DE LEON.pdf

Adequacy of Microbial
Indicators of Faecal
Contamination in Tropical
Recreational Waters
Shervon De Leon, Lyndon Robertson,
Smail Mahdi, Marc Lavoie

Thank You !!
• CEHP –AML, WINDREF
• Soufriere Marine Management Authority
(Newton Eristhee)
• UNEP, IWCAM (Christopher Corbin & Vincent
Sweeney)
• Graduate Studies
• Shakira Grannum & Julia Welch
• Dr. Francis Lopez
• BCS staff esp. Nicole Artherly & Michelle
Mendes
• Family & Friends

Adequacy of Microbial
Indicators of Faecal
Contamination in Tropical
Recreational Waters

Background
• Need to determine water quality (WQ)
received much attention after the 1855
Salmonella outbreak (Snow & Budd,
1855)
• To determine WQ, simple, reliable &
rapid methods for detection &
enumeration of microorganisms (MO)
are necessary

• Pathogens are not detected easily & routine
culturing not recommended
• Methods developed to determine presence
of other faecal microorganisms (FM)
• Absence of these FM means pathogens
unlikely to be present
• Concept of “indicator organisms” was
introduced in 1892 and is the basis for most
microbial water quality standards

Indicator Organisms:
• Leclerc et al. (2001) defined indicators as „„. . .
organisms whose detection at prescribed levels
would point to a certain probability of pathogenic
organisms of similar physiological and ecological
character also being present, albeit at much lower
levels than those of the markers‟‟.
• “A group of organisms that indicates the presence
of faecal contamination, such as the bacterial
groups, thermotolerant coliforms or E. coli. Hence,
they only infer that pathogens may be present.”
(WHO, 2001)

• present when pathogens are present
and are therefore usually used to
estimate the presence of pathogens
• these organisms are then indicators of
potential human health risk

Characteristics of an Ideal Indicator
Organism
• exclusively of faecal origin
• present when faecal contamination is present
• occurs in equal or greater numbers than associated
pathogens
• exhibits same or greater survival characteristics in
treatment processes and in the environment than
associated pathogens
• must not proliferate to any great extent in the environment
• detection, enumeration and identification should be simple,
reliable, quick and inexpensive

Faecal Indicators Organisms include:
• Coliform bacteria
• Total Coliforms
• Faecal Coliforms
• Escherichia coli
• Enterococci
• Clostridium perfringens
• Bacteroides spp.

Gastrointestinal pathogens associated with
enteric disease outbreaks are largely from the
Enterobacteriaceae family & include:
• Salmonella
• Shigella
• Yersinia enterocolitica
• Enterotoxigenic E. coli

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Other enteric pathogens often
found in contaminated water
• Bacteria (Vibrio cholerae & Campylobacter
jejuni)
• Viruses (enteroviruses)
• Protists (Giardia, Cryptosporidium)

The Problem

The Problem
“No ideal WQI exists”
• WQIs are sometimes detected when no
pathogens are present in the environment
• Regrowth & persistence of indicator
organisms in the environment

Rationale
• Region‟s heavy dependence on (sun, sea & sand)
tourism as a major revenue generating industry,
therefore water quality monitoring is important
• Indicator organisms & water quality standards
developed & based on research done in temperate
regions
• Increasing body of research showing traditional
indicator may not be suitable for use, especially in
tropics
- persist in environment (soil & water)
- found in areas that are considered pristine

“Experts highlighted the importance of
conducting national and regional
epidemiological studies to assess the
suitability of microbial indicators
currently used to evaluate the
microbiological quality of recreational
waters.”
UNEP 2006 workshop report

Objectives
• To determine the adequacy of coliform
bacteria and enterococci for use as faecal
pollution indicators in tropical recreational
waters
• To evaluate the adequacy of other
bacterial indicators like Clostridium
perfringens and Bacteroides spp.

Specific Objectives
• To assess the recreational water quality in Barbados
and the Windward Islands using coliforms, E.coli
and enterococci
• To establish correlations between FC, E. coli,
enterococci, C. perfringens, Bacteroides and faecal
human pathogens in tropical recreational water
• To determine Sensitivity, Specificity and Accuracy of
each indicator
• To determine and propose the most suitable
microbial indicators for recreational water quality
monitoring

Methodology

Collection of Samples
Analysis of Samples
Compilation of Results
Analysis of Data
Conclusions &
Recommendations

Sampling
• Samples collected in accordance with
Standard Methods 2005.
• All samples were stored at 4 o C and analyzed
within 24hrs of being taken

Barbados

Dominica

St. Lucia

Grenada

St. Vincent

Table 1. Standard Methods (2005) for pathogens & indicators.
Bacterial pathogen
Salmonella
Shigella
Vibrio cholerae
Yersinia enterocolitica
Microbial Indicators
Method of Detection
9260 B
9260 E
9260 H
9260 K
Method of Detection
E. coli USEPA Method # 1603
Enterococci USEPA Method # 1600
Faecal Coliform
9222 D
C. perfringens Bisson & Cabelli
(1978) modified by
Armon & Payment
(1988)
Bacteroides sp. was isolated by recently developed methods (Linroy Christian,
Ph.D. Thesis).

Membrane Filtration

Data Compilation

Data Analysis
• Compare results with US EPA standards
• Correlations determined using IBM SPSS 19
• Spearman‟s correlation
• Holm‟s tests of significance by Olkin-Finn Z
statistic method (Cheung and Chan 2004)
• Calculate Sensitivity, Specificity & Accuracy

Number of true negatives
• SPECIFICITY = ------------------------------------------------------- X 100
Number of TN + Number of FP
Number of True Positives
• SENSITIVITY = --------------------------------------------------------- X 100
Number of TP + Number of FN
Number of TP + Number of TN
• ACCURACY = ------------------------------------------------------------ X 100
Where:
# of TP + # of TN + # of FP + # of FN
• True positives (TP) = pathogens present when IO above prescribed
level
• True negatives (TN) = pathogen absent when IO below prescribed
level
• False positives (FP) = IO above prescribed level but pathogen absent
• False negative (FN) = IO below prescribed level but pathogen present

Results

Water Quality
• Total of 713 marine and 127 fresh water
samples collected
• 164 marine from Barbados,
• 184 marine & 75 fresh from St. Lucia,
• 181 marine & 27 fresh from Dominica,
• 96 marine & 16 fresh from Grenada, and
• 88 marine & 9 fresh from St. Vincent.

Frequency at which the sample counts were
above the proposed limits for marine waters 1,2
Barbados St. Lucia Dominica Grenada St. Vincent
Number of samples 164 184 181 96 88
Faecal coliform
One day value (800
CFU/100ml) 3 (2%) 30 (16%) 30 (17%) 6 (6%) 2 (2%)
Escherichia coli
One day value (235
CFU/100ml)
Enterococci
One day value (104
CFU/100ml)
5 (3%) 28 (15%) 26 (14%) 4 (4%) 13 (15%)
14 (9%) 16 (9%) 16 (9%) 7 (7%) 3 (3%)
Clostridium
perfringens
Geometric mean (5
CFU/100ml)
95 (58%) 87 (47%) 78 (43%) 32 (33%) 39 (44%)
1
USEPA 2003
2
Fujioka, 2001

Frequency at which the sample counts were
above the proposed limits for Fresh waters 1,2
St. Lucia Dominica Grenada St. Vincent
Number of samples 75 27 16 9
Feacal coliforms
One day value (800
CFU/100ml)
Escherichia coli
One day value (235
CFU/100ml)
Enterococci
One day value (104
CFU/100ml)
Clostridium
perfringens
Geometric mean (50
CFU/100ml)
31 (41%) 16 (59%) 4 (25%) 3 (33%)
28 (37%) 15 (56%) 4 (25%) 4 (44%)
33 (44%) 8 (30%) 6 (38%) 4 (44%)
9 (12%) 5 (19%) 1 (6%) 3 (33%)
1
USEPA 2003
2
Fujioka, 2001

Occurrence of Pathogens in relation to indicator levels
in the marine & fresh waters of Barbados and the
Windward Islands
Indicators Pathogens in Marine Water Pathogens in Fresh Water
Feacal coliforms≥800 CFU/100ml
Faecal coliform < 800 CFU/100ml
59%
41%
60%
17%
Escherichia coli ≥235 CFU/100ml
Escherichia coli < 235 CFU/100ml
64%
40%
53%
21%
Enterococci ≥104 CFU/100ml
Enterococci < 104 CFU/100ml
Clostridium perfringens ≥ 50
CFU/100ml
Clostridium perfringens < 50
CFU/100ml
Bacteroides spp. ≥ 1 CFU/100ml
Bacteroides spp. < 1 CFU/100ml
71%
39%
3%
34%
69.2%
24.6%
45%
28%
11%
24%
62%
18%

Correlations between Indicators and Pathogens in
Marine Water (N = 713). All values (Sigma twotailed)
were significant at p < 0.05.
Faecal
coliform
Faecal
coliform E. coli Enterococci
C.
perfringens
Bacteroides
Pooled
Pathogens
1.000 .901 .712 .528 .491 .364
E. coli .901 1.000 .743 .509 .503 .376
Enterococci .712 .743 1.000 .510 .479 .419
C.
perfringens
Bacteroides
spp.
Pooled
Pathogens
.528 .509 .510 1.000 .532 .450
.491 .503 .479 .532 1.000 .482
.364 b .376 b,c .419 a,c .450 a .482 a 1.000
a,b,c
Values with the same letter are not statistically significantly different

Correlations between Indicator Organisms and
Between Indicators and Pathogens in Fresh
Water (N = 127)
Faecal
coliform
Faecal
Bacteroides Pooled
coliform E. coli Enterococci C. perfringens spp. Pathogens
1.000 .929 .788 .615 .551 .500
E. coli .929 1.000 .816 .620 .587 .467
Enterococci .788 .816 1.000 .565 .506 .281
C. perfringens .615 .620 .565 1.000 .774 .571
Bacteroides
spp.
Pooled
Pathogens
.551 .587 .506 .774 1.000 .511
.500 a .467 a .281 b .571 a .511 a 1.000
a,b,c
Values with the same letter are not statistically significantly different

Marine Water
Fresh Water
Sensitivity, Specificity & Accuracy
Analyses
Matrix Faecal coliforms E. coli enterococci C. perfringens Bacteroides
spp.
Sensitivity 10% 13.1% 15.2% 8.3% 46.9%
Specificity 95.5% 94.8% 96.1% 98.4% 83.9%
Accuracy 63.5% 64.3% 65.9% 64.6% 68.1%
Sensitivity 75.6% 66.7% 53.3% 31.1% 68.9%
Specificity 74.4% 72.1% 66.3% 96.5% 77.9%
Accuracy 74.8% 70.2% 61.8% 74% 74.8%

Discussion
Does not take into account viruses & protists
Correlation coefficients not statistically
significantly different but close to significance
C. perfringens less closely correlated to
pathogens than Bacteroides spp. but more
easily detected

Conclusions &
Recommendations
More analyses to further confirm these results
Clostridium perfringens be adopted and used alongside E.
coli & enterococci
Develop more easy-to-use techniques for Bacteroides
Epidemiology study using traditional and proposed
indicators
Survival rate comparisons: indicators vs pathogens

Questions Please?

Percentage true and false positives and
negative in fresh and marine samples
Marine Water
Fresh Water
Parameter TN FN TP FP TN FN TP FP
Faecal coliforms 60% 34% 4% 3% 49% 8% 26% 17%
E. coli 59% 33% 5% 3% 47% 11% 23% 18%
Enterococci 60% 32% 6% 2% 44% 16% 18% 22%
C. perfringens 62% 34% 3% 1% 63% 24% 11% 2%
Bacteroides spp. 48% 22% 20% 9% 51% 11% 24% 15%
Where:
True positives (TP) = pathogens present when IO above prescribed level
True negatives (TN) = pathogen absent when IO below prescribed level
False positives (FP) = IO above prescribed level but pathogen absent
False negative (FN) = IO below prescribed level but pathogen present