Download E. coli

•Maryland Department of
the Environment
•Delaware Department of
Natural Resources and
Environmental Control
•Salisbury University
Drs. Elichia Venso and Mark Frana
Bacterial Source Tracking Laboratory

To determine levels of fecal indicator organisms (E.
coli & Enterococcus) in beach sediment and water
samples at 1 Delaware and 3 Maryland sampling
sites

To investigate survival and/or regrowth of fecal
indicator organisms found in sediment and water
samples

To determine if an association was present between
bacterial density in beach sand and in beach water

To select for and identify potential bacterial
pathogens in these same water and sediment
samples

4 sampling locations:
 Maryland
 Assateague Island (bay side)
 Granary Creek (Wye River tributary)
 Sandy Point

Delaware
 Delaware Shore

12 month project: Jan–Dec 2008

Sample collections:



Monthly (Jan-Apr; Oct – Dec)
Bimonthly (May – Sep)
Samples collected at each study site:



Water at knee depth
Sediment at water sampling location (“wet”)
Sediment in foreshore between high tide line and
water’s edge (“dry”)

Collection data sheets included information on:

Water
 Temperature
 Conductivity
 Dissolved Oxygen
 pH
 Salinity

Sediment
 Temperature
 Note: Analysis was conducted on one set of samples for
nitrogen, phosphorus, pH, etc.

Other
 Air Temperature and Direction
 Weather
 Tide
 Unusual observations

Water:

Analysis of triplicate water samples to enumerate the fecal
indicators using Colilert ®-18 Most Probable Number analysis
for the detection of E. coli and Enterolert™ for the detection of
Enterococci

Membrane filtration onto selective media for the isolation of
potential pathogens

Sediment:




Determination of moisture content
Agitation of 10 grams in 50 ml of extraction buffer for 30
minutes using a wrist-action shaker , then allowed to settle for
30 min
Membrane filtration of supernatant for enumeration of E. coli
and Enterococci and identification of potential pathogens.
Regrowth:

Incubation of select sediment and water samples at 4°C, 21°C,
and 37 °C, followed by enumeration of E. coli and Enterococcus
MacConkey Sorbitol
E. coli O157:H7
CIN
Yersinia
CAMPY CSM
Campylobacter
XLT4: BG:
Salmonella
SS Agar
Salmonella/Shigella


Carbon Source
Utitilization using
Biolog©
Polymerase Chain
Reaction (PCR)-Midi
Labs©


DNA amplification
Sequence matching
E. coli Mean Densities (MPN/100 ml or 100 g)
Location
Water
Wet
Dry
AI
181
56
338
DS
7
204
164
GC
206
383
5594
SP
82
90
101
Enterococci Mean Densities (MPN/100 ml or 100 g)
Location
Water
Wet
Dry
AI
6
77
251
DS
6
15
69
GC
35
170
820
SP
34
72
235
Assateague: E. coli
Bacteria/100mls or grams
2500
12000
2000
5180
1500
1000
500
0
Water
Wet Sed
Dry Sed
Bacteria/100mls or grams
Assateague: Enterococci
2500
2000
170000
24000
1500
1000
500
0
Water
Wet Sed
Dry Sed
Bacteria/100mls or grams
Delaware Shore: E. coli
1600
1400
1200
1000
800
600
400
200
0
Water
Wet Sed
Dry Sed
Bacteria/100mls or grams
Delaware Shore: Enterococci
1600
1400
1200
1000
800
600
400
200
0
Water
Wet Sed
Dry Sed
Granary Creek: E. coli
Bacteria/100mls or grams
1600
5140
1400
7130
14000
1200
23000
1000
800
600
400
200
0
Water
Wet Sed
Dry Sed
Granary Creek: Enterococci
Bacteria/100ml or grams
1600
1400
5840
9290
1200
3100
2130
1000
3500
2100
800
600
400
200
0
Water
Wet Sed
Dry Sed
Bacteria/100mls or grams
Sandy Point: E. coli
1600
1400
1200
1000
800
600
400
200
0
5440
2490
5770
Water
Wet Sed
Dry Sed
Sandy Point: Enterococci
Bacteria/100mls or grams
1600
1400
110000
1200
1000
800
600
400
200
0
Water
Wet Sed
Dry Sed
Sandy Point Water: E. coli
250
MPN/100 mls
200
150
4oC
100
21oC
37oC
50
0
Granary Creek Water: Enterococci
250
MPN/100mls
200
150
4oC
21oC
100
37oC
50
0
0 hr
24hr
48hr
72hr
96hr
120 hr 144 hr 168 hr 192 hr
Granary Creek dry sediment
cfu/100 dry grams
2500
2000
1500
GC dry 4oC
GC dry 21oC
1000
GC dry 37oC
500
0
0 hr 24 hr 48 hr 72 hr 96 hr 120
hr
144
hr
168
hr
192 day 9 day day day
hr
10 11 12
Assateague wet sediment
300
cfu/100 dry grams
250
200
AI wet 4oC
150
AI wet 21oC
100
AI wet 37oC
50
0
0 hr
24 hr
48 hr
21°C
4°C
30000
30000
25000
25000
20000
20000
15000
GC dry
10000
GC wet
5000
15000
10000
GC dry
5000
GC wet
0
30000
0 hr
24 hr
48 hr
72 hr
96 hr
120 hr
144 hr
168 hr
192 hr
day 9
day 10
day 11
day 12
day 13
day 13
day 12
day 11
day 10
day 9
192 hr
168 hr
144 hr
120 hr
96 hr
72 hr
48 hr
24 hr
0 hr
0
37°C
25000
20000
15000
GC dry
10000
GC wet
5000
0
0 hr 24 48 72 96 120 144 168 192 day day day day day
hr hr hr hr hr hr hr hr 9 10 11 12 13

Density in water versus in wet and dry sediments:
 E. coli:
 Enterococci

49% at SP
29% at GC
Density in water versus in wet and dry sediments and
% moisture:
 E. coli:
 Enterococci:

72% at AI
58% at AI
82% at AI
92% at DS
99% at GC
61% at GC
100% at SP
50% at SP
Density in water versus in dry sediment and %
moisture:
 Enterococci:
92% DS
35% at GC
25% at SP



Percentage of experiments with regrowth in water:
 E. coli:
47% at AI 27% at DS 73% at GC
67% at SP
 Enterococci:
33% at GC 27% at SP
Percentage of experiments with regrowth in wet sediment:
 E. coli:
67% at GC 20% at SP
 Enterococci:
33% at GC 20% at SP
Percentage of experiments with regrowth in dry sediment:
 E. coli:
33% at GC 20% at SP
 Enterococci:
27% at GC 20% at AI and SP

One “potential” pathogen with
a definitive ID:


Sandy Point Wet Sediment
 Vibrio furnissii
Six additional potential
pathogens, but with low
confidence ID:

Granary Creek Water
 E. coli O157:H7 (2)
 Shigella dysenteriae (2)

Sandy Point Wet Sediment
 E. coli O157:H7
 Shigella flexneri

Other Genera identified:
 Aeromonas
 Citrobacter
 Enterobacter
 Klebsiella
 Proteus
 Pseudomonas
 Serratia
 Shewanella

Detectable indicator bacterial densities were found in 47%
of 408 assays.

The highest fecal indicator counts were found in sediment
samples from Granary Creek.

The lowest fecal indicator counts were found at the
Delaware Shore site.

Regrowth/survivability data was measurable at all four
sites, although survival was relatively short-term for
samples held at 21 °C and 37 °C as compared to samples
held at 4 °C.

Only one potential pathogen was definitively identified.

MDE
Kathy Brohawn
William Beatty
John “Rusty”
McKay
 Heather Morehead
 Kathy Bassett
 Sarah Harvey
 Ann McManus

DNREC

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
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
John Pingree
Debbie Rouse
Glenn King
Salisbury University


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Lesley Frana
Annie Adkins
Chris Labe
Isha Choudhary
Mary Vendetti,
Leo Cabrera
Cloe Manarinjara
Megan Robison