High numbers of Staphylococcus aureus at three bathing beaches in South Florida.

While the value of Staphylococcus aureus as an indicator for non-enteric diseases is unclear, understanding its prevalence in recreational beaches would prove useful, given its pathogenic potential. Staphylococcus aureus levels were evaluated in sand and seawater at three beaches during one year. To elucidate possible S. aureus sources or colonization trends, distribution in sand was analyzed at Hollywood Beach. Staphylococcus aureus levels fluctuated throughout the study with highest average densities detected in dry sand (3.46 × 105 CFU/g, Hobie Beach), particularly at beaches with high human density. Patchy distribution marked hotspots of human use and/or possible bacterial re-growth. Data from a brief epidemiological survey indicated a very slight association between beach usage and skin conditions; suggesting high S. aureus levels in sand may not necessarily constitute major health risks. Because the possibility of disease transmission exists, particularly to children and immuno-compromised beach-goers, periodic surveying of highly frequented beaches seems warranted.

Photoresponse in the heterotrophic marine dinoflagellate Oxyrrhis marina.

Expressed rhodopsins were detected by proteomic analysis in an investigation of potential signal receptors in the cell membrane of the marine heterotrophic dinoflagellate Oxyrrhis marina (CCMP604). We inferred these to be sensory rhodopsins, a type of G-protein-coupled receptor trans-membrane signaling molecule. Because phototactic behavior based on sensory rhodopsins has been reported in other protists, we investigated the photosensory response of O. marina. This dinoflagellate exhibited strongest positive phototaxis at low levels (2-3 µE/m(2)/s) of white light when the cells were previously light adapted and well fed. Positive phototaxis was also found for blue (450 nm), green (525 nm), and red (680 nm) wavelengths. In a further test, O. marina showed significantly greater phototaxis toward concentrated algal food illuminated by blue light to stimulate red chlorophyll-a autofluorescence in the prey, compared with using bleached algae as prey. Concentration of a cytoplasmic downstream messenger molecule, cyclic adenosine monophosphate, a component of the signaling pathway of G-protein-coupled receptor molecules, rapidly increased in O. marina cells after exposure to white light. In addition, treatment with hydroxylamine, a rhodopsin signaling inhibitor, significantly decreased their phototactic response. Our results demonstrate that a heterotrophic marine dinoflagellate can orient to light based on rhodopsins present in the outer cell membrane and may be able to use photosensory response to detect algal prey based on chlorophyll autofluorescence.

Using inhibitors to investigate the involvement of cell signaling in predation by marine phagotrophic protists.

Phagotrophic protists are major consumers of microbial biomass in aquatic ecosystems. However, biochemical mechanisms underlying prey recognition and phagocytosis by protists are not well understood. We investigated the potential roles of cell signaling mechanisms in chemosensory response to prey, and in capture of prey cells, by a marine ciliate (Uronema sp.) and a heterotrophic dinoflagellate (Oxyrrhis marina). Inhibition of protein kinase signal transduction biomolecules caused a decrease in both chemosensory response and predation. Inhibition of G-protein coupled receptor signaling pathways significantly decreased chemosensory response but had no effect on prey ingestion. Inhibitor compounds did not appear to affect general cell health, but had a targeted effect. These results support the idea that cell signaling pathways known in other eukaryotic organisms are involved in feeding behavior of free-living protists.

Prevalence and distribution of fecal indicator organisms in South Florida beach sand and preliminary assessment of health effects associated with beach sand exposure.

Fecal indicator levels in nearshore waters of South Florida are routinely monitored to assess microbial contamination at recreational beaches. However, samples of sand from the surf zone and upper beach are not monitored which is surprising since sand may accumulate and harbor fecal-derived organisms. This study examined the prevalence of fecal indicator organisms in tidally-affected beach sand and in upper beach sand and compared these counts to levels in the water. Since indicator organisms were statistically elevated in sand relative to water, the study also considered the potential health risks associated with beach use and exposure to sand. Fecal coliforms, Escherichia coli, enterococci, somatic coliphages, and F(+)-specific coliphages were enumerated from sand and water at three South Florida beaches (Ft. Lauderdale Beach, Hollywood Beach, and Hobie Beach) over a 2-year period. Bacteria were consistently more concentrated in 100g samples of beach sand (2-23 fold in wet sand and 30-460 fold in dry sand) compared to 100ml samples of water. Somatic coliphages were commonly recovered from both sand and water while F(+)-specific coliphages were less commonly detected. Seeding experiments revealed that a single specimen of gull feces significantly influenced enterococci levels in some 3.1m(2) of beach sand. Examination of beach sand on a micro-spatial scale demonstrated that the variation in enterococci density over short distances was considerable. Results of multiple linear regression analysis showed that the physical and chemical parameters monitored in this study could only minimally account for the variation observed in indicator densities. A pilot epidemiological study was conducted to examine whether the length of exposure to beach water and sand could be correlated with health risk. Logistic regression analysis results provided preliminary evidence that time spent in the wet sand and time spent in the water were associated with a dose-dependent increase in gastrointestinal illness.

The application of peptide nucleic acid probes for rapid detection and enumeration of eubacteria, Staphylococcus aureus and Pseudomonas aeruginosa in recreational beaches of S. Florida.

A novel chemiluminescent in situ hybridization technique using peptide nucleic acids (PNA) was adapted for the detection of bacteria in beach sand and recreational waters in South Florida. The simultaneous detection and enumeration of eubacteria and the novel indicators, Staphylococcus aureus and Pseudomonas aeruginosa, was achieved within 6-8 h of processing. Following 5 h of incubation on TSA, soybean peroxidase-labeled peptide nucleic acid probes (Boston Probes, Boston, MA) targeting species-specific 16S rRNA sequences of P. aeruginosa and S. aureus were used to hybridize microcolonies of the target species in-situ. In addition, a universal probe for 16S rRNA sequences was used to target the eubacteria. Probes were detected after a light generating reaction with a chemiluminescent substrate and their presence recorded on Polaroid film. The probes showed limited cross-reactivity with mixed indigenous bacteria extracted from seawater and sand by shaking with phosphate-buffered saline (PBS). Specificity and cross-reactivity was tested on the reference bacterial genera Pseudomonas, Staphylococcus, Vibrio, Shigella, Salmonella, Acinetobacter, Enterobacter, Escherichia and Citrobacter. These tests confirmed that the probes were specific for the microorganisms of interest and were unaffected by high salt levels. The results of the PNA chemiluminescent in situ hybridization were compared with traditional plate count methods (PCM) for total 'freshwater' eubacteria, S. aureus and P. aeruginosa. Counts of eubacteria and S. aureus were comparable with numbers obtained from traditional plate counts but levels of P. aeruginosa were higher with PNA than with PCM. It is possible that PNA is more sensitive than PCM because it can detect microcolonies on the agar surface that never fully develop with the plate count method. We conclude that the in situ hybridization technique used here represents an important potential tool for the rapid monitoring of novel indicator organisms in beaches and recreational waters.