Biofouling communities contribute significantly to aquatic ecosystem productivity and biogeochemical cycling. Our knowledge of the distribution, composition, and activities of these microbially dominated communities is limited compared to other components of estuarine ecosystems. This study investigated the temporal stability and change of the dominant phylogenetic groups of the domain Bacteria in estuarine biofilm communities. Glass slides were deployed monthly over 1 year for 7-day incubations during peak tidal periods in East Sabine Bay, Fla. Community profiling was achieved by using 16S rRNA genes and terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes in combination with ribotyping, cloning, and sequencing to evaluate diversity and to identify dominant microorganisms. Bacterial community profiles from biofilms grown near the benthos showed distinct periods of constancy within winter and summer sampling periods. Similar periods of stability were also seen in T-RFLP patterns from floating biofilms. Alternating dominance of phylogenetic groups between seasons appeared to be associated with seasonal changes in temperature, nutrient availability, and light. The community structure appeared to be stable during these periods despite changes in salinity and in dissolved oxygen.Biofilms develop on all surfaces in aquatic environments and are defined as matrix-enclosed microbial populations adherent to each other and/or surfaces (1, 32). A substantial part of the microbial activity in nature is associated with surfaces (12). Surface association (biofouling) is an efficient means for bacteria to proliferate in both favorable and sometimes hostile environments. By adopting a sessile mode of life, microbes can achieve several advantages over their planktonic counterparts (38), including the ability to capture and concentrate nutrients from the water column in the existing exopolysaccharide matrix, cometabolic interactions with neighboring microorganisms (17), and resistance to harmful chemicals (2) and environmental stress.Biofilm-associated microbes, because of their ubiquity, diverse metabolic capabilities, and high enzymatic activity, play a crucial role in biogeochemical cycling. Direct observations show that biofilm-associated organisms (photo-and heterotrophic) account for a major part of ecosystem processes, both numerically and metabolically (12). Biofilm communities in nature play a key role in the production and degradation of organic matter, the degradation of environmental pollutants, and the cycling of limiting nutrients.Biofilm formation and persistence in estuarine environments is governed by a suite of complex physical, chemical, and biological processes (4,44,45). Many of these parameters can vary significantly over different time scales. For example, nutrient availability can vary over diel light cycles, daily tidal cycles, and with rainfall events and seasonal change. Patterns in particulate and dissolved nutrient input to estuarine systems may influence shifts in biofilm bact...
We examined ultraviolet radiation (UVR)-induced DNA damage in marine micro-organisms collected from surface seawater along a latitudinal transect in the Central Pacific Ocean from 70°N to 68°S. Samples were collected predawn and incubated under ambient UVR in transparent incubators at in situ temperatures until late afternoon at which time they were filtered into primarily bacterioplankton and eukaryotic fractions. Cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts [(6-4)PDs] were quantified in DNA extracts using radioimmunoassays. UVB was lowest in the polar regions and highest near the equator and correlations between UVB and DNA damage were observed. The eukaryotic fraction showed significant CPDs across the entire transect; (6-4)PDs were detected only in the tropics. The bacterial fraction showed no accumulation of (6-4)PDs at any latitude, although residual (6-4)PDs were observed. Bacterial cell volumes were greatest in the subArctic and northern temperate latitudes and lower in the tropics and southern hemisphere, a unique observation that parallels Bergmann's rule. A strong negative correlation was observed between cell volume and CPDs. The environmental impact of solar UVR on marine micro-organisms in the open ocean is complex and our results suggest that several factors such as DNA repair, cell size, temperature, salinity, nutrients and species composition are important in determining relative sensitivity.
Significance and Impact of the Study: Whether in the context of microbial ecology or in an industrial context, many questions in microbiology are linked to microbial viability. As cultivation of micro-organisms can be long or may not be possible, fast methods to assess the numbers of live cells are in great demand. We present here a straightforward strategy to determine the relative proportions of intact cells. The PCR-based rapid method is expected to be useful where relative information is sufficient (e.g. for comparing the effect of different antimicrobial treatments on known numbers of micro-organisms) or when the presence of PCR inhibitors does not allow absolute quantification. KeywordsCryptosporidium parvum, live-dead distinction, propidium monoazide, viability. AbstractThe fast analysis of relative proportions of live and dead cells can be of great value whether for comparing inactivation efficiencies of different biocidal treatments or for monitoring organisms of interest in environmental samples. We introduce here a straightforward method to determine the percentage of intact cells based on treatment of samples with the viability dye propidium monoazide (PMA). PMA selectively enters membrane-damaged cells and suppresses their PCR detection through modification of their DNA. The study was performed using Cryptosporidium parvum oocysts as a model although the principle should be applicable to other organisms. Validation was performed with defined mixtures of live and heat-killed oocysts and by exposing oocysts to a heat stress gradient. The method correctly indicated increasingly lower proportions of intact cells with increasing temperatures. When comparing the loss of membrane integrity of UV-killed (40 mJ cm À2 ) oocysts during storage in nonsterile tap water, results suggested that integrity declines slowly (over weeks) and at a rate comparable to non-UV-exposed oocysts. For all experiments, the amplification of longer DNA sequences was found beneficial. In the UV experiment, longer amplicons revealed not only higher sensitivity in excluding membrane-damaged oocysts, but also in excluding DNA with UV-induced damage.
Cryptosporidium spp., Giardia spp., and members of Microsporidia are enteropathogenic parasites of humans and animals, producing asymptomatic to severe intestinal infections. To circumvent various impediments associated with current detection methods, we tested a method providing multistage purification and separation in a single, confined step. Standard real-time PCR was used as a detection method. Samples spiked with C. parvum and G. intestinalis were split for comparison to standard Method 1623. Results were equivalent to immunomagnetic procedures for Cryptosporidium, and Giardia. Overall percent recovery for Cryptosporidium with Method 1623 averaged 26.89% (std 21.44%; min = 0%; max = 73%) and was similar but less variable for qPCR method at an estimated average of 27.67 (std 17.65%; min = 5%; max = 63%). For Giardia, Method 1623 had an overall average recovery of 27.11% (std 17.98%; min = 1%; max = 58%), while multistage purification and qPCR had an estimated lower overall recovery at 18.58% (std 13.95%; min = 0%; max = 35%). Microsporidia were also readily detected with an estimated recovery of 46.81% overall (std 17.66%; min = 18%; max = 70%) for E. intestinalis and 38.90% (std 14.36%; min = 13%; max = 62%) for E. bieneusi.
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