1. Staley, C., T. Kaiser, M.L. Gidley, I.C. Enochs, P.R. Jones, K.D. Goodwin, C.D. Sinigalliano, M.J. Sadowsky, and C.L. Chun. A next-generation sequencing approach to characterize the impacts of land-based sources of pollution on the microbiota of southeast Florida coral reefs. Applied and Environmental Microbiology, 83(10):e03378-16, doi:10.1128/AEM.03378-16 2017


    Coral reefs are dynamic ecosystems known for decades to be endangered due, in large part, to anthropogenic impacts from land-based sources of pollution (LBSP). In this study, we utilized an Illumina-based next-generation sequencing approach to characterize prokaryotic and fungal communities from samples collected off the southeast coast of Florida. Water samples from coastal inlet discharges, oceanic outfalls of municipal wastewater treatment plants, treated wastewater effluent before discharge, open ocean samples, and coral tissue samples (mucus and polyps) were characterized to determine relationships between microbial communities in these matrices and those in reef water and coral tissues. Significant differences in microbial communities were noted among all sample types but varied between sampling area. Contamination from outfalls was found to be the greatest potential source of LBSP influencing native microbial community structure among all reef samples, although pollution from inlets was also noted. Notably, reef water and coral tissue communities were found to be more greatly impacted by LBSP at southern reefs, which also experienced the most degradation during the course of the study. Results of this study provide new insights into how microbial communities from LBSP can impact coral reefs in Southeast Florida and suggest that wastewater outfalls may have a greater influence on the microbial diversity and structure of these reef communities than do contaminants carried in runoff although the influence of runoff and coastal inlet discharge on coral reefs are still substantial.

  2. Haugland, R.A., S. Siefring, M. Varma, K.H. Oshima, M. Sivaganesan, Y. Cao, M. Raith, J. Griffith, S.B. Weisberg, R.T. Noble, A.D. Blackwood, J. Kinzelman, T. Anan’eva, R.N. Bushon, E.A. Stelzer, V.J. Harwood, K.V. Gordon, and C. Sinigalliano. Multi-laboratory survey of qPCR enterococci analysis method performance in U.S. coastal and inland surface waters. Journal of Microbiological Methods, 123:114-125, doi:10.1016/j.mimet.2016.01.017 2016


    Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are several methodological options. Here we evaluated how three method permutations, type of mastermix, sample extract dilution, and use of controls in results calculation affect method reliability among multiple laboratories with respect to sample interference. Multiple samples from each of 22 sites representing an array of habitat types were analyzed using EPA Method 1611 and 1609 reagents with full strength and five-fold diluted extracts. The presence of interference was assessed three ways: using sample processing and PCR amplifications controls; consistency of results across extract dilutions; and relative recovery of target genes from spiked enterococci in water samples compared to control matrices with acceptable recovery defined as 50 to 200%. Method 1609, which is based on an environmental mastermix, was found to be superior to Method 1611, which is based on a universal mastermix. Method 1611 had over a 40% control assay failure rate with undiluted extracts and a 6% failure rate with diluted extracts. Method 1609 failed in only 11% and 3% of undiluted and diluted extracts analyses. Use of sample processing control assay results in the delta–delta Ct method for calculating relative target gene recoveries increased the number of acceptable recovery results. Delta–delta tended to bias recoveries from apparent partially inhibitory samples on the high side which could help in avoiding potential underestimates of enterococci, an important consideration in a public health context. Control assay and delta–delta recovery results were largely consistent across the range of habitats sampled, and among laboratories. The methodological option that best balanced acceptable estimated target gene recoveries with method sensitivity and avoidance of underestimated enterococci densities was Method 1609 without extract dilution and using the delta–delta calculation method. The applicability of this method can be extended by the analysis of diluted extracts to sites where interference is indicated but, particularly in these instances, should be confirmed by augmenting the control assays with analyses for target gene recoveries from spiked target organisms.

  3. Symonds, E.M., C. Sinigalliano, M. Gidley, W. Ahmed, S.M. McQaig-Ulrich, and M. Breitbart. Fecal pollution along the southeastern coast of Florida and insight into the use of pepper mild mottle virus as an indicator. Journal of Applied Microbiology, 121(5):1469-1481, doi:10.1111/mam.13252 2016


    In 2014, bimonthly surface water samples were collected from inlets, exposed to runoff and septic seepage, and coastal sites, exposed to ocean outfalls. Analysis of culturable enterococci and a suite of microbial source tracking (MST) markers (BacHum, CowM2, DogBact, HF183, HPyV, PMMoV) revealed fecal pollution, primarily of human origin, at all sites. Since PMMoV was detected more frequently than other MST markers, the process limits of quantification (undiluted to 10−2 dilution) and detection (10−2 dilution) for the RT-qPCR method were determined by seeding untreated wastewater into the coastal waters. Simulated quantitative microbial risk assessment, employing human norovirus as a reference pathogen, calculated a 0·286 median risk of gastrointestinal illness associated with the PMMoV limit of detection. This study demonstrated that PMMoV is an important domestic wastewater-associated marker that should be included in the MST toolbox; therefore, future studies should thoroughly investigate the health risks associated with its detection and quantification in environmental waters.

  4. Campbell, A.M., J. Fleisher, C. Sinigalliano, J.R. White, and J.V. Lopez. Dynamics of marine bacterial community diversity of the coastal waters of the reefs, inlets, and wastewater outfalls of southeast Florida. Microbiology Open, 4(3):390-408, doi:10.1002/mbo3.245 2015


    Coastal waters adjacent to populated southeast Florida possess different habitats (reefs, oceanic inlets, sewage outfalls) that may affect the composition of their inherent microbiomes. To determine variation according to site, season, and depth, over the course of 1 year, we characterized the bacterioplankton communities within 38 nearshore seawater samples derived from the Florida Area Coastal Environment (FACE) water quality survey. Six distinct coastal locales were profiled – the Port Everglades and Hillsboro Inlets, Hollywood and Broward wastewater outfalls, and associated reef sites using culture-independent, high-throughput pyrosequencing of the 16S rRNA V4 region. More than 227,000 sequences helped describe longitudinal taxonomic profiles of marine bacteria and archaea. There were 4447 unique operational taxonomic units (OTUs) identified with a mean OTU count of 5986 OTUs across all sites. Bacterial taxa varied significantly by season and by site using weighted and unweighted Unifrac, but depth was only supported by weighted Unifrac, suggesting a change due to presence/absence of certain OTUs. Abundant microbial taxa across all samples included Synechococcus, Pelagibacteraceae, Bacteroidetes, and various Proteobacteria. Unifrac analysis confirmed significant differences at inlet sites relative to reef and outfalls. Inlet-based bacterioplankton significantly differed in greater abundances of Rhodobacteraceae and Cryomorphaceae, and depletion of SAR406 sequences. This study also found higher counts of Firmicutes, Chloroflexi, and wastewater associated SBR1093 bacteria at the outfall and reef sites compared to inlet sites. This study profiles local bacterioplankton populations in a much broader context, beyond culturing and quantitative PCR, and expands upon the work completed by the National Oceanic and Atmospheric Administration FACE program.

  5. Carsey, T.P., S.J. Stamates, C.M. Featherstone, N. Amornthammarong, J.R. Bishop, C.J. Brown, A. Campbell, H.L. Casanova, M.L. Gidley, M. Kosenko, R.M. Kotkowski, J.V. Lopez, C.D. Sinagalliano, L.A. Visser, and J.-Z. Zhang. Broward County coastal ocean water quality study, 2010-2012. NOAA Technical Report, OAR-AOML-44, 217 pp., doi:10.7289/V5TD9VCC 2015


    Researchers with the Ocean Chemistry and Ecosystems Division of NOAA’s Atlantic Oceanographic and Meteorological Laboratory conducted 12 monthly cruises in two separate track lines off of Broward County, Florida, from November 2010 through January 2012. The cruise tracks were designed to provide information on three categories of the coastal ocean: (1) the vicinity of the Broward and Hollywood treated-wastewater outfalls; (2) the vicinity of the Hillsboro and Port Everglades inlets; and (3) the interstitial areas in between. Sampling took place from aboard the NOAA R/V Hildebrand using a conductivity-temperature-depth (CTD)/rosette for water samples and water column profiles and appropriately located acoustic Doppler current profiler (ADCP) instruments for ocean current information. Measured discrete parameters included location, depth, salinity, temperature, pH, oxygen saturation (dissolved oxygen, DO), oxidation-reduction (redox) potential (ORP), chlorophyll-a, phaeopigments, total suspended solids (TSS), nitrate (NO3), nitrite (NO2), ammonium (NH4), silicate (Si), orthophosphate (PO4), total dissolved nitrogen (TDN), total dissolved phosphorus (TDP), particulate carbon (PC), particulate phosphorus (PP), particulate nitrogen (PN), and dissolved organic carbon (DOC). CTD profile data included depth, turbidity, ORP, DO, pH, chlorophyll-a, salinity, temperature, and density. A variety of microbiological entities were measured, including fecal indicator bacteria (FIB), selected waterborne pathogens, and molecular microbial source tracking (MST) markers. Community bacterial metagenomic profiles were also generated for selected sample sites. Quality controls of nutrient sample analyses were obtained following National Environmental Laboratory Accreditation Conference (NELAC)-certified procedures. The data obtained present a view of the coastal ocean as having a low “background” concentration of most analytes, interrupted by elevated concentrations near the outfalls and inlets whose excess concentrations decreased rapidly away from the point sources. The waters were found to be oligotrophic, with no evidence of bloom events. A major upwelling event was observed on August 11, 2011, where a ~10°C temperature drop was observed near the southernmost portion of the sampled area.

  6. Kopf, A., M. Bicak, R. Kottmann, J. Schnetzer, I. Kostadinov, K. Lehmann, A. Fernandez-Guerra, C. Jeanthon, E. Rahav, M. Ullrich, A. Wichels, G. Gerdts, P. Polymenakou, G. Kotoulas, R. Siam, R.Z. Abdallah, E.C. Sonnenschein, T. Cariou, F. O’Gara, S. Jackson, S. Orlic, M. Steinke, J. Busch, B. Duarte, I. Cacador, J. Canning-Clode, O. Bobrova, V. Marteinsson, E. Reynisson, C.M. Loureiro, G.M. Luna, G.M. Quero, C.R. Loscher, A. Kremp, M.E. DeLorenzo, L. Ovreas, J. Tolman, J. LaRoche, A. Penna, M. Frischer, T. Davis, K. Barker, C.P. Meyer, S. Ramos, C. Magalhaes, F. Jude-Lemeilleur, M.L. Aguirre-Macedo, S. Wang, N. Poulton, S. Jones, R. Collin, J.A. Fuhrman, P. Conan, C. Alonso, N. Stambler, K. Goodwin, et al. The ocean sampling day consortium. GigaScience, 4:27, doi:10.1186/s13742-015-0066-5 2015


    Ocean Sampling Day was initiated by the EU-funded Micro B3 (Marine Microbial Biodiversity, Bioinformatics, Biotechnology) project to obtain a snapshot of the marine microbial biodiversity and function of the world’s oceans. It is a simultaneous global mega-sequencing campaign aiming to generate the largest standardized microbial data set in a single day. This will be achievable only through the coordinated efforts of an Ocean Sampling Day Consortium, supportive partnerships and networks between sites. This commentary outlines the establishment, function and aims of the Consortium and describes our vision for a sustainable study of marine microbial communities and their embedded functional traits.

  7. Joyner, J., D. Wanless, C.D. Sinigalliano, and E.K. Lipp. Use of quantitative real-time PCR for direct detection of Serratia marcescens in marine and other aquatic environments. Applied and Environmental Microbiology, 80(5):1679-1683, doi:10.1128/AEM.02755-13 2014


    Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml−1 and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml−1. This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.

  8. Ebentier, D.L., K.T. Hanley, Y. Cao, B.D. Badgley, A.B. Boehm, J.S. Ervin, K.D. Goodwin, M. Gourmelon, J.F. Griffith, P.A. Holden, C.A. Kelty, S. Lozach, C. McGee, L.A. Peed, M. Raith, H. Ryu, M.J. Sadowsky, E.A. Scott, J. Santo Domingo, A. Schriewer, C.D. Sinigalliano, O.C. Shanks, L.C. Van De Werfhorst, D. Wang, S. Wuertz, and J.A. Jay. Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods. Water Research, 47(18):6839-6848, doi:10.1016/j.watres.2013.01.060 2013


    Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing the standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (Bsteri, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories.

  9. Hower, S., M.C. Phillips, M. Brodsky, A. Dameron, M.A. Tamargo, N.C. Salazar, C.R. Jackson, J.B. Barrett, M. Davidson, J. Davis, S. Mukherjee, R.Y. Ewing, M.L. Gidley, C.D. Sinigalliano, L. Johns, F.E. Johnson, O. Adebanjo, and L.W.R. Plano. Clonally related methicillin-resistant Staphylococcus aureus isolated from short-finned pilot whales (Globicephala macrorhynchus), human volunteers, and a bayfront cetacean rehabilitation facility. Microbial Ecology, 65(4):1024-1038, doi:10.1007/s00248-013-0178-3 2013


    In May of 2011, a live mass stranding of 26 short-finned pilot whales (Globicephala macrorhynchus) occurred in the lower Florida Keys. Five surviving whales were transferred from the original stranding site to a nearby marine mammal rehabilitation facility where they were constantly attended to by a team of volunteers. Bacteria cultured during the routine clinical care of the whales and necropsy of a deceased whale included methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). In order to investigate potential sources or reservoirs of MSSA and MRSA, samples were obtained from human volunteers, whales, seawater, and sand from multiple sites at the facility, nearby recreational beaches, and a canal. Samples were collected on 3 days. The second collection day was 2 weeks after the first, and the third collection day was 2 months after the last animal was removed from the facility. MRSA and MSSA were isolated on each day from the facility when animals and volunteers were present. MSSA was found at an adjacent beach on all three collection days. Isolates were characterized by utilizing a combination of quantitative real-time PCR to determine the presence of mecA and genes associated with virulence, staphylococcal protein A typing, staphylococcal cassette chromosome mec typing, multilocus sequence typing, and pulsed field gel electrophoresis (PFGE). Using these methods, clonally related MRSA were isolated from multiple environmental locations as well as from humans and animals. Non-identical but genetically similar MSSA and MRSA were also identified from distinct sources within this sample pool. PFGE indicated that the majority of MRSA isolates were clonally related to the prototype human strain USA300. These studies support the notion that S. aureus may be shed into an environment by humans or pilot whales and subsequently colonize or infect exposed new hosts.

  10. Layton, B.A., Y. Cao, D.L. Ebentier, K. Hanley, E. Balleste, J. Brandao, M. Byappanahalli, R. Converse, A.H. Farnleitner, J. Gentry-Shields, M.L. Gidley, M. Gourmelon, C.S. Lee, J. Lee, S. Lozach, T. Madi, W.G. Meijer, R. Noble, L. Peed, G.H. Reischer, R. Rodrigues, J.B. Rose, A. Schriewer, C.D. Sinigalliano, S. Srinivasan, J. Stewart, L.C. Van De Werfhorst, D. Wang, R. Whitman, S. Wuertz, J. Jay, P.A. Holden, A.B. Boehm, O. Shanks, and J.F. Griffith. Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study. Water Research, 47(18):6897-6908, doi:10.1016/j.watres.2013.05.060 2013


    A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, B. thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman, HumM2, and M. smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: (1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; (2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc.); and (3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman was found to be the most effective marker of human fecal contamination in this California-based study.

  11. Plano, L.R.W., T. Shibata, A.C. Garza, J. Kish, J.M. Fleisher, C.D. Sinigalliano, M.L. Gidley, K. Withum, S.M. Elmir, S. Hower, C.R. Jackson, J.B. Barrett, T. Cleary, M. Davidson, J. Davis, S. Mukherjee, L.E. Fleming, and H.M. Solo-Gabriele. Human-associated methicillin-resistant Staphylococcus aureus from a subtropical recreational marine beach. Microbial Ecology, 65(4):1039-1051, doi:10.1007/s00248-013-0216-1 2013


    Reports of Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) detected in marine environments have occurred since the early 1990s. This investigation sought to isolate and characterize S. aureus from marine waters and sand at a subtropical recreational beach, with and without bathers present, in order to investigate possible sources and to identify the risks to bathers of exposure to these organisms. During 40 days over 17 months, 1,001 water and 36 intertidal sand samples were collected by either bathers or investigators at a subtropical recreational beach. Methicillin-sensitive S. aureus (MSSA) and MRSA were isolated and identified using selective growth media and an organism-specific molecular marker. Antimicrobial susceptibility, staphylococcal cassette chromosome mec (SCCmec) type, pulsed-field gel electrophoresis (PFGE) pattern, multi-locus sequence type (MLST), and staphylococcal protein A (spa) type were characterized for all MRSA. S. aureus was isolated from 248 (37%) bather nearby water samples at a concentration range of <2-780 colony forming units per ml, 102 (31%) ambient water samples at a concentration range of <2-260 colony forming units per ml, and 9 (25%) sand samples. Within the sand environment, S. aureus was isolated more often from above the intertidal zone than from intermittently wet or inundated sand. A total of 1334 MSSA were isolated from 37 sampling days and 22 MRSA were isolated from ten sampling days. Seventeen of the 22 MRSA were identified by PFGE as the community-associated MRSA USA300. MRSA isolates were all SCCmec type IVa, encompassed five spa types (t008, t064, t622, t688, and t723), two MLST types (ST8 and ST5), and 21 of 22 isolates carried the genes for Panton–Valentine leukocidin. There was a correlation (r = 0.45; p = 0.05) between the daily average number of bathers and S. aureus in the water; however, no association between exposure to S. aureus in these waters and reported illness was found. This report supports the concept that humans are a potential direct source for S. aureus in marine waters.

  12. Schriewer, A., K.D. Goodwin, C.D. Sinigalliano, A.M. Cox, D. Wanless, J. Bartkowiak, D.L. Ebentier, K.T. Hanley, J. Ervin, L.A. Deering, O.C. Shanks, L.A. Peed, W.G. Meijer, J.F. Griffith, J. Santo Domingo, J.A. Jay, P.A. Holden, and S. Wuertz. Performance evaluation of canine-associated Bacterioidales assays in a multi-laboratory comparison study. Water Research, 47(18):6909-6920, doi:10.1016/j.watres.2013.03.062 2013


    The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.

  13. Sinigalliano, C.D., J.S. Ervin, L.C. Van De Werfhorst, B.D. Badgley, E. Balleste, J. Bartkowiak, A.B. Boehm, M. Byappanahalli, K.D. Goodwin, M. Gourmelon, J. Griffith, P.A. Holden, J. Jay, B. Layton, C. Lee, J. Lee, W.G. Meijer, R. Noble, M. Raith, H. Ryu, M.J. Sadowsky, A. Schriewer, D. Wang, D. Wanless, R. Whitman, S. Wuertz, and J.W. Santo Domingo. Multi-laboratory evaluations of the performance of Catellicoccus marimammalium PCR assays developed to target gull fecal sources. Water Research, 47(18):6883-6896, doi:10.1016/j.watres.2013.02.059 2013


    Here we report results from a multi-laboratory (n=11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR® Green qPCR method, and two TaqMan® qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.

  14. Carsey, T.P., S.J. Stamates, N. Amornthammarong, J.R. Bishop, F. Bloetscher, C.J. Brown, J.F. Craynock, S.R. Cummings, W.P. Dammann, J. Davis, C.M. Featherstone, C.J. Fischer, K.D. Goodwin, D.E. Meeroff, J.R. Proni, C.D. Sinigalliano, P.K. Swart, and J.-Z. Zhang. Boynton Inlet 48-hour sampling intensives: June and September 2007. NOAA Technical Report, OAR-AOML-40, 43 pp., 2012


    Researchers with the Ocean Chemistry Division of NOAA’s Atlantic Oceanographic and Meteorological Laboratory performed two 48-hour intensive studies of the water flowing through the Boynton Inlet at Boynton Beach, Florida, during June and September 2007. These studies were conducted in support of the Florida Area Coastal Environment (FACE) program. Academic partners who also participated in the effort included colleagues with the University of Miami’s Cooperative Institute for Marine and Atmospheric Studies and the Rosenstiel School of Marine and Atmospheric Science, Florida Atlantic University’s Laboratories for Engineered Environmental Solutions, and the Applied Research Center of Florida International University. Sampling was performed from the southern boardwalk at Boynton Beach during the June intensive and the Boynton Beach Inlet Bridge during the September intensive. The sampling strategy was designed to collect water samples over four complete tidal cycles for each intensive; these data would be employed to quantify the total flux of nearshore-source entities into the coastal waters. The first sampling event was conducted on June 4-6, 2007, and the second was conducted on September 26-28, 2007. Data collected include nutrients (silicate, orthophosphate, ammonium, nitrite+nitrate), isotope ratios of nitrogen, the presence or absence of selected biological indicators (Escherichia coli, enterococci, and total coliform), and physical parameters that included pH, salinity, total suspended solids, and turbidity. Critical to this study was the continuous in situ flow rate measurements obtained via an acoustic Doppler current profiler (ADCP) mounted on the north side of the inlet. This report presents the data gathered from the two sampling intensives. The data reported herein suggest that inlets are important contributors of nutrient and microbiological loads to the coastal zone. The overall view presented is that the lagoon input into Boynton Inlet may be substantial but is also highly variable.

  15. Shanks, O.C., M. Sivaganesan, L. Peed, C.A. Kelty, A.D. Blackwood, M.R. Greene, R.T. Noble, R.N. Bushon, E.A. Stelzer, J. Kinzelman, T. Anan'eva, C. Sinigalliano, D. Wanless, J. Griffith, Y. Cao, S. Weisberg, V.J. Harwood, C. Staley, K.H. Oshima, M. Varma, and R.A. Haugland. Interlaboratory comparison of real-time PCR protocols for quantification of general fecal indicator bacteria. Environmental Science and Technology, 46(2):945-953, doi:10.1021/es2031455 2012


    The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol.

  16. Abdelzaher, A.A., M.E. Wright, C. Ortega, A.R. Hasan, T. Shibata, H.M. Solo-Gabriele, J. Kish, K. Withum, G. He, S.M. Elmir, J.A. Bonilla, T.D. Bonilla, C.J. Palmer, T.M. Scott, J. Lukasik, V.J. Harwood, S. McQuaig, C.D. Sinigalliano, M.L. Gidley, D. Wanless, L.R.W. Plano, A.C. Garza, X. Zhu, J.R. Stewart, J.W. Dickerson, H. Yampara-Iquise, C. Carson, J.M. Fleisher, and L.E. Fleming. Daily measures of microbes and human health at a non-point source marine beach. Journal of Water and Health, 9(3):443-457, doi:10.2166/wh.2011.146 2011

    Abstract: Studies evaluating the relationship between microbes and human health at non-point source beaches are necessary for establishing criteria which would protect public health while minimizing economic burdens. The objective of this study was to evaluate water quality and daily cumulative health effects (gastrointestinal, skin, and respiratory illnesses) for bathers at a non-point source subtropical marine recreational beach in order to better understand the inter-relationships between these factors and hence improve monitoring and pollution prevention techniques. Daily composite samples were collected, during the Oceans and Human Health Beach Exposure Assessment and Characterization Health Epidemiologic Study conducted in Miami (Florida, USA) at a non-point source beach, and analyzed for several pathogens, microbial source tracking markers, indicator microbes, and environmental parameters. Analysis demonstrated that rainfall and tide were more influential, when compared to other environmental factors and source tracking markers, in determining the presence of both indicator microbes and pathogens. Antecedent rainfall and F+ coliphage detection in water should be further assessed to confirm their possible association with skin and GI illness outcomes, respectively. The results of this research illustrate the potential complexity of beach systems characterized by non-point sources, and how more novel and comprehensive approaches are needed to assess beach water quality for the purpose of protecting bather health.

  17. Carsey, T.P., C.M. Featherstone, K.D. Goodwin, C.D. Sinigalliano, S.J. Stamates, J.-Z. Zhang, J. Proni, J.R. Bishop, C.J. Brown, M. Adler, P.L. Blackwelder, and H. Alsayegh. The Boynton-Delray coastal water quality monitoring program. NOAA Technical Report, OAR-AOML-39, 177 pp., 2011


    This report discusses a sequence of six cruises in the vicinity of the Boynton-Delray (South Central) treated-wastewater plant outfall plume (26°27'43"N, 80°2'32"W), the Boynton Inlet (26°32'43"N, 80°2'30"W), and the Lake Worth Lagoon, Palm Beach County, Florida. The sampling cruises took place on June 5-6, 2007; August 28-29, 2007; October 18-19, 2007; February 14 and 18, 2008; May 19-20, 2008; and July 11-13, 2008. Water was sampled at 18 locations at the surface, middle, and near the seafloor (where there was sufficient depth) for a total of 45 samples; these samples were analyzed for a variety of nutrients and related parameters. The water sampling unit contained a conductivity-temperature-depth (CTD) instrument from which data were obtained at each sampling site. Synchronal ocean current data were measured by a nearby acoustic Doppler current profiler (ADCP) instrument.

  18. Gooch-Moore, J., K.D. Goodwin, C. Dorsey, R.D. Ellender, J.B. Mott, M. Ornelas, C.D. Sinigalliano, R. Vincent, D. Whiting, and S.H. Wolfe. New USEPA water quality criteria by 2012: GOMA concerns and recommendations. Journal of Water and Health, 9(4):718-733, doi:10.2166/wh.2011.156 2011


    The Gulf of Mexico Alliance (GOMA) was tasked by the five Gulf State Governors to identify major issues affecting the Gulf of Mexico (GoM) and to set priorities for ameliorating these problems. One priority identified by GOMA is the need to improve detection methods for water quality indicators, pathogens and microbial source tracking. The United States Environmental Protection Agency (USEPA) is tasked with revising water quality criteria by 2012; however, the locations traditionally studied by the USEPA are not representative of the GoM and this has raised concern about whether or not the new criteria will be appropriate. This paper outlines a number of concerns, including deadlines associated with the USEPA Consent Decree, which may prevent inclusion of research needed to produce a well-developed set of methods and criteria appropriate for all regulated waters. GOMA makes several recommendations including ensuring that criteria formulation use data that include GoM-specific conditions (e.g., lower bather density, nonpoint sources), that rapid-testing methods be feasible and adequately controlled, and that USEPA maintains investments in water quality research once the new criteria are promulgated in order to assure that outstanding scientific questions are addressed and that scientifically defensible criteria are achieved for the GoM and other regulated waterbodies.

  19. Plano, L.R.W., A. Garza, T. Shibata, S.M. Elmir, J. Kish, C.D. Sinigalliano, M.L. Gidley, G. Miller, K. Withum, L.E. Fleming, and H.M. Solo-Garbriele. Shedding of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus from adult and pediatric bathers in marine waters. BMC Microbiology, 11:5, 10 pp., doi:10.1186/1471-2180-11-5 2011

    Abstract: Staphylococcus aureus including methicillin resistant S. aureus, MRSA, are human colonizing bacteria that commonly cause opportunistic infections primarily involving the skin in otherwise healthy individuals. These infections have been linked to close contact and sharing of common facilities such as locker rooms, schools, and prisons. Waterborne exposure and transmission routes have not been traditionally associated with S. aureus infections. Coastal marine waters and beaches used for recreation are potential locations for the combination of high numbers of people with close contact and, therefore, could contribute to the exposure to and infection by these organisms. The primary aim of this study was to evaluate the amount and characteristics of the shedding of methicillin sensitive S. aureus, MSSA and MRSA by human bathers in marine waters. Nasal cultures were collected from bathers, and water samples were collected from two sets of pools designed to isolate and quantify MSSA and MRSA shed by adults and toddlers during exposure to marine water. A combination of selective growth media and biochemical and polymerase chain reaction analysis was used to identify and perform limited characterization of the S. aureus isolated from the water and the participants. Twelve of 15 MRSA isolates collected from the water had identical genetic characteristics as the organisms isolated from the participants exposed to that water, while the remaining 3 MRSA were without matching nasal isolates from participants. The amount of S. aureus shed per person corresponded to 105 to 106 CFU per person per 15-minute bathing period, with 15 to 20% of this quantity testing positive for MRSA. This is the first report of a comparison of human colonizing organisms with bacteria from human exposed marine water attempting to confirm that participants shed their own colonizing MSSA and MRSA into their bathing milieu. These findings clearly demonstrate that adults and toddlers shed their colonizing organisms into marine waters and, therefore, can be sources of potentially pathogenic S. aureus and MRSA in recreational marine waters. Additional research is needed to evaluate recreational beaches and marine waters as potential exposure and transmission pathways for MRSA.

  20. Shah, A.H., A.M. Abdelzaher, M. Phillips, R. Hernandez, H.M. Solo-Gabriele, J. Kish, G. Scorzetti, J.W. Fell, M.R. Diaz, T.M. Scott, J. Lukasik, V.J. Harwood, S. McQuaig, C.D. Sinigalliano, M.L. Gidley, D. Wanless, A. Agar, J. Lui, J.R. Stewart, L.R.W. Plano, and L.E. Fleming. Indicator microbes correlate with pathogenic bacteria, yeast, and helminthes in sand at a subtropical recreational beach site. Journal of Applied Microbiology, 110(6):1571-1583, doi:10.1111/j.1365-2672.2011.05013.x. 2011

    Abstract: Research into the relationship between pathogens, fecal indicator microbes, and environmental factors in beach sand has been limited, yet vital to the understanding of the microbial relationship between sand and the water column and to the improvement of criteria for better human health protection at beaches. The objectives of this study were to evaluate the presence and distribution of pathogens in various zones of beach sand (subtidal, intertidal, and supratidal) and to assess their relationship with environmental parameters and indicator microbes at a non-point source subtropical marine beach. In this exploratory study in subtropical Miami (Florida, USA), beach sand samples were collected and analyzed over the course of 6 days for several pathogens, microbial source tracking markers, and indicator microbes. An inverse correlation between moisture content and most indicator microbes was found. Significant associations were identified between some indicator microbes and pathogens (such as nematode larvae and yeasts in the genus Candida), which are from classes of microbes that are rarely evaluated in the context of recreational beach use. Results indicate that indicator microbes may predict the presence of some of the pathogens, in particular helminthes, yeasts, and the bacterial pathogen Staphylococcus aureus including methicillin-resistant forms. Indicator microbes may thus be useful for monitoring beach sand and water quality at non-point source beaches. The presence of both indicator microbes and pathogens in beach sand provides one possible explanation for human health effects reported at non-point sources beaches.

  21. Solo-Gabriele, H.M., A.B. Boehm, T.M. Scott, and C.D. Sinigalliano. Microbial source tracking in beaches and coastal environments. In Microbial Source Tracking: Methods, Applications, and Case Studies, E. Hagedorn, A. Blanch, and V.J. Harwood (eds.). Springer Press, 451-484, 2011

    Abstract: This chapter summarizes the rationale for using microbial source tracking (MST) methods at beach sites and coastal water bodies, as MST methods are especially useful for evaluating waters impacted by nonpoint sources of pollution. This chapter also describes the most common traditional and alternative MST markers used at beach sites. Two case studies are presented that describe the use of both biological/chemical MST methods and physical MST methods for identifying sources of microbes at two marine beach sites in the USA, one located on the west coast (California) and the other located on the east coast (Florida). The chapter closes with a discussion and recommendations concerning the utility and application of MST tools at beach sites impacted by nonpoint-source pollution. Although this chapter focuses on marine beaches, an incredible wealth of MST data has been gathered at freshwater beaches (Byappanahalli et al., 2006; Harwood et al., 2005; Jenkins et al., 2005; Scott et al., 2002; Stapleton et al., 2009; Whitman and Nevers, 2003; Whitman et al., 2004), and a comprehensive review of beach studies merits the inclusion of MST work within freshwater systems. The use of MST in freshwater systems is further discussed in chapters 18, 19, and 21.

  22. Abdelzaher, A.M., M.E. Wright, C. Ortega, H.M. Solo-Gabriele, G. Miller, S. Elmir, X. Newman, P. Shih, J.A. Bonilla, T.D. Bonilla, C.J. Palmer, T. Scott, J. Lukasik, V.J. Harwood, S. McQuaig, C. Sinigalliano, M. Gidley, L.R.W. Plano, X. Zhu, J.D. Wang, and L.E. Fleming. Presence of pathogens and indicator microbes at a non-point source subtropical recreational marine beach. Applied and Environmental Microbiology, 76(3):724-732, doi:10.1128/AEM.02127-09 2010

    Abstract: Swimming in ocean water, including ocean water at beaches not impacted by known point sources of pollution, is an increasing health concern. This study was an initial evaluation of the presence of indicator microbes and pathogens and the association among the indicator microbes, pathogens, and environmental conditions at a subtropical, recreational marine beach in south Florida impacted by non-point sources of pollution. Twelve water and eight sand samples were collected during four sampling events at high or low tide under elevated or reduced solar insolation conditions. The analyses performed included analyses of fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli, enterococci, and Clostridium perfringens), human-associated microbial source tracking (MST) markers (human polyomaviruses [HPyVs] and Enterococcus faecium esp gene), and pathogens (Vibrio vulnificus, Staphylococcus aureus, enterovirus, norovirus, hepatitis A virus, Cryptosporidium spp., and Giardia spp.). The enterococcus concentrations in water and sand determined by quantitative PCR were greater than the concentrations determined by membrane filtration measurement. The FIB concentrations in water were below the recreational water quality standards for three of the four sampling events, when pathogens and MST markers were also generally undetectable. The FIB levels exceeded regulatory guidelines during one event, and this was accompanied by detection of HPyVs and pathogens, including detection of the autochthonous bacterium V. vulnificus in sand and water, detection of the allochthonous protozoans Giardia spp. in water, and detection of Cryptosporidium spp. in sand samples. The elevated microbial levels were detected at high tide and under low-solar-insolation conditions. Additional sampling should be conducted to further explore the relationships between tidal and solar insolation conditions and between indicator microbes and pathogens in subtropical recreational marine waters impacted by non-point source pollution.

  23. Carsey, T.P., H. Casanova, C. Drayer, C. Featherstone, C. Fischer, K. Goodwin, J. Proni, A. Saied, C. Sinigalliano, J. Stamates, P. Swart, and J.-Z. Zhang. FACE outfalls survey cruise: October 6-19, 2006. NOAA Technical Report, OAR-AOML-38, 130 pp. (CD-ROM), 2010


    In October 2006, a cruise of the Florida Area Coastal Environment (FACE) program was conducted aboard the NOAA RV Nancy Foster. The cruise visited coastal sites in the vicinity of six treated wastewater boils in south Florida. The outfalls included in this study were those for the South Central, Boca Raton, Broward, Hollywood, Miami-Dade North, and Miami-Dade Central wastewater treatment plants. The boils and associated down-current plumes were studied to produce a data set of ocean currents, ocean chemistry, and microbiology. Seawater was analyzed for nitrate + nitrite, nitrite, ammonia, orthophosphate, and silicate. Samples down-current of each boil were collected by a conductivity-temperature-depth (CTD) rosette at three depths--near-surface, mid-depth, and near-bottom--and from three transects--inshore of the boil, approximately in line with the boil, and further offshore of the boil. Overall, surface samples showed the highest nutrient concentrations versus samples taken at other depths; surface samples taken nearest the boil showed the highest nutrient concentrations in comparison to other samples collected in the vicinity of the outfall. The only exception was Si, which had a maximum observed concentration at an inlet sampling site. The outfall plume was found to be dynamic, irregular, and mainly in the upper 10 m of the water column. Samples were analyzed for a variety of microbes; the detection frequency was higher for the southern boils compared to the northern boils. The CTD data indicated a tendency toward better defined and deeper thermoclines at the 20-30 m depth in the deeper (more offshore) casts; the thermocline showed a tendency to shoal at 10-20 m and become less well defined in more inshore casts.

  24. Fleisher, J.M., L.E. Fleming, H.M. Solo-Gabriele, J.K. Kish, C.D. Sinigalliano, L.W. Plano, S.M. Elmir, J.D. Wang, K.F. Withum, T. Shibata, M.L. Gidley, A. Abdelzaher, G. He, C. Ortega, X. Zhu, M.D. Wright, J.A. Hollenbeck, and L.C. Backer. The BEACHES Study: Health effects and exposures from nonpoint source microbial contaminants in subtropical recreational marine waters. International Journal of Epidemiology, 39(5):1291-1298, doi:10.1093/ije/dyq084 2010

    Abstract: Microbial water quality indicators, found in high concentrations in sewage, are used to determine whether water is safe for recreational purposes. Recently, concerns have been raised about the appropriate use of indicators to regulate recreational water bodies, particularly non-point source sub/tropical recreational marine waters. A group of 1303 adult regular bathers were randomly assigned to bather and non-bather groups, with subsequent follow-up for reported illness, in conjunction with an ongoing extensive environmental sampling of indicator organisms (enterococcus) and pathogens. After controlling for non-water related risk factors/possible confounders, bathers were 1.75 times (95% confidence interval = 0.94-3.26; p = 0.078) more likely to report gastrointestinal illness relative to non bathers; bathers were 4.46 times (0.99-20.97; p = 0.051) more likely to report acute febrile respiratory illness; and bathers were 5.51 times (2.68-11.36; p = 0.<0.0001) more likely to report skin illness relative to non-bathers after seven days of follow up from beach exposure. There was an apparent dose-response relationship between bather exposure to increasing levels of enterococcus and risks of reported skin and gastrointestinal illness, and some evidence of increased risk of respiratory illness among bathers relative to nonbathers. There was a possible threshold effect for gastrointestinal illness at approximately 40 CFU/100 mL of enterococcus; however, no threshold effect was observed for skin illness. Human exposures to and health risks from microbial pollution may occur even in a non-point source recreational marine environment.

  25. Shibata, T., H.M. Solo-Gabriele, C.D. Sinigalliano, M.L. Gidley, L.R.W. Plano, J.M. Fleisher, J.D. Wang, S.M. Elmir, G. He, M.E. Wright, A.M. Abdelzaher, C. Ortega, D. Wanless, A.C. Garza, J. Kish, T. Scott, J. Hollenbeck, L.C. Backer, and L.E. Fleming. Evaluation of conventional and alternative monitoring methods for a recreational marine beach with nonpoint source of fecal contamination. Environmental Science and Technology, 44(21):8175-8181, doi:10.1021/es100884w 2010


    The objectives of this work were to compare enterococci (ENT) measurements based on the membrane filter, ENT(MF) with alternatives that can provide faster results including alternative enterococci methods (e.g., chromogenic substrate (CS), and quantitative polymerase chain reaction (qPCR)), and results from regression models based upon environmental parameters that can be measured in real-time. ENT(MF) were also compared to source tracking markers (Staphylococcus aureus, Bacteroidales human and dog markers, and Catellicoccus gull marker) in an effort to interpret the variability of the signal. Results showed that concentrations of enterococci based upon MF (p < 0.01). The correlations between MF and CS (r = 0.58, p < 0.01) were stronger than between MF and qPCR (r ≤ 0.36, p < 0.01). Enterococci levels by MF, CS, and qPCR methods were positively correlated with turbidity and tidal height. Enterococci by MF and CS were also inversely correlated with solar radiation but enterococci by qPCR was not. The regression model based on environmental variables provided fair qualitative predictions of enterococci by MF in real-time, for daily geometric mean levels, but not for individual samples. Overall, ENT(MF) was not significantly correlated with source tracking markers with the exception of samples collected during one storm event. The inability of the regression model to predict ENT(MF) levels for individual samples is likely due to the different sources of ENT impacting the beach at any given time, making it particularly difficult to to predict short-term variability of ENT(MF) for environmental parameters.

  26. Sinigalliano, C.D., J.M. Fleisher, M.L. Gidley, H.M. Solo-Gabriele, T. Shibata, L.R.W. Plano, S.M. Elmir, D. Wanless, J. Bartkowiak, R. Boiteau, K. Withum, A.M. Abdelzaher, G. He, C. Ortega, X. Zhu, M.E. Wright, J. Kish, J. Hollenbeck, T. Scott, L.C. Backer, and L.E. Fleming. Traditional and molecular analyses for fecal indicator bacteria in non-point source subtropical recreational marine waters. Water Research, 44(13):3763-3772, doi:10.1016/j.watres.2010.04.026 2010

    Abstract: The use of enterococci as the primary fecal indicator bacteria (FIB) for the determination of recreational water safety has been questioned, particularly in sub/tropical marine waters without known point sources of sewage. Alternative FIB (such as the Bacteroidales group) and alternative measurement methods (such as rapid molecular testing) have been proposed to supplement or replace current marine water quality testing methods which require culturing enterococci. Moreover, environmental parameters have also been proposed to supplement current monitoring programs. The objective of this study was to evaluate the health risks to humans from exposure to subtropical recreational marine waters with no known point source. The study reported symptoms between one set of human subjects randomly assigned to marine water exposure with intensive environmental monitoring compared with other subjects who did not have exposure. In addition, illness outcomes among the exposed bathers were compared to levels of traditional and alternative FIB (as measured by culture-based and molecular-based methods) and compared to easily measured environmental parameters. Results demonstrated an increase in self-reported gastrointestinal, respiratory and skin illnesses among bathers versus non-bathers. Among the bathers, a dose-response relationship by logistic regression modeling was observed for skin illness, where illness was positively related to enterococci enumeration by membrane filtration (odds ratio = 1.46 [95% confidence interval = 0.97-2.21] per increasing log10 unit of enterococci exposure) and positively related to 24 hour antecedent rain fall (1.04 [1.01-1.07] per increasing millimeters of rain). Acute febrile respiratory illness was inversely related to water temperature (0.74 [0.56-0.98] per increasing degree of water temperature). There were no significant dose response relationships between report of human illness and any of the other FIB or environmental measures. Therefore, for non-point source subtropical recreational marine waters, this study suggests that humans may be at increased risk of reported illness, and that the currently recommended and investigational FIB may not track gastrointestinal illness under these conditions; the relationship between other human illness and environmental measures is less clear.

  27. Carsey, T., K.D. Goodwin, J. Hendee, J.R. Proni, C. Sinigalliano, J. Stewart, J.-Z. Zhang, N. Amornthammarong, J. Craynock, S. Cummings, P. Dammann, C. Featherstone, J. Stamates, and K. Sullivan. A glimpse of the Florida Area Coastal Environment (FACE) program. Proceedings, 11th International Coral Reef Symposium, Ft. Lauderdale, FL, July 7-11, 2008. International Society for Reef Studies, 559-563, 2009

    Abstract: The Florida Area Coastal Environment (FACE) research program gathers a variety of data related to water inputs into the coastal zone of southeast Florida. The water inputs studied include treated wastewater discharges, inlet flows, and upwelling events. Measurements include currents, nutrients, microbial contaminants, and stable isotopes. This report provides a glimpse of the data collected in this program. Data collected from the Boynton inlet point to the significance of this discharge as a source of nutrient and microbiological loads to coastal waters and demonstrate the importance of accounting for all major discharges in order to fully understand the impact of land use and water management decisions on coastal resources.

  28. Elmir, S.M., T. Shibata, H.M. Solo-Gabriele, C.D. Sinigalliano, M.L. Gidley, G. Miller, L.R.W. Plano, J. Kish, K. Withum, and L.E. Fleming. Quantitative evaluation of enterococci and bacteroidales released by adults and toddlers in marine water. Water Research, 43(18):4610-4616, doi:10.1016/j.watres.2009.07.006 2009


    Traditionally, the use of enterococci has been recommended as the fecal indicator bacteria of choice for testing marine recreational water quality, and prior studies have shown that bathers shed large numbers of enterococci into the water. The current study expands upon prior research by evaluating shedding from both toddlers and adults, and by the expansion of measurements to include enterococci shedding via three different methods (membrane filter (MF), chromogenic substrate (CS), and quantitative polymerase chain reaction (qPCR)) and shedding of alternative fecal indicator bacteria (Bacteroidales human markers UCD and HF8 via qPCR). Two sets of experiments were conducted. The first experiment consisted of two groups of 10 adults who bathed together in a large pool. The second study consisted of 14 toddlers who bathed individually in a small pool which allowed for sand recovery. Sand recovery was used to estimate the amount of sand transported on the bodies of toddlers and to estimate the number of fecal indicator bacteria released from this sand. The numbers of estimated enterococci shed per adult ranged from 1.8 x 104 to 2.8 x 106 CFU, from 1.9 x 103 to 4.5 x 106 MPN, and from 3.8 x 105 to 5.5 x 106 GEU based on the MF, CS, and qPCR methods, respectively. The estimated numbers of Bacteroidales human markers ranged from 1.8 x 104 to 1.3 x 106 for UCD, and ranged from the below detection limit to 1.6 x 105 for HF8. The estimated amount of sand transported per toddler (n = 14) into the water column after sand exposure was 8 ± 6 g on average. When normalizing the numbers of enterococci shed from toddlers via sand by the 3.9 body surface area ratio, the differences between toddlers and adults were insignificant. Contributions of sands to the total enterococci (MF) shed per toddler was 3.7 ± 4.4% on average. Although shedding via beach sand may contribute a small fraction of the microbial load during initial bathing, it may have a significant role if bathers go to water repetitively after sand exposure.

  29. Goodwin, K.D., L. Matragrano, D. Wanless, C.D. Sinigalliano, and M.J. LaGier. A preliminary investigation of fecal indicator bacteria, human pathogens, and source tracking markers in beach water and sand. Environmental Research Journal, 2(4):395-417, 2009

    Abstract: Data suggesting that fecal indicating bacteria may persist and/or regrow in sand has raised concerns that fecal indicators may become uncoupled from sources of human fecal pollution. To investigate this possibility, wet and dry beach sand, beach water, riverine water, canal water, and raw sewage samples were screened by PCR for certain pathogenic microbes and molecular markers of human fecal pollution. The targets included in this study were human specific Bacteroides (HF8 marker), human-specific enterococci (esp gene), Staphylococcus aureus, Escherichia coli 0157:H7, Campylobacter jejuni, and adenovirus. Sewage samples were also tested for Salmonella species. The results were compared to concentrations of enterococci, Escherichia coli, and Bacteroides species, as determined by membrane filtration methods. Molecular analysis yielded positive results for human specific Bacteroides, and S. aureus, in samples of raw sewage. Two of the environmental samples were positive for human specific Bacteroides and one was positive for S. aureus. The PCR screen was negative for other samples and targets, despite exceedance of EPA single sample guidelines for recreational waters on several of the sample dates (5/11 dates). However, estimates of the number of cells delivered to the PCR reaction suggested that few of the samples met the detection limit of the PCR reaction due to a variety of factors. The analysis indicated a need to improve nucleic acid processing in order to enable better delivery of DNA to downstream molecular methods.

  30. Rosario, K., E.M. Symonds, C.D. Sinigalliano, J. Stewart, and M. Breitbart. Pepper mild mottle virus as an indicator of fecal pollution. Applied and Environmental Microbiology, 75(22):7261-7267, doi:10.1128/​AEM.00410-09 2009

    Abstract: Accurate indicators of fecal pollution are needed in order to minimize public health risks associated with wastewater contamination in recreational waters. However, the bacterial indicators currently used for monitoring water quality do not correlate with the presence of pathogens. Here we demonstrate that the plant pathogen Pepper mild mottle virus (PMMoV) is widespread and abundant in wastewater from the United States, suggesting the utility of this virus as an indicator of human fecal pollution. Quantitative PCR was used to determine the abundance of PMMoV in raw sewage, treated wastewater, seawater exposed to wastewater, and fecal samples and/or intestinal homogenates from a wide variety of animals. PMMoV was present in all wastewater samples at concentrations greater than 1 million copies per milliliter of raw sewage. Despite the ubiquity of PMMoV in human feces, this virus was not detected in the majority of animal fecal samples tested, with the exception of chicken and seagull samples. PMMoV was detected in four out of six seawater samples collected near point sources of secondary treated wastewater off southeastern Florida, where it co-occurred with several other pathogens and indicators of fecal pollution. Since PMMoV was not found in nonpolluted seawater samples and could be detected in surface seawater for approximately 1 week after its initial introduction, the presence of PMMoV in the marine environment reflects a recent contamination event. Together, these data demonstrate that PMMoV is a promising new indicator of fecal pollution in coastal environments.

  31. Scorzetti, G., L.E. Brand, G.L. Hitchcock, K.S. Rein, C.D. Sinigalliano, and J.W. Fell. Multiple simultaneous detection of harmful algal blooms (HABs) through a high throughput bead array technology, with potential use in phytoplankton community analysis. Harmful Algae, 8(2):196-211, doi:10.1016/j.hal.2008.05.003 2009

    Abstract: As an alternative to traditional, morphology-based methods, molecular techniques can provide detection of multiple species within the HAB community and, more widely, the phytoplankton community in a rapid, accurate and simultaneous qualitative analysis. These methods require detailed knowledge of the molecular diversity within taxa in order to design efficient specific primers and specific probes able to avoid cross-reaction with non-target sequences. Isolates from Florida coastal communities were sequence-analyzed and compared with the GenBank database. Almost 44% of the genotypes obtained did not match any sequence in GenBank, showing the existence of a large and still unexplored biodiversity among taxa. Based on these results and on the GenBank database, we designed 14 species-specific probes and 4 sets of specific primers. Multiple simultaneous detection was achieved with a bead array method based on the use of a flow cytometer and color-coded microspheres, which are conjugated to the developed probes. Following a parallel double PCR amplification, which employed universal primers in a singleplex reaction and a set of species-specific primers in multiplex, detection was performed in a cost effective and highly specific analysis. This multi-format assay, which required less than 4 h to complete from sample collection, can be expanded according to need. Up to 100 different species can be identified simultaneously in a single sample, which allows for additional use of this method in community analyses extended to all phytoplankton species. Our initial field trials, which were based on the 14 species-specific probes, showed the co-existence and dominance of two or more species of Karenia during toxic blooms in Florida waters.

  32. Sinigalliano, C.D., J. Winshell, M.A. Guerrero, G. Scorzetti, J.W. Fell, R.W. Eaton, L. Brand, and K.S. Rein. Viable cell sorting of dinoflagellates by multiparametric flow cytometry. Phycologia, 48(4):249-257, doi:10.2216/08-51.1 2009

    Abstract: Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

  33. Proni, J.R., S.J. Stamates, T.P. Carsey, J.-Z. Zhang, C.D. Sinigalliano, and K.F. Sullivan. Acoustic methods for water mass delineation in coastal marine ecosystems. Proceedings, Ninth European Conference on Underwater Acoustics (ECUA 2008), Paris, France, June 29-July 4, 2008. European Acoustics Association, Volume 1, 237-242, 2008

    Abstract: Acoustical methods play an important role in identifying sources of nutrient to coral reef ecosystems in the South Florida coastal waters. Nutrient fluxes into the coastal ocean are associated with distinct water masses such as inlet discharge plumes, wastewater outfall discharge plumes, and upwelling of deep oceanic water. Various nutrient-bearing water masses can be identified by water column acoustic backscatter profiles, obtained via either shipborne instrumentation or in-situ instrumentation. Such multidimensional images of water masses can be used to optimize the design of chemical and biological sampling efforts. Examples of water mass imaging will be presented, as well as the use of such images in the design of water quality sampling programs.

  34. Stewart, J.R., R.J. Gast, R.S. Fujioka, H.M. Solo-Gabriele, J.S. Meschke, L.A. Amaral-Zettler, E. Del Castillo, M.F. Polz, T.K. Collier, M.S. Strom, C.D. Sinigalliano, P.D.R. Moeller, and A.F. Holland. The coastal environment and human health: Microbial indicators, pathogens, sentinels and reservoirs. Environmental Health, 7(Suppl. 2):S3, 14 pp., doi:10.1186/1476-069X-7-S2-S3 2008

    Abstract: Innovative research relating oceans and human health is advancing our understanding of disease-causing organisms in coastal ecosystems. Novel techniques are elucidating the loading, transport and fate of pathogens in coastal ecosystems, and identifying sources of contamination. This research is facilitating improved risk assessments for seafood consumers and those who use the oceans for recreation. A number of challenges still remain and define future directions of research and public policy. Sample processing and molecular detection techniques need to be advanced to allow rapid and specific identification of microbes of public health concern from complex environmental samples. Water quality standards need to be updated to more accurately reflect health risks and to provide managers with improved tools for decision-making. Greater discrimination of virulent versus harmless microbes is needed to identify environmental reservoirs of pathogens and factors leading to human infections. Investigations must include examination of microbial community dynamics that may be important from a human health perspective. Further research is needed to evaluate the ecology of non-enteric water-transmitted diseases. Sentinels should also be established and monitored, providing early warning of dangers to ecosystem health. Taken together, this effort will provide more reliable information about public health risks associated with beaches and seafood consumption, and how human activities can affect their exposure to disease-causing organisms from the oceans.

  35. Sinigalliano, C.D., M.B. Gidley, T. Shibata, D. Whitman, T.H. Dixon, E. Laws, A. Hou, D. Bachoon, L. Brand, L. Amaral-Zettler, R.J. Gast, G.F. Steward, O.D. Nigro, R. Fujioka, W.Q. Betancourt, G. Vithanage, J. Mathews, L.E. Fleming, and H.M. Solo-Gabriele. Impacts of Hurricanes Katrina and Rita on the microbial landscape of the New Orleans area. Proceedings of the National Academy of Sciences, 104(21):9029-9034, doi:10.1073/pnas.0610552104 2007


    Floodwaters in New Orleans from Hurricanes Katrina and Rita were observed to contain high levels of fecal indicator bacteria and microbial pathogens, generating concern about long-term impacts of these floodwaters on the sediment and water quality of the New Orleans area and Lake Pontchartrain. We show here that fecal indicator microbe concentrations in offshore waters from Lake Pontchartrain returned to prehurricane concentrations within two months of the flooding induced by these hurricanes. Vibrio and Legionella species within the lake were more abundant in samples collected shortly after the floodwaters had receded compared with samples taken within the subsequent three months; no evidence of a long-term hurricane-induced algal bloom was observed. Giardia and Cryptosporidium were detected in canal waters. Elevated levels of fecal indicator bacteria observed in sediment could not be solely attributed to impacts from floodwaters, as both flooded and nonflooded areas exhibited elevated levels of fecal indicator bacteria. Evidence from measurements of Bifidobacterium and bacterial diversity analysis suggest that the fecal indicator bacteria observed in the sediment were from human fecal sources. Epidemiologic studies are highly recommended to evaluate the human health effects of the sediments deposited by the floodwaters.