing leaves by determining bacterial and fungal biomass and fungal reproduction.     

Hatching and earliest larval stages of the priapulid worm Priapulus caudatus

Abstract. Here we describe the hatching and morphology of the earliest larval stages of the priapulid worm Priapulus caudatus for the first time. The hatching larva differs considerably from previously described larvae not only in its general body shape but also in its lack of a proper lorica including the typical lorica tubuli. Furthermore, no mouth opening or pharyngeal teeth have formed as yet, and the number and arrangement of scalids differ from that of later larvae. The hatching larva molts and emerges as the first lorica larva. This larva partially resembles earlier described lorica larvae, but there are a number of important differences; the first lorica larva is smaller, and the mouth opening as well as pharyngeal teeth are still yet to form. The second lorica larva is equipped with four rings of pharyngeal teeth; it shows striking similarity to the previously described larva of P. caudatus , i.e., the larva-type 2 , only differing in the scalid pattern. We conclude that the first two larval stages of P. caudatus have not been described previously. We suggest that discrepancies between the earliest lorica larvae described here and in earlier publications might depend on sub-speciation or ecophenotypic modification of larvae collected from different localities. Our findings highlight the importance of studying the development of non-model organisms such as priapulids under controlled laboratory conditions.     

Effect of environmental stress on clonal structure of <Emphasis Type="Italic">Eucypris virens</Emphasis> (Crustacea,Ostracoda)

Environmental stress imposes strong natural selection on clonal populations, promoting evolutionary change in clonal structure. Environmental stress may also lead to reduction in population size, which together with clonal selection may reduce genotypic diversity of the local populations. We examined how clonal structure in wild-collected samples of two parthenogenetic populations of the freshwater ostracod Eucypris virens responded to hypersalinity and starvation, and the combination of the two stressors. We applied the stress treatments in a factorial design for one generation. When 60% of the individuals per experimental unit had died, post-experimental clonal structure was compared to that of the start of the experiment, which reflected the field conditions. We used five polymorphic allozyme loci as genotype markers. All stress treatments reduced survival compared to the control treatment. In the population “Rivalazzetto”, we observed a reduction of clonal richness in the control treatment, with the initially dominant clone maintaining dominance. This may have resulted from interclonal competition and clone-specific survival under the different laboratory conditions. Clonal richness remained high in the salinity treatment while it was reduced in the combined stress and starvation treatments. In the population “Fornovo”, clonal richness reduced in all treatments including control, while the salinity and combined stress treatment reduced clonal evenness. The clone dominating at the start of the experiment increased in frequency in all treatments, but the change in clonal structure during the experiment was more pronounced in this population. These results suggest that in some conditions an intermediate level of environmental stress may lessen the decline in genetic diversity by strong inter-clonal competition. Moreover, the variation in clonal structure among the stress treatments and distinct genetic backgrounds indicates that more general predictions of stress effects on clonal structure may be difficult.     

Current distribution and ecology of the critically endangered <Emphasis Type="Italic">Valencia letourneuxi</Emphasis> in Greece

The current population status of the critically endangered Greek valencia Valencia letourneuxi (Sauvage, 1880), is presented, based on a thorough survey of low-altitude aquatic systems of Western Greece. The species’ historical geographical range appears to have been reduced, with the westernmost and southernmost populations being currently extinct or near extinction respectively, and the remaining ones in a vulnerable state. According to univariate and multivariate analysis, V. letourneuxi exhibits habitat specificity, with a strong affinity towards spring-fed wetlands with clear waters and rich surface vegetation, being thus vulnerable to human-induced habitat modifications and hydrological changes. The impact of the above threats, as well as of the introduction of alien species such as the mosquitofish Gambusia holbrooki is discussed.     

Quenching of triplet state fluorophores for studying diffusion-mediated reactions in lipid membranes

An approach to study bimolecular interactions in model lipid bilayers and biological membranes is introduced, exploiting the influence of membrane-associated electron spin resonance labels on the triplet state kinetics of membrane-bound fluorophores. Singlet-triplet state transitions within the dye Lissamine Rhodamine B (LRB) were studied, when free in aqueous solutions, with LRB bound to a lipid in a liposome, and in the presence of different local concentrations of the electron spin resonance label TEMPO. By monitoring the triplet state kinetics via variations in the fluorescence signal, in this study using fluorescence correlation spectroscopy, a strong fluorescence signal can be combined with the ability to monitor low-frequency molecular interactions, at timescales much longer than the fluorescence lifetimes. Both in solution and in membranes, the measured relative changes in the singlet-triplet transitions rates were found to well reflect the expected collisional frequencies between the LRB and TEMPO molecules. These collisional rates could also be monitored at local TEMPO concentrations where practically no quenching of the excited state of the fluorophores can be detected. The proposed strategy is broadly applicable, in terms of possible read-out means, types of molecular interactions that can be followed, and in what environments these interactions can be measured.     

Feeding ecology of the critically endangered Valencia letourneuxi (Valenciidae)

Valencia letourneuxi is a critically endangered freshwater fish in urgent need of targeted conservation. Little information is however available on its life history and feeding ecology; therefore, food resource utilization by V. letourneuxi was studied in its most abundant known Greek population of Chiliadou stream. The diet of this population appears to be dominated by microcrustaceans, Dipteran larvae, Acari, and Mollusca. Its feeding is highly dependent on seasonal prey availability and diversity, with niche overlap being low only between winter and the rest of the seasons, indicating that only during winter its diet differs significantly in relation to the other seasons. There are no significant sex- and size-related dietary shifts. This V. letourneuxi population is characterized by a generalist feeding strategy and appears to consist mostly of individuals with broad niches. Its generalist feeding pattern and dietary flexibility permits it to fully exploit this very diverse and rich habitat and may account for the high local abundance of this population.     

Persistent negative effects of pesticides on biodiversity and biological control potential on European farmland

During the last 50 years, agricultural intensification has caused many wild plant and animal species to go extinct regionally or nationally and has profoundly changed the functioning of agro-ecosystems. Agricultural intensification has many components, such as loss of landscape elements, enlarged farm and field sizes and larger inputs of fertilizer and pesticides. However, very little is known about the relative contribution of these variables to the large-scale negative effects on biodiversity. In this study, we disentangled the impacts of various components of agricultural intensification on species diversity of wild plants, carabids and ground-nesting farmland birds and on the biological control of aphids.In a Europe-wide study in eight West and East European countries, we found important negative effects of agricultural intensification on wild plant, carabid and bird species diversity and on the potential for biological pest control, as estimated from the number of aphids taken by predators. Of the 13 components of intensification we measured, use of insecticides and fungicides had consistent negative effects on biodiversity. Insecticides also reduced the biological control potential. Organic farming and other agri-environment schemes aiming to mitigate the negative effects of intensive farming on biodiversity did increase the diversity of wild plant and carabid species, but – contrary to our expectations – not the diversity of breeding birds.We conclude that despite decades of European policy to ban harmful pesticides, the negative effects of pesticides on wild plant and animal species persist, at the same time reducing the opportunities for biological pest control. If biodiversity is to be restored in Europe and opportunities are to be created for crop production utilizing biodiversity-based ecosystem services such as biological pest control, there must be a Europe-wide shift towards farming with minimal use of pesticides over large areas.     

Geographic and molecular variation in a natural plant transgene

A PCR based survey of Festuca ovina plants from populations around the southern part of the Baltic Sea demonstrates both geographic and molecular variation in the enzyme gene PgiC2, horizontally transferred from a Poa-species. Our results show that PgiC2—a natural functional nuclear transgene—is not a local ephemeral phenomenon but is present in a very large number of individuals. We find also that its frequency is geographically variable and that it appears in more than one molecular form. The chloroplast variation in the region does not indicate any distinct subdivision due to different colonization routes after the last glaciation. Our data illustrate the geographic and molecular variation that may occur in natural populations with a polymorphic, unfixed transgene affected by diverse kinds of mutational and evolutionary processes.