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Environmental Microbiology and Molecular Microbial Source

Tracking Hollywood & Broward Coastal

Water Quality Monitoring Chris Sinigalliano Atlantic Oceanographic &

Meteorological Laboratory

with Thomas Carsey,

Jack Stamates, and Maribeth Gidley

Oct 18, 2013

The Florida Area Coastal Environment (FACE) Program at NOAA-AOML

• Assessment of discharges to coastal waters

• Includes wastewater outfalls, coastal inlets, and upwelling characterized for nutrient concentrations & microbiological water quality

• Fate and transport of land-based sources of pollution impacting coral reefs, coastal ecosystem health, and public health of coastal communities

Microbiological Metrics of Ecosystem Health

• Exposure to land-based sources of microbial contaminants:

• Human-source pathogens from sewage/septic discharge

• Measured by proxy with fecal indicator bacteria (Enterococci)

• Measured by genetic human-host fecal bacteria markers

• Measured by proxy with sewage/septic chemical markers

(sucralose)

• Measured by direct detection of selected pathogens

• Pathogens from terrestrial Urban Stormwater Runoff

• Measured by proxy with genetic markers for domestic animal fecal

bacteria (dogs)

• Total bacterial community structure, diversity, and abundance:

• Total Bacterial Community Next-Generation Sequencing and

Community Metagenomic analysis

• Similarity and differences between Inlet, Outfall, and Reef waters?

• Evidence of reef water community structures influenced by LBSP?

• Direct detection of pathogens in the community sequencing data?

Fecal Indicator Assays:

•too slow (>18 hr)

• it is unclear if the correct thing is being monitored (axioms of fecal indicator theory violated – environmental persistence)

•no pathogen detection

•no ability to source track (source tracking needed to guide remediation)

The Potential of Biotechnology

species-specific ID

rapid

sensitive

high-throughput

allows sample storage

culture & microscope

independent

simultaneous information on multiple targets

Source-tracking of fecal indicator hosts

Quantitative Real-Time

Polymerase Chain Reaction

(qPCR):

Next Generation Community

Genetic Sequencing and

Metagenomics Analysis:

A suite of genomics technologies and bioinformatics tools

Extremely high Throughput (> 100,000 individiual sequence IDs per sample by Illumina)

Provides diversity and abundance data for entire populations

Generates large population databases that can be screened for wide variety of specific targets (like pathogens)

Hollywood and Broward Coastal Water Quality Monitoring

Measured Microbial Parameters:

• Live Enterococci fecal indicator bacteria by IDEXX EnteroLert &

by EPA method 1600 MF plate counts (i.e. traditional methods)

• Cryptosporidium and Giardia (Oo)cysts (protozoan pathogens)

by EPA method 1623 immunomagnetic capture and

immunoflourescent microscopy

Molecular Parameters:

• Quantitative real-time PCR to detect specific genes for:

• Total Enterococci (general fecal indicator)

• Total Bacteroidales (alternate general fecal indicator)

• Human-host-source Bacteroidales (sewage/septic marker)

• Dog-host-source Bacteroidales (stormwater runoff marker)

• Sucralose – human-source chemical marker for sewage/septic

• Next-Generation Sequencing for Bacterial Community

Metagenomic Characterizaton (454-pyrosequencing & Illumina)

FACE Hollywood/Broward WQ Monitoring –

Viable Enterococci by IDEXX

EnteroLert™

HW4 = Hollywood outfall

HW5 = 500meters north of

outfall

HW9 = reef tract

HW14 = Port Everglades Inlet

BR1 = North of Inlet

BR10 = Broward outfall

BR11 = 500meters north of outfall

BR7 = reef tract

BR13 = Hillsboro Inlet

BR14 = Intercoastal South of Inlet

BR15 = Intercoastal North of Inlet

EPA 2012 exposure criteria:

1) Geomean ≤ 35 per100mL

2) Geomean ≤ 30 per 100mL


Total Enterococci by qPCR (EPA “entero1”

assay)


HW5 = 500meters north of outfall

HW9 = reef tract





BR7 = reef tract




Boxplots of all cumulative monthly cruise data showing the decrease in abundance with distance from the outfall for total Enterococci (dead, dormant, and live) as measured by the Entero1A qPCR assay at the Hollywood outfall (right) and Broward outfall (left).

FACE Hollywood/Broward WQ Monitoring – Total Enterococci by qPCR (EPA “entero1” assay)


Total Bacteroidales by qPCR (EPA

“GenBac3” assay)



HW9 = reef tract





BR7 = reef tract




FACE Hollywood/Broward WQ Monitoring – Human-Source

Bacteroidales by qPCR (“BacHum-

UCD” assay)



HW9 = reef tract





BR7 = reef tract




FACE Hollywood/Broward WQ Monitoring – Human-Source

Bacteroidales by qPCR (“EPA HF183

Taqman” assay)



HW9 = reef tract





BR7 = reef tract




Boxplots of all cumulative monthly cruise data showing the decrease in abundance with distance from the outfall for Human-host-specific Bacteroidales human fecal marker (combined data from both the BacHum-UCD and HF-183 qPCR assays) at the Hollywood outfall (right) and Broward outfall (left).

FACE Hollywood/Broward WQ Monitoring – Human-Source Bacteroidales by qPCR ( both “EPA HF183

Taqman” and “BacHum-UCD assays combined)


Canine-Source Bacteroidales by

qPCR (“AOML Dog-Bac” assay)



HW9 = reef tract





BR7 = reef tract





Protozoan Pathogens: Cryptosporidium and

Giardia (Oo)cysts

Cryptosporidium and Giardia (Oo)cyst enumeration by

Immunomagnetic Capture & Immunofluorescence Microscopy (EPA

method 1623), & potential viability assessment by DAPI fluorescent

counter-staining

Boxplots of all cumulative monthly cruise data for Hollywood and Broward outfalls showing the relative abundance of pathogenic protozoan (oo)cysts in 100 Liter volume samples from the outfall expression boil surface waters.

Hollywood Beach Microbial Source Tracking

• Collected water and sand samples by AOML on

9/13 & 9/15

• Viable culture assays

• Culturable enterococci by EPA method 1600

• Culturable Bacterodales by BBE plate counts

• MST qPCR

• General Enterococci (EPA entero1 assay)

• General Bacteroidales (EPA GenBac3 assay)

• Human-host-source Bacteroidales (Kildare BacHum-

UCD)

• Dog-host-source Bacteroidales (AOML DogBac)

• Gull/Pelican-host-source Catellicoccus marimammalium

(AOML Gull2 assay)

Hollywood

Beach MST

Study Sites

Hollywood Beach Microbial Source Tracking

Summary of Results

• Culture Assays: • 2 samples in exceedance of single-grab limits

• Jefferson St. and Harrison St. showed high levels of viable Bacteroidales on 9/15

• qPCR MST Assays: • Minnesota St. Beach site showed elevated human-source

marker on 9/13

• Southern sites from Jefferson St.to New York St. showed low level of human marker on 9/15

• Only low-levels of dog fecal marker observed even at Custer St. dog beach

• Many sites showed elevated to high levels of gull fecal contamination – larges numbers of gulls and pigeons also observed during sampling at southern sites.

• Populations of Enterococci were observed in the sand for many samples

Sample sites for Next-Generation Community Sequencing and & Metagenomic Analaysis of Total Bacterial Populations

Water samples from: Jan, April, July, Nov

(A= surface, C=bottom)

• BR14 – Hillsboro Inlet

• BR10 – Broward Outfall

• BR7 – reef site

• HW14 – Port Everglades Inlet

• HW9 – reef site

• HW 4 – Hollywood Outfall

Rarefaction Plot of Sequence Sample IDs for Hollywood Sites

More

Diversity

Less

Diversity

Increasing ratios of unique “Operational Taxonomic Units” (OTUs) vs. total number of sequences

detected indicates increasing diversity of unique sequence taxa

Rarefaction Plot of Sequence Sample IDs for Broward Sites

Increasing ratios of unique “Operational Taxonomic Units” (OTUs) vs. total number of sequences

detected indicates increasing diversity of unique sequence taxa

More

Diversity

Less

Diversity

Similarity of Bacterial Community Composition

Similarity of Bacterial Taxon Abundance

Other Pathogens Detected to date in Bacterial Community Sequence Data from 454-Pyrosequencing

• HW14 – Port Everglades

• Staphylococcus aureus

• Serratia marcescens (3 strains)

• Campylobacter jejuni

• HW4 – Hollywood Outfall

• Staphylococcus aureus (3

strains)


• Campylobacter mucosalis

• Helicobacter pullorum

• HW9 – Reef


• Campylobacter concisus

• Vibrio parahaemolyticus

• BR14 – Hillsboro Inlet



• Campylobacter concisus

• Helicobacter pullorum

• BR10 – Broward Outfall

• Serratia marcescens (2 strains)

• BR7 – Reef

• Salmonella enterica (subspp.

enterica and arizonae)

• Klebsiella pneumoniae

• Vibrio parahaemolyticus

• Vibrio vulnificus

Figure 113: Relative abundance of fecal indicator bacteria and microbial source tracking markers with distance from the Hollywood Outfall during the “HYTEX” Hollywood Dye Tracer cruise of July 9, 2012.

Hollywood Dye Tracer Cruise

(HYTEX) July 9, 2012

Figure 115: Relative abundance of fecal indicator bacteria and microbial source tracking markers, as well as Sucralose, with distance from the Broward Outfall during the “BOTEX1” Broward Dye Tracer cruise of November 8, 2012.

Map of 1st Broward Dye Tracer Experiment (BOTEX1) cruise of November 7, 2013 showing the locations and concentrations of the artificial sweetener Sucralose (i.e. “SPLENDA®”) as a human-source fecal excretion marker in relation to distance from the Broward outfall. Site BR numbers on this map correspond with the CTD cast numbers for this cruise . Sucralose concentrations in natural waters in excess of 100 ng/L have been suggested in some literature to potentially reflect impairment of those waters by sewage and/or septage sources.

Relative abundance of fecal indicator bacteria and microbial source tracking markers, as well as Sucralose, with distance from the Broward Outfall during the “BOTEX2” Broward Dye Tracer cruise of November 29, 2012. Note anomalously high signals for CTD cast #9 – perhaps an eddy?

BR9

Map of 2nd Broward Tracer Experiment (BOTEX2) cruise of November 28, 2013 showing the location and concentration of artificial sweetener sucralose excretion marker in relation to the outfall boil. Sucralose concentrations in ng/L are shown in yellow text and site IDs are shown in white text. Sucralose values in coastal waters over 100 ng/L might be considered impaired by human excretion (i.e. sewage/septage). Site BR site numbers correspond with the CTD cast numbers. CTD cast BR9 showed anomalously high values for both sucralose and other fecal source tracking markers.

SUMMARY: MICROBIOLOGY

• Elevated concentrations of fecal indicator bacteria and human-specific

microbial source tracking markers were associated with inlets and outfalls

and were primarily confined to surface samples and most commonly

decreased to relative background levels within 1-2 km of the discharge

source, with a large decrease in abundance within the first 0.2 km

• Coastal waters contained low concentrations of viable enterococci.

Concentrations were highest near inlets and outfalls, but diluted rapidly to

near detection limits within 1 km.

• Enterococci appeared to be more abundant near the outfalls during winter

months. No such significant seasonal trends were observed for coastal

inlets.

• The bacterial particulates discharged from the outfalls appear to be

predominately dead or dormant based upon viable enterococci tests.

SUMMARY: MICROBIOLOGY (cont’d) • Coastal inlets appear to contribute more potentially infectious bacterial

agents to the coastal waters than the outfalls. During this study, the Hillsboro

inlet appeared to be the highest contributor of live fecal bacteria to the

immediate coastal region.

• The coastal inlets were also observed to periodically discharge high levels of

live fecal indicator bacteria and human-specific microbial source tracking

markers, as well as dog-specific source tracking markers.

• Elevated concentrations of canine-specific microbial source tracking markers

were primarily associated with inlets, but also occasionally with outfalls. It is

likely this may represent, at least in part, an influence of urban runoff on the

region. Very occasionally canine fecal marker was detected in waters of reef

tract sites, especially when inlet levels were extremely high.

• No significant levels of pathogenic protozoan (oo)cysts were observed in the

surface expression boils of either the Hollywood or Broward outfalls for

samples collected during this study, but for one exception.

SUMMARY: MICROBIOLOGY (cont’d)

• Locations more distant from the inlets and outfalls generally had significantly

lower concentrations of fecal indicator bacteria, molecular source tracking

markers, and chemical human-specific fecal marker. However, some farther-

field samples had elevated concentrations of one or more marker indicating

sewage-associated particulates.

• These results suggest that sometimes elevated levels of sewage-associated

bacteria may travel farther than was commonly observed in this study. This

farther-field transport appears relatively rare and primarily constrained to the

surface waters .

• The results observed did not indicate if such bacterial particulates can move

down to the reef tract. The study design sampled only water, and actual

coral tissue was not tested in this study for the presence of these land-

based-source microbial contaminants. This represents an important

information gap that we hope to address with future studies by conducting

molecular analysis of bacterial communities from coral tissue and mucus.

SUMMARY: MICROBIOLOGY (cont’d) • Elevated concentrations of the artificial sweetener Sucralose (i.e.

“Splenda”® ), as a soluble chemical marker of human-specific fecal

contamination, were associated with outfalls and were primarily confined to

surface samples and also decreased rapidly with distance from the outfall,

with the most rapid decrease within the first 0.5 km. While Sucralose was

detected at some level within all the water samples it was tested with, the

concentrations of this human-specific chemical fecal marker dropped below

100ng/L within 1 km of the outfall

• Additional regional studies would be useful, including an assessment of

corals to measure if and how much they are assimilating land-based-source

microbial pollutants (including pathogens) into the microbial assemblage of

their tissues and near-coral waters.

• The community sequencing data is an important value-added component of

this study that will be incorporated into international genomic observatory

databases such as the Earth Biome Project, and be available for continued

metagenomic analysis by researchers world-wide, thus providing on-going

new insights from these same sequence data sets.

If you have questions or would like more information, please contact

Dr. Chris Sinigalliano

PI, Environmental Microbiology Program

National Oceanic & Atmospheric Administration

Atlantic Oceanographic & Meteorological Laboratory

4301 Rickenbacker Causeway, Miami, FL 33149

Office phone: 305-361-4384

Cellular: 954-801-7789

Email: [email protected]

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