Coliphage as an Indicator of Fecal Pollution in Marine Waters: Assay, Validation, and Application

Escherichia coli, the preferred bacterial indicator for fecal pollution in fresh waters, does not conform to the concept of an indicator microorganism because it is rapidly killed or inactivated by seawater. This series of papers investigated the value of coliphage, a virus which infects E. coli, as an indicator of pollution in saline waters. In order to be an accurate indicator an organism must (1) be ubiquitous in wastewater, (2) survive and be detectable at least as long as the harmful organisms, and (3) be easy to isolate and identify. A review of the literature determined that coliphage were more resistant than the common bacterial indicators to physico-chemical factors such as inorganic ions, temperature, heavy metals, nutrients, and antibiotics. Coliphage correlation with their bacterial hosts and similarities in behavior to the pathogenic viruses make them both bacterial and viral indicators. Various culture media and host culture strains were investigated for maximum plaque forming unit (pfu) production. Two way analysis of variance showed that selection of a suitable host was of paramount importance. While selection of the culture medium was significant, it was of lesser importance. Host strain ATCC 13706 and tryptic soy agar gave the highest recovery of pfu\u27s. One ml log phase E.coli host culture, five ml of water sample or dilution, and five ml of culture media (maintained at 44.5 C) were combined in a sterile screw cap tube, mixed, poured into a sterile 100 X 15 mm petri dish, and incubated at 35 C. Plaque forming units were counted after 24 hrs. and expressed per 100 ml of sample. The method proved repeatable; the titer of frozen phage aliquots declined slightly over 77 days but, the slope of the trend was not significantly different from zero at the 0.10 level (r = 0.55). These repeated analyses were done with different batches of media and hosts and represent a test of total method repeatability. Bench studies utilizing a decimal dilution series of sewage contaminated freshwater and uncontaminated seawater showed that both coliform and coliphage closely follow a theoretical dilution curve immediately after dilution with seawater. However, coliform bacteria die off at a higher rate than coliphage at higher salinities over time. Field validation studies in fresh and brackish water (\u3c10 \u3eppt) compared coliphage with total and fecal coliforms (n = 53) and gave correlation coefficients of 0.98 and 0.91 respectively. The regression equation for these samples was: log coliphage = 0.983 (log total coliform) -1.001 The combined total coliform/coliphage relationship at 68 saltwater (\u3e10 ppt) stations yielded a correlation coefficient of 0. 45. Coliphage are a logical choice for a fecal indicator in marine waters since their titers are closely related to total and fecal coliform in freshwater, survive much better than coliforms in seawater, and they can be enumerated by a simple method which is not subject to salinity artifacts. The constant relation of coliphage and coliforms in freshwater indicate a possible link to current water quality standards based on total or fecal coliforms. Since coliphage pfu are a rather constant 8 - 10 % of total coliform cfu in low salinity waters where coliform inactivation is less severe, a coliphage titer of 80 – 100 pfu per 100 ml in seawater may indicate water quality equivalent to that indicated by a coliform count of 1000 cfu per 100 mI. This could aid in the interpretation of coliphage data relative to current coliform-based water quality codes. Monitoring of sanitary water quality in Bell Channel Bay, Bahamas, during repair of a sewer plant showed that following chlorination and diversion of the effluent to a deep well, total coliform declined rapidly below detection limits. Coliphage remained easily detectable ten days later. Two canals and two marinas on Biscayne Bay were assayed for coliphage to compare sanitary water quality related to point and non-point source pollution. The Biscayne Canal was impacted by periodic upstream sewage spills, while the Little River displayed chronic contamination along its length by liveaboard boats or sewer leaks. Coliphage were shown to persist six days longer than coliform after a sewage spil l was tracked in the Canal. The liveaboard Dinner Key marina displayed low-level, spotty contamination with no seasonal pattern. King\u27s Bay marina was free of detectable fecal contamination during the study. The use of coliphage allowed the assessment and monitoring of fecal contamination in marine waters where coliform bacteria were not suitable

Fine grained compositional analysis of Port Everglades Inlet microbiome using high throughput DNA sequencing

Background Similar to natural rivers, manmade inlets connect inland runoff to the ocean. Port Everglades Inlet (PEI) is a busy cargo and cruise ship port in South Florida, which can act as a source of pollution to surrounding beaches and offshore coral reefs. Understanding the composition and fluctuations of bacterioplankton communities (“microbiomes”) in major port inlets is important due to potential impacts on surrounding environments. We hypothesize seasonal microbial fluctuations, which were profiled by high throughput 16S rRNA amplicon sequencing and analysis. Methods & Results Surface water samples were collected every week for one year. A total of four samples per month, two from each sampling location, were used for statistical analysis creating a high sampling frequency and finer sampling scale than previous inlet microbiome studies. We observed significant differences in community alpha diversity between months and seasons. Analysis of composition of microbiomes (ANCOM) tests were run in QIIME 2 at genus level taxonomic classification to determine which genera were differentially abundant between seasons and months. Beta diversity results yielded significant differences in PEI community composition in regard to month, season, water temperature, and salinity. Analysis of potentially pathogenic genera showed presence of Staphylococcus and Streptococcus. However, statistical analysis indicated that these organisms were not present in significantly high abundances throughout the year or between seasons. Discussion Significant differences in alpha diversity were observed when comparing microbial communities with respect to time. This observation stems from the high community evenness and low community richness in August. This indicates that only a few organisms dominated the community during this month. August had lower than average rainfall levels for a wet season, which may have contributed to less runoff, and fewer bacterial groups introduced into the port surface waters. Bacterioplankton beta diversity differed significantly by month, season, water temperature, and salinity. The 2013–2014 dry season (October–April), was warmer and wetter than historical averages. This may have driven significant differences in beta diversity. Increased nitrogen and phosphorous concentrations were observed in these dry season months, possibly creating favorable bacterial growth conditions. Potentially pathogenic genera were present in the PEI. However their relatively low, non-significant abundance levels highlight their relatively low risk for public health concerns. This study represents the first to sample a large port at this sampling scale and sequencing depth. These data can help establish the inlet microbial community baseline and supplement the vital monitoring of local marine and recreational environments, all the more poignant in context of local reef disease outbreaks and worldwide coral reef collapse in wake of a harsh 2014–16 El Niño event

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

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. aureussources 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

Spatial Distribution of Petroleum Hydrocarbons in Sediment Cores from Blind Pass, St. Pete Beach, Florida

One hundred and one sediment cores were collected to characterize the spatial distribution of petroleum hydrocarbons within and just outside Blind Pass, St. Pete Beach, Florida. Twenty-five percent of the cores exhibited levels of petroleum hydrocarbons above detection limits of the gas chromatograph/flame ionization detector (GC/FID) (0.01 mg/Kg), but at generally low concentrations. Petroleum hydrocarbon speciation studies of these samples (gas chromatography/mass spectroscopy [GC/MS]) indicate above-detection level (1 μg/Kg) petroleum hydrocarbons are similar to the non-volatile petroleum hydrocarbons found in a Bouchard 155 reference sample collected after the 1993 oil spill in the area, but are in a much degraded and weathered state. Individual petroleum hydrocarbons were, in all but one case, below the threshold effective level (TEL) described in the literature (MacDonald, 1994). The petroleum hydrocarbons were primarily found at 100-300 cm depth in Blind Pass cores. Above-detection level petroleum hydrocarbons were generally found in samples from cores in the center of the channel, near the edges of the shoal, and just outside of Blind Pass. A second mixture of hydrocarbons, primarily phthalates, ketones, and ether, was found at relatively shallow core depths (0-99 cm) in the Mid- and North End Channel cores. These suggest a separate source of contamination, possibly storm water runoff. The fuel fluorescence detector (FFD) probe was investigated for its ability to detect petroleum hydrocarbons in marine sediments. When analyzed with the FFD, all sediments from the cores produced peaks of fluorescence, but none above the background levels of Blind Pass native sediments. All but two samples analyzed by GC/FID were below the detection limits (100 ppm) of the FFD. These samples were found in dark-colored sediments. The combination of the detection limits of the instrument, sediment color, and the degraded nature of the heavier weight petroleum hydrocarbons may have resulted in fluorescence outputs below background levels. These studies demonstrate that the distribution of petroleum hydrocarbons within Blind Pass sediments is generally low and patchy. However, 25% of the cores exhibited levels above detection using GC/FID/MS. These cores could be subjected to individual speciation studies which indicate generally below TEL levels and an association of some, but not all, with the 1993 oil spill in Blind Pass. Appendix A provides photographs and tables for sediment subsamples which exhibited total petroleum hydrocarbon concentrations above detection limits, while Appendix B presents the results from fuel fluorescence detector probe analyses. A discussion of the results of the study in relation to sediment quality guidelines and soil cleanup target level guidance documents is included as Appendix C. Some preliminary results using the above techniques on core samples from the nearby John’s Pass are presented in Appendix D

Cortical and amygdalar Lewy body burden in Parkinson's disease patients with visual hallucinations

Visual hallucinations (VH) are among the most common non-motor complications of Parkinson's disease (PD). A few studies on their etiopathogenesis have suggested involvement of cortical and amygdalar areas. In order to investigate the possible association between extranigral Lewy Body (LB) distribution across cortical and amygdalar regions and the presence of VH in PD brain donors, we conducted a clinico-pathological comparison of 10 PD patients with VH vs 10 closely matched PD patients without VH. The LB burden was significantly higher across the amygdala and the frontal, temporal and parietal cortical areas in patients with VH . Although our results suggest significant extranigral involvement, the precise etiopathologic mechanisms responsible for the development of VH need further clarification

Mutation screening of mitofusin 2 in Charcot-Marie-Tooth disease type 2

Charcot-Marie-Tooth (CMT) disease is among the most common inherited neurological disorders. Mutations in the gene mitofusin 2 ( MFN2 ) cause the axonal subtype CMT2A, which has also been shown to be associated with optic atrophy, clinical signs of first motor neuron involvement, and early onset stroke. Mutations in MFN2 account for up to 20–30% of all axonal CMT type 2 cases. To further investigate the prevalence of MFN2 mutations and to add to the genotypic spectrum, we sequenced all exons of MFN2 in a cohort of 39 CMT2 patients. We identified seven variants, four of which are novel. One previously described change was co-inherited with a PMP22 duplication, which itself causes the demyelinating form CMT1A. Another mutation was a novel in frame deletion, which is a rare occurrence in the genotypic spectrum of MFN2 characterized mainly by missense mutations. Our results confirm a MFN2 mutation rate of ~ 15–20% in CMT2