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Microbial Indicators of Fecal Pollution: Recent Progress and Challenges in Assessing Water Quality



Purpose of review: Fecal contamination of water is a major public health concern. This review summarizes recent developments and advancements in water quality indicators of fecal contamination. Recent findings: This review highlights a number of trends. First, fecal indicators continue to be a valuable tool to assess water quality and have expanded to include indicators able to detect sources of fecal contamination in water. Second, molecular methods, particularly PCR-based methods, have advanced considerably in their selected targets and rigor, but have added complexity that may prohibit adoption for routine monitoring activities at this time. Third, risk modeling is beginning to better connect indicators and human health risks, with the accuracy of assessments currently tied to the timing and conditions where risk is measured. Research has advanced although challenges remain for the effective use of both traditional and alternative fecal indicators for risk characterization, source attribution and apportionment, and impact evaluation.
Microbial Indicators of Fecal Pollution: Recent Progress
and Challenges in Assessing Water Quality
David A. Holcomb
&Jill R. Stewart
#The Author(s) 2020
Purpose of Review Fecal contamination of water is a major public health concern. This review summarizes recent developments
and advancements in water quality indicators of fecal contamination.
Recent Findings This review highlights a number of trends. First, fecal indicators continue to be a valuable tool to assess water
quality and have expanded to include indicators able to detect sources of fecal contamination in water. Second, molecular
methods, particularly PCR-based methods, have advanced considerably in their selected targets and rigor, but have added
complexity that may prohibit adoption for routine monitoring activities at this time. Third, risk modeling is beginning to better
connect indicators and human healthrisks, with the accuracy of assessments currently tied to the timing and conditions where risk
is measured.
Summary Research has advanced although challenges remain for the effective use of both traditional and alternative fecal
indicators for risk characterization, source attribution and apportionment, and impact evaluation.
Keywords Escherichia coli .Environmental antimicrobial resistance .Fecal indicator bacteria .Microbial source tracking .
qPCR .Water quality
Fecal contamination of water continues to be a major public
health concern, with new challenges necessitating a renewed
urgency in developing rapid and reliable methods to detect
contamination and prevent human exposures. Aging sewer
infrastructure in the USA and elsewhere will require rapid
methods to assess fecal contamination of water [1,2,3].
The number of extreme weather events including flooding
events is forecast to increase with climate change and has been
associated with contamination of water resources [46]. Also,
the increasing threat of antimicrobial resistance is making it all
the more important to lower the rates of infections across the
globe, especially infections that require antibiotic treatment,
and to identify environments contaminated with antibiotic-
resistant pathogens [79].
Fecal indicator bacteria have been used for over 150 years to
indicate fecal contamination of water and associated health
risks (Table 1). The latter half of the nineteenth century saw
the discovery of waterborne disease transmission, perhaps most
famously in the analysis of drinking water systems by John
Snow during the 1854 London cholera outbreak and the isola-
tion of Vibrio cholerae by Robert Koch in 1884 (though first
identified in 1854 by Filippo Pacini) [1012]. Recognition that
sewage contamination of water sources spreads diseases such
as cholera and typhoid necessitated a means by which to ascer-
tain the presence of sewage in drinking water. Coliform bacte-
ria, a group of typically harmless Gram-negative bacteria that
constitute part of the natural gut microbiota in humans and other
warm-blooded animals, provided a simple and reasonably reli-
able tool for diagnosing sewage pollution in drinking water
samples owing to their high concentrations in sewage and ease
of culture [13,14]. A growing concern about fecal pollution in
the wider environment and the potential for human exposure to
enteric pathogens through additional environmental pathways,
This article is part of the Topical Collection on Water and Health
*Jill R. Stewart
Department of Epidemiology, Gillings School of Global Public
Health, University of North Carolina at Chapel Hill, 135 Dauer Dr.,
Chapel Hill, NC 27599-7435, USA
Department of Environmental Sciences and Engineering, Gillings
School of Global Public Health, University of North Carolina at
Chapel Hill, 135 Dauer Dr., Chapel Hill, NC 27599-7431, USA
Published online: 15 June 2020
Current Environmental Health Reports (2020) 7:311–324
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
especially recreational and foodborne exposure routes, encour-
aged subsequent efforts to assess fecal contamination in an
increasing variety of environmental matrices [15,16]. Feces
can contain a wide range of pathogens, which when introduced
to the environment may persist for varying amounts of time,
often at concentrations too low for reliable detection but still
hazardous to human health [1719]. Furthermore, conventional
methods of enteric pathogen detection are generally time-con-
suming, expensive, and often insensitive even in fresh feces
[20]. The use of fecal indicator bacteria (FIB) like the fecal
coliform Escherichia coli to suggest the presence of hazardous
fecal pollution therefore continues to be a valuable tool to assess
water quality.
Limitations of the fecal indicator paradigm have long been
acknowledged [2123]. Researchers have identified many
challenges and limitations to the effective use of both tradi-
tional and alternative fecal indicators to characterize risk,
identify sources, and evaluate interventions [2426].
Arguably, one of the most significant limitations is the incon-
sistent relationships between FIB occurrence, enteric patho-
gens, and health risks [25,27]. In settings with high rates of
enteric infection and inadequate fecal waste management,
E. coli in drinking water (but not other coliforms) has often
been associated with increased risk of illness [2830].
Similarly, the health risks of recreational uses of surface wa-
ters have been found to increase with FIB density, but gener-
ally only at locations with known human fecal inputs or under
high-risk conditions, such as following precipitation or the
removal of physical barriers [25,3135]. The FIB found to
correlate with health risks also vary widely by site [32]. The
co-occurrence of enteric pathogens and FIB in ambient waters
is inconsistent at best [27,36], and commonly used FIB are
known to persist and grow in the environment [3742].
In this paper, we review recent progress in the quest for
improved indicators of fecal contamination in water. We sum-
marize recent advances in alternative indicators with a focus
on microbial source tracking markers. We also recognize the
advances in molecular methods that are increasingly being
used to detect fecal contamination of water and to identify
sources of contamination. Improvements in detection capabil-
ities, analytical sensitivity, and data quality are discussed
along with barriers that must be overcome for wider adoption.
We review efforts to characterize health hazards associated
with fecal contamination, and we distinguish the timing and
conditions when indicators appear best suited to identify risks
to human health. Finally, we identify opportunities for contin-
ued improvements in the use of indicator organisms to assess
environmental fecal pollution and to safeguard human health.
Alternative Indicators
Many alternative fecal microbes have been proposed to ad-
dress the limitations of traditional FIB as indicators of fecal
pollution [21,22,43]. Better surrogates that share environ-
mental fate and transport mechanisms with pathogens of
concernparticularly coliphages, viruses that infect E. coli
bacteria, and obligate anaerobes, thought to have host speci-
ficity and to derive exclusively from recent fecal
contaminationhave frequently been identified as potential
alternative indicators expected to better represent risks to hu-
man health [4446]. Health risks from exposure to ambient
fecal contamination are largely a function of the specific path-
ogens present and their concentrations in the exposure matrix,
which is strongly influenced by the source of the fecal pollu-
tion [4751]. Enteric viruses that primarily derive from human
Table 1 Indicators of fecal contamination for water quality assessments
Indicator Example targets Applications Stage of
Fecal indicator bacteria
culture-based detection
E. coli, enterococci Hazard identification, regulatory
Fecal indicator bacteria
molecular detection
E. coli, enterococci Hazard identification, regulatory
Fecal indicator viruses Coliphages, Bacteroides bacteriophages Assess risk from enteric viruses Late
Human-associated MST
Bacteroides HF183, HumM2, PMMoV,
Determine source of contamination Middle
Animal-associated MST
BacCow, BacCan, avian GFD, Pig-2-Bac Determine source of contamination Middle
Index pathogens Noroviruses, rotaviruses, Salmonella spp.,
Campylobacter spp., Cryptosporidium spp.
Risk assessment Middle
(AMR) bacteria
ESBL E. coli, MAR E. coli, MRSA Assess environmental antimicrobial
Antimicrobial resistance
genes (ARGs)
intI, mcr-1,tnpA,sul1, tetW, tetM, qepA,bla
Assess environmental antimicrobial
PMMoV pepper mild mottle virus, ESBL E. coli extended-spectrum β-lactamase-producing E. coli,MAR E. coli multiple antibioticresistant E. coli,
MRSA methicillin-resistant Staphylococcus aureus
312 Curr Envir Health Rpt (2020) 7:311–324
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sources, notably human noroviruses, drive the global burden
of gastrointestinal illness, including from recreational water
exposures [2,5256]. Highly persistent and infectious proto-
zoan parasites are shed at high rates by livestock [2,5759],
and avian sources introduce bacterial pathogens like
Campylobacter spp.and non-typhoidal Salmonella spp., par-
ticularly the poultry common in domestic environments in
low- and middle-income countries (LMIC) where frequent
exposure is likely [57,6064]. Given the differences in human
health risks and appropriate mitigation strategies for fecal pol-
lution from different sources, there is a need to identify not
only the presence of fecal contamination but also its origins.
Because traditional FIB cannot discriminate between fecal
sources, host-associated fecal microbes have been the subject
of extensive research in recent years for use as indicators of
source-attributable fecal pollution, an approach known as mi-
crobial source tracking (MST) [23,43].
Microbial Source Tracking
Numerous host-associated organisms and gene markers have
been identified for identifying sources of fecal pollution in
water, none of which has demonstrated perfect source sensi-
tivity and specificity [46]. Numerous MST markers target
members of the order Bacteroidales,manyofwhichareobli-
gate anaerobes and abundant constituents of the gut microbi-
ota in warm-blooded animals, including one of the most com-
mon and earliest-proposed human-associated molecular
markers, HF183 [43,46,65,66]. Because MST targets spe-
cific constituents of the gut microbiota, the diagnostic perfor-
mance of each MST marker can vary substantially between
populations. Validation of existing MST markers for use in
new geographic locations is increasingly standard practice
[24], with recently published MST validation studies conduct-
ed in Australia [67,68], Bangladesh [69,70], Costa Rica [71],
India [72], Japan [73,74], Mozambique [75], New Zealand
[76], Nepal [77,78], Singapore [79], Thailand [80], and the
USA [81,82], and a global evaluation of markers in sewage
from 13 countries on 6 continents [83]. Potential MST
markers continue to be identified, most notably human-
associated crAssphage, a bacteriophage infecting
Bacteroides intestinalis recently discovered to be an abundant,
globally distributed constituent of the human gut virome
[8489]. Human-associated E. coli markers, long-desired for
their direct correspondence to a common FIB used for regu-
latory purposes, have also been developed [9092], though
they may lack the analytical sensitivity for effective use in
ambient waters [93]. The identification of new markers is
increasingly supported by advances in sequencing technology
and bioinformatics [84,9496], and next-generation sequenc-
ing (NGS)based MST approaches continue to be refined
[97]. Although highly dependent on fecal library composition
(the collection of metagenomic sequences from known fecal
sources that informs source identification algorithms)
[97101], NGS-MST has the potential to identify finer dis-
tinctions between sources, as demonstrated by a study in
Kenya that distinguished between fecal contamination from
young children and adults [102]. The recent introduction of
more affordable and portable long-read sequencing platforms,
while currently error prone, promises to accelerate the use of
sequencing to characterize fecal contamination [103,104].
MST proponents typically advocate a toolbox approach
to fecal source attribution that combines multiple MST
markers, detection methods, and sampling strategies in recog-
nition of the limitations of any single MST marker to reliably
and conclusively characterize fecal pollution [105107]. Two
toolbox constituents recently receiving much attention in the
literature are pepper mild mottle virus (PMMoV), a plant virus
infecting Capsicum species acquired by humans from dietary
sources, and crAssphage, both viruses that hold promise as
human-associated viral surrogates owing to their global distri-
bution in sewage at densities typically much higher than other
viruses [2,71,74,78,108110]. Nonetheless, Bacteroides
HF183 and its variants have arguably consolidated their role
as the default tool for human source tracking [43], featuring
consistently high concentrations in sewage globally [83], fre-
quent detection in surface waters [61,93,110112], standard-
ized protocols [81,113], and validated multiplex assays [89,
114]. However, the diagnostic performance of HF183 and
most other human-associated markers has typically been poor
in highly contaminated settings in many low- and middle-
income countries (LMIC) [58,69,70,72,75,115], with the
exception of high sensitivity to child feces in urban Kenya
Successful identification of non-human fecal sources may
best demonstrate the value of MST for informing management
and research priorities. Unlike human-associated markers, an-
imal fecal markers (e.g., livestock-associated BacCow and
canine-associated BacCan, both with Bacteroidales targets,
and avian-associated GFD, which targets the genus
Helicobacter) have performed well in LMIC settings and have
repeatedly identified livestock as major sources of fecal con-
tamination and pathogens in the domestic environment,
supporting recent calls for renewed emphasis on animal waste
management [58,116]. Likewise, MST investigations can im-
pact management and mitigation programs by determining
that wildlife, livestock, or pets contributed substantially to
fecal pollution in certain watersheds and beaches [81,117,
118,119]. The forensic potential of MST was demonstrated
during a 2019 Campylobacter outbreak in Norway, which was
attributed to non-human sources, most likely horses, using a
combination of FIB, MST, and direct pathogen detection
[120]. MST has also been used to identify sources of antimi-
crobial resistance, the environmental dimensions of which re-
main poorly understood [121,122]. Similar investigations are
likely to increasingly carry legal implications, for instance, by
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implicating animal agriculture industries in unpermitted sur-
face water pollution [13,123,124].
Molecular Methods: Challenges and Advances
The historic infeasibility of comprehensive direct pathogen
detection in environmental waters continues to motivate the
use of fecal indicators, which have traditionally been detected
using culture-based methods. Growing FIB from water sam-
ples on selective media is routine and relatively inexpensive
but generally requires a minimum of 18 hours to obtain re-
sults, by which time conditions at the sampling location may
have dramatically changed [125127]. Furthermore, the obli-
gate anaerobes and host-specific viruses proposed as alterna-
tive indicators for MST are often not amenable to laboratory
culture [43]. A range of alternative detection methods contin-
ue to be developed and are the subject of several recent com-
prehensive reviews [97,128131], with real-time polymerase
chain reaction (qPCR) and related molecular methods that
infer the presence of fecal microbes from their genetic material
experiencing particularly widespread adoption [43].
By bypassing culture, samples can be analyzed by qPCR in as
few as three hours or stabilized for transport and extended
storage prior to analysis [126], However, this analysis may
detect residual signals from organisms that are not viable or
infectious at the time of collection [132]. Although gene
markers must be pre-specified, qPCR (alongside reverse-
transcription PCR (RT-PCR) for RNA markers) provides a
consistent approach for detecting targets ranging from FIB
to viruses, human mitochondrial DNA, and genes conferring
pathogenicity or antimicrobial resistance [27,43,133]. qPCR
assays can also be multiplexed to detect a limited number of
targets in a single reaction. Furthermore, the recent develop-
ment of qPCR array cards that enable simultaneous detection
of dozens of gene targets in a single sample demonstrates the
growing feasibility of direct detection of a comprehensive set
of enteric pathogens alongside functional genes and fecal in-
dicators [134142].
Analytical Sensitivity
Although qPCR is a sensitive method relative to culture and
conventional PCR [20,143], it is vulnerable to interference
from other substances common in environmental waters that
can reduce the availability of target DNA or inhibit polymer-
ase function, limiting assay sensitivity [144]. Strategies to
mitigate matrix interference include sample dilution or chem-
ical treatment, nucleic acid purification, inhibition-resistant
reagents, and the use of multiple processing and internal
controls to both identify inhibited samples and competitively
bind interfering substances [144146]. Such approaches in-
crease the complexity, expense, and time requirements for
analysis, and physical removal of inhibitors through dilution
or purification also reduces target DNA, providing
diminishing returns to analytical sensitivity [147,148].
Complete abatement of qPCR inhibition is likely unrealistic;
nevertheless, recent efforts to standardize qPCR procedures
for water quality assessment suggest that a set of existing
mitigation practices is sufficient to render matrix interference
a manageable nuisance in most applications [81,144,149].
Increasing adoption of digital PCR (dPCR), a quantitative
PCR approach robust to inhibition, will likely further alleviate
the challenge of inhibition for routine molecular detection of
fecal microbes [61,114,128,150152].
Improved assay sensitivity offers little benefit if the target
is unlikely to be present in the test sample due to low ambient
concentrations. While simulation studies indicate that the con-
centrations at which bacterial indicators represent elevated
risk of illness are well above the limits of detection [153],
enteric viruses, protozoan parasites, and some alternative in-
dicators (e.g., coliphages) commonly require larger sample
volumes for reliable capture, necessitating concentration
methods to obtain test sample volumes that can be accommo-
dated by the chosen detection method [44,128]. Filtration
approaches that allow simultaneous concentration of a wide
range of organisms are increasingly used to process samples,
including as part of automated large-volume samplers, prior to
culture or molecular detection [128,154156]. Ultrafiltration
techniques in particular have demonstrated reasonably effi-
cient and consistent recovery for a variety of organisms, water
types, and sample volumes, providing a natural complement
to multitarget arrays, and increasingly appear to be the default
concentration approach for many applications [154,
157161]. Co-concentration of qPCR inhibitors during ultra-
filtration is a concern, but effective inhibition mitigation has
been demonstrated by further processing of the concentrate
prior to analysis [160,162164].
Data Quality
Generalized data reporting guidelines notwithstanding [165], dif-
ferences in analytical procedures and data handling practices
were identified as major sources of variability in a
multilaboratory comparison study of primarily qPCR-based
MST approaches [24,166]. Substantial effort has been devoted
in recent years to the development and implementation of stan-
dardized protocols and quality control metrics for fecal indicator
assessment by qPCR [113,167,168]. A notable feature shared
by these protocols and other recent recommendations for im-
proved reliability of molecular detection methods is a reliance
on numerous controls throughout the procedure [128,137].
While the use of positive and negative controls is standard for
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most analytical techniques, requirements outlined in standard-
ized qPCR protocols generally include multiple serial dilutions
of standard reference material to construct calibration curves,
sample processing controls (SPC), method extraction blanks
(MEB), internal amplification controls (IAC), and no template
controls (NTC), with two or three replicates of each sample,
control, and standard dilution series concentration analyzed on
each instrument run [113]. Each additional reference or control
material must be obtained, prepared, stored, and used in the
appropriate manner, increasing per-sample costs and introducing
considerable complexity and opportunity for user error. In a
recent large demonstration project, some laboratories without
extensive previous qPCR experience struggled to achieve ade-
quate quality control despite receiving method-specific instru-
mentation, materials, and training. Even laboratories with sub-
stantial qPCR experience regularly failed to meet data quality
criteria in this study [149]. The compounding complexity re-
quired for reliable results suggests that qPCR in its current form
may be unsuitable for routine monitoring purposes except in
particularly well-resourced laboratories that regularly process
sufficient sample numbers to warrant the equipment, properly
maintain assay materials, and ensure sustained institutional ex-
perience. By contrast, culture-based FIB detection has grown
increasingly accessible following recent efforts to develop low-
cost field tests that can be performed with minimal equipment at
ambient temperatures [125,169,170]. While still requiring sub-
stantial resources and expertise, dPCR requires fewer controls
and precise reference materials than qPCR because it is robust to
matrix interference and offers absolute quantification, features
which may position dPCR to be increasingly adopted for general
use [171]. Both up-front and per-reaction costs are considerably
higher for dPCR compared to qPCR, but the improved
multiplexing performance, fewer required control reactions,
and greater precision of dPCR present opportunities to mitigate
differences in per-sample costs [114,143,172,173].
Health Relevance and Protection
In addition to revealing fecal pollution and elucidating its
sources, fecal indicators are widely used to characterize health
hazards in waters potentially impacted by fecal contamination.
This approach has proven somewhat effective in drinking wa-
ter and for recreational exposures during wet weather or near
point sources of fecal pollution [25,28,35]. A recent review
found increased likelihood of co-detection of fecal indicators
and enteric pathogens in recreational waters under similar
conditions [27]. However, relationships between fecal indica-
tors and gastrointestinal illness have mostly not been observed
in waters impacted by non-point source pollution [25,33,
174], despite well-documented risks to swimmers [31].
In the absence of consistent empirical relationships, quan-
titative microbial risk assessment (QMRA) has been used to
estimate the health implications of various indicators intro-
duced by different fecal sources [153,175]. Notably, thresh-
old concentrations at which MST markers correspond to in-
creased risk of illness have been estimated in several QMRA
studies; these thresholds are comfortably above the typical
limits of detection, suggesting that the markers are highly
likely to be detected should their associated pathogens be
present at hazardous levels [2,3,49,56,62,176].
Indicator-based risk assessment requires defining the relation-
ships between the indicator concentration and the index patho-
gens selected for consideration, typically a subset of pathogens
expected to account for the majority of the risk and for which
dose-response relationships have been characterized [55,175]. A
substantial body of research characterizing processes affecting
indicator-pathogen relationships has culminated in the recent
publication of several comprehensive reviews and meta-
analyses of the occurrence, transport, and persistence of indica-
tors and common index pathogens in fecal waste streams and
surface water [17,27,177179]. Associations between indica-
tors and pathogens in surface water have been largely inconsis-
tent, although empirical determination of these relationships is
challenged by the limitations of direct pathogen detection; asso-
ciations are more commonly observed among more frequently
detected organisms [27,36]. Microbial occurrence is more con-
sistent in feces and particularly in sewage, which smooths the
high individual variability in fecal microbe shedding by
representing the combined fecal inputs of populations [177,
178,180182]. Despite less frequent detection of alternative
indicators in recreational waters [27], high concentrations of
multiple human-associated markers have been reported world-
wide in both raw and biologically treated wastewater [83].
Meta-analyses have also found high coliphage and norovirus
densities in raw sewage around the world [178,180]. A wide
range of pathogens have frequently been detected in stormwater,
though with greater variability and typically at lower concentra-
tions than in sewage [179].
Upon introduction to the environment, microbial contami-
nants are subject to highly variable dispersal and decay process-
es [17]. Differential transport and decay of indicators and path-
ogens reduce associations between them that may have been
present at the source, but the numerous factors affecting envi-
ronmental fate and transport were previously poorly understood
[24]. Many studies have since investigated the persistence of
different organisms under various conditions, often using seeded
mesocosms [17]. A recent QMRA study incorporated a meta-
analysis of decay rates and found that the risk represented by a
particular concentration of sewage-derived HF183 increased
with time because it decayed faster than norovirus, the principal
driver of risk [2]. Conversely, another QMRA found that failing
to account for differential decay overestimated the risk posed by
animal fecal sources but did not meaningfully affect the risk
from human sources, which in this study was dominated by
viruses with similar decay characteristicsashuman-associated
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markers [49]. However, the multitude of factors that affect mi-
crobial fate and transport calls into question the generalizability
of such assessments given the wide spatial and temporal varia-
tion in natural conditions, particularly across organisms and fe-
cal sources [17,183].
Applications and Recommendations
The use of FIB for fecal contamination assessment continues to
have many applications and has expanded with the broad adop-
tion of MST approaches. Routine monitoring of surface waters is
widely conducted in order to assess regulatory compliance, char-
acterize water quality trends, and provide timely warnings to
protect public health [184189]. Specific investigations, often
supplemented with historical monitoring data, may be conducted
to inform management strategies and remediation efforts and to
evaluate the impacts of infrastructure, policy, and practices [59,
118,179,190193]. Forensic applications are increasingly pur-
sued to assign responsibility for fecal pollution, largely enabled
by wider adoption of MST approaches and molecular detection
methods [13,69,70,106,117,120,123].
Despite their widespread use, evidence for the suitability of
indicators in evaluative applications remains mixed and ap-
pears to vary depending on the timing and conditions under
which they are applied. Under favorable conditions that pro-
vide more proximate connections between indicators and their
sources (e.g., near wastewater outfalls or in household drink-
ing water), indicator abundance may be associated with in-
creased risk of illness that one would expect with elevated
fecal loads [25,29,194]. Interventions that directly impact
sources, such as gull deterrence at beaches, may also be
reflected in indicator concentrations [118]. Contamination
through less direct processes, such as non-point source pollu-
tion, is subject to the numerous factors affecting microbial fate
and transport, which may account for the large temporal var-
iability often observed in FIB concentrations and the lack of
association with illness [25,127,195]. Such variability limits
the amount of information conveyed by individual observa-
tions, requiring much larger datasets to disentangle trends in
indicator occurrence from the inherent variance in indicator
measurements [188,189]. These limitations are especially
pronounced when anticipated effects are indirect and small
relative to typical indicator concentrations, which may be
maintained in part by other sources and pathways of contam-
ination [138,192,193,196]. The outsized influence of pre-
cipitation on microbial concentrations may obscure less dra-
matic dynamics in many systems [195]. Furthermore, clear
long-term indicator trends do not necessarily represent con-
comitant changes in pathogen hazards [157].
The increasing feasibility of comprehensive direct path-
ogen detection suggests that situations demanding a high
degree of confidence about the presence of hazardous fecal
contamination may be best served by assaying pathogens
directly, utilizing concentration methods to improve sensi-
tivity as appropriate [128]. The possibility of false nega-
tives due to temporal and spatial variability, while partially
addressable through strategies such as composite sam-
pling, nevertheless suggests that general fecal indicators
should continue to be assessed to complement direct path-
ogen detection efforts. Despite the recent introduction of
procedures to simultaneously quantify multiple FIB, MST,
and pathogen genes in under 4 hours [139], the expense,
necessary expertise, and rapid pace of change likely pre-
clude the routine application of direct pathogen detection
for some time to come. Meanwhile, protecting public
health in recreational waters remains an important (and
legally mandated) goal. High-traffic beaches with
established daily microbial water quality testing programs
and dedicated laboratory facilities are likely to benefit from
implementing rapid FIB qPCR monitoring with same-day
notification [126,197]. As such beaches are often located
near large urban areas and impacted by human sources,
they may further benefit from instead implementing simul-
taneous monitoring of FIB and human-associated markers
by duplex dPCR to establish time trends in human-source
contamination at little additional cost [112,114]. Although
associations between human-associated markers and gas-
trointestinal illness are generally lacking [32,174], their
regular application across multiple human-impacted loca-
tions may provide useful information to prioritize remedi-
ation efforts [111,112].
Locations that host fewer recreators, have limited monitoring
resources and sampling frequency, or are impacted by non-point
sources, for which generalizable relationships between indicators
and risk are lacking, are unlikely to realize similar benefits from
adopting rapid molecular monitoring while incurring substantial
additional expense, complexity, and opportunity for error [149,
198,199]. Rather, supplementing existing FIB monitoring pro-
grams with predictive modeling may present a more feasible
approach for expanding the scope of microbial water quality
assessment in the numerous surface waters for which monitoring
resources are limited [200,201]. Precipitationperhaps the most
consistent factor in recreational water quality, reliably increasing
ambient fecal microbe concentrations and the risk of illness
likewise tends to drive predictive FIB model outcomes [119,179,
195,202204]; for many applications, providing recreational
guidance on the basis of recent precipitation may well present
the most reliable method for protecting public health [110].
The value of fecal indicators as investigative tools to identify
fecal pollution has been reaffirmed and expanded with the
broad adoption of MST approaches, despite imperfect
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sensitivity and specificity [58,81,110,118,119]. Also, the
literature on many technical aspects of fecal indicators and
their applications has notably matured, as demonstrated by
the recent publication of several comprehensive reviews [2,
17,25,27,44,144,153]. The improved understanding of
microbial dynamics and detection approaches has supported
the development of more nuanced and robust procedures for
characterizing fecal pollution. Nevertheless, this body of work
also serves to emphasize the incredible complexity and vari-
ability of fecal microbes in the environment and reinforces the
challenges to their effective use.
Major challenges remain in source apportionment, risk
characterization, and impact evaluation. Additional research
is needed to further refine indicators of fecal contamination
and to add tools to the toolbox appropriate for emerging chal-
lenges. New indicators are needed to detect antimicrobial-
resistant bacteria and resistance genes in water samples and
to link environmental antimicrobial resistance to health risks.
Better risk characterizations are needed to improve risk
modeling and to expand the timing and conditions under
which these models can reliably predict threats to human
health. Also, empirical models that identify associations be-
tween indicators and co-measured predictors, particularly
rainfall, can likely alleviate some of the sample burden asso-
ciated with water quality assessments. Direct pathogen detec-
tion is becoming more feasible than in previous years and is
likely to be more of a focus for water quality tests in the future.
Together, these advances are improving water quality assess-
ments and identifying appropriate actions to safeguard public
health across the globe.
Acknowledgments David Holcomb received support from the National
Institute of Environmental Health Sciences (T32ES007018). Jill Stewart
was supported by NSF grant 1316318 as part of the joint NSF-NIH-
USDA Ecology and Evolution of Infectious Diseases program.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflicts of
Human and Animal Rights and Informed Consent This article does not
contain any studies with human or animal subjects performed by any of
the authors.
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... Additionally, several studies have reported the detection of E. coli, S. aureus, and L. monocytogenes in unpasteurized soft cheeses in the USA [2,8]. These contaminants pose a problem, because contamination of the food with enteric pathogens [48,49]. Additionally, the antibiotic resistance profiles of E. coli have been used as indicators for the emergence and spread of AMR in foods [50]. ...
... The densities of bacteria like E. coli and S. aureus are used as microbiological indicators to determine the acceptability and safety of cheese [9]. High bacterial densities of fecal indicators, like E. coli, also suggest the possible contamination of the food with enteric pathogens [48,49]. Additionally, the antibiotic resistance profiles of E. coli have been used as indicators for the emergence and spread of AMR in foods [50]. ...
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Dairy foods are a staple in Lebanon, a low- and middle-income country that has been experiencing serious challenges to food safety and antimicrobial stewardship among other issues. The microbiological acceptability of dairy products has been of increasing concern. This is partially due to the failing economy and prolonged power outages that affect the quality of raw material and disrupt the dairy cold chain, respectively. Therefore, we assessed the microbiological acceptability of Akkawi, a popular white-brined cheese in Lebanon. For this purpose, we quantified the densities of Escherichia coli (a fecal indicator) and Staphylococcus aureus in cheeses collected from Lebanese retail stores. Additionally, we evaluated the antibiotic resistance profiles of the E. coli isolated from the cheese. E. coli and S. aureus were detected in 40 (80%) and 16 (32%) of the 50 cheese samples, respectively. Notably, 40 (80%) and 16 (32%) of the samples exceeded the maximum permissible limit of E. coli and S. aureus, respectively. A high percentage of the 118 E. coli isolated from the cheeses showed resistance to clinically and agriculturally important antibiotics, while 89 (75%) isolates were classified as multidrug-resistant (MDR). Given that Akkawi can be consumed without cooking, our findings highlight serious food safety and antimicrobial resistance problems that require immediate interventions.
... In this paper, we describe and report the test results from a small prototype pulsed ultraviolet recirculation system for purification of seawater artificially contaminated with high levels of Escherichia coli, Staphylococcus aureus, Bacillus cereus, Candida albicans and Salmonella typhimurium. For example, Escherichia coli and Salmonella species were specifically chosen because they are used as index pathogens of faecal contamination for water quality assessment (Holcomb and Stewart, 2020). Candida albicans was chosen because it is representative of a clinical yeast occurring in wastewater (Babič et al. 2017). ...
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The increase in pathogen levels in seawater threatens the safety of entire aquatic ecosystems. Foodborne pathogens can potentially accumulate in shellfish, especially in filter feeders such as bivalves, requiring an efficient depuration process before consumption. Alternative approaches to promote a cost-efficient purge at depuration plants are urgently needed. A small prototype pulsed ultraviolet (PUV) light recirculation system was designed, and its depuration potential was tested in a seawater matrix artificially contaminated with high levels of microbial pathogens Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Bacillus cereus and Candida albicans. The analysis of treatment parameters including voltage, number of pulses and duration of treatment was performed to ensure the highest reduction in contaminant levels. Optimal PUV disinfection was attained at 60 pulses/min at 1 kV for 10 min (a UV output of 12.9 J/cm2). All reductions were statistically significant, and the greatest was observed for S. aureus (5.63 log10), followed by C. albicans (5.15 log10), S. typhimurium (5 log10), B. cereus (4.59 log10) and E. coli (4.55 log10). PUV treatment disrupted the pathogen DNA with the result that S. aureus, C. albicans and S. typhimurium were not detectable by PCR. Regulations were reviewed to address the applicability of PUV treatment as a promising alternative to assist in the reduction of microbial pathogens at depuration plants due to its high efficiency, short treatment period, high UV dose and recirculation system as currently employed in shellfish depuration plants.
... Biological factors of water are measured by the presence of pollution indicators of organisms, e.g., Total Germ (e.g., Total Bacteria, Viruses, Salmonella spp.), Coliforms (both Fecal and Total), Protozoa and Algae (Wilhm and Dorris 1968;Holcomb and Stewart 2020). These are important parameters of water potability. ...
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Surface water is heavily exposed to contamination as this is the ubiquitous source for the majority of water needs. This situation is exaggerated by the excessive population, heavy industrialization, rapid urbanization, and improper sanitation. Comprehensive measurement and knowledge extraction of surface water quality is therefore pivotal for ensuring safe and hygienic water use. Consequently, surface water quality profiling has received remarkable academic attention in recent decades that produces an ample amount of research results. This study, therefore, conducts a comprehensive systematic literature review to summarize and structure the existing literature and to identify current research trends and hotspots. Reported results suggest that the terrain of fresh surface water includes 13 distinct water sources that are predominantly used in 5 sectors. These sectors often cause the water pollution in the form of industrial effluents, agricultural runoffs, and domestic sewage. For profiling the water quality, around 23 Water Quality Index (WQI) models, and 10 Pollution Index (PI) models are used in research. These models often use a number of water quality parameters. This study reports an exhaustive taxonomy of 69 prominent quality parameters in three categories which will support their adoption for these models. Finally, the limitations of the current manual water quality measurement approaches are summarized to propose a set of seven requirements for the tech- intensive water quality profiling research and system development.
... Contrastingly, in the wetlands corresponding to the southern basins, where the influence of "El Centenario" is limited, the NSF-WQI shows better water quality (medium quality) and the bacterial concentration drastically dropped in both climatic periods, compared with the central south of the lake. The PCA illustrated that wetlands are associated with waters of higher transparency and lower levels of variables related to contamination, such as fecal coliforms and BOD5 [58]. The wetlands are the section of the lake where the anthropogenic influence is most limited. ...
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In the Yucatán Peninsula, anthropogenic activities such as urbanization and the final disposal of solids and wastewater critically impact aquatic systems. Here, we evaluated the anthropo-genic-related environmental alteration of Lake La Sabana, located in the northern limits of one of the main cities of the Mexican Caribbean. We evaluated lake water quality, using physical, chemical, and microbiological indicators, and heavy metals in surficial sediment and fish tissue to evaluate the potential environmental risk. Multivariate analyses revealed that environmental conditions in La Sabana are spatially and temporally heterogeneous. Medium to bad water quality was determined within different basins by the National Sanitation Foundation water quality index, related to the degree of anthropogenic influence at each zone. The central-south zones displayed critical mi-crobiological values largely exceeding national standards. Heavy metals in sediment and fish tissue such as Zn and Hg were relatively low, but Hg concentrations threaten the ecological environment. Incipient wastewater treatment and its final disposal in La Sabana are mainly responsible for the changes in the trophic status and availability of nutrients, which in turn may have promoted changes in the biological structure and aquatic plant invasions. Lake La Sabana can be considered a model of the potential and sequential effects of anthropogenic alterations in the oligotrophic karst tropical aquatic systems in the Yucatan Peninsula.
... Contrastingly, in the wetlands corresponding to the southern basins, where the influence of "El Centenario" is limited, the NSF-WQI shows better water quality (medium quality) and the bacterial concentration drastically dropped in both climatic periods, compared with the central south of the lake. The PCA illustrated that wetlands are associated with waters of higher transparency and lower levels of variables related to contamination, such as fecal coliforms and BOD5 [58]. The wetlands are the section of the lake where the anthropogenic influence is most limited. ...
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In the Yucatán Peninsula, anthropogenic activities such as urbanization and final dispose of solid and wastewaters critically impact aquatic systems. Here, we evaluated the anthropogenic-related environmental alteration of Lake La Sabana, located in the northern limit of one of the main cities of the Mexican Caribbean. We evaluate lake water quality using physical, chemical, and microbiological indicators, and heavy metals in surficial sediments and fish tissue to evaluate the potential environmental risk. Multivariate analyses reveal that environmental conditions in La Sabana are spatial and temporal heterogeneous. Medium to bad water quality was determined within basins by the NSFWQI, related with the degree of anthropogenic influence. The center-south zones display critical microbiological values largely exceeding national standards. Heavy metals in sediments (Zn>Hg) and fish tissue (Hg) were relatively low, but Hg concentration threat the ecological environment. Incipient wastewater treatment and final dispose in La Sabana is the main responsible of changes in the trophic status and nutrients availability, which in turn may have promoted changes in the biological structure and aquatic plant invasions. Lake La Sabana can be considered a model of the potential sequential effects of the anthropogenic alterations in oligotrophic karts tropical aquatic systems in Yucatán Peninsula.
Background: Water, sanitation, and hygiene (WASH) improvements are promoted to reduce diarrhoea in low-income countries. However, trials from the past 5 years have found mixed effects of household-level and community-level WASH interventions on child health. Measuring pathogens and host-specific faecal markers in the environment can help investigate causal pathways between WASH and health by quantifying whether and by how much interventions reduce environmental exposure to enteric pathogens and faecal contamination from human and different animal sources. We aimed to assess the effects of WASH interventions on enteropathogens and microbial source tracking (MST) markers in environmental samples. Methods: We did a systematic review and individual participant data meta-analysis, which included searches from Jan 1, 2000, to Jan 5, 2023, from PubMed, Embase, CAB Direct Global Health, Agricultural and Environmental Science Database, Web of Science, and Scopus, of prospective studies with water, sanitation, or hygiene interventions and concurrent control group that measured pathogens or MST markers in environmental samples and measured child anthropometry, diarrhoea, or pathogen-specific infections. We used covariate-adjusted regression models with robust standard errors to estimate study-specific intervention effects and pooled effect estimates across studies using random-effects models. Findings: Few trials have measured the effect of sanitation interventions on pathogens and MST markers in the environment and they mostly focused on onsite sanitation. We extracted individual participant data on nine environmental assessments from five eligible trials. Environmental sampling included drinking water, hand rinses, soil, and flies. Interventions were consistently associated with reduced pathogen detection in the environment but effect estimates in most individual studies could not be distinguished from chance. Pooled across studies, we found a small reduction in the prevalence of any pathogen in any sample type (pooled prevalence ratio [PR] 0·94 [95% CI 0·90-0·99]). Interventions had no effect on the prevalence of MST markers from humans (pooled PR 1·00 [95% CI 0·88-1·13]) or animals (pooled PR 1·00 [95% CI 0·97-1·03]). Interpretation: The small effect of these sanitation interventions on pathogen detection and absence of effects on human or animal faecal markers are consistent with the small or null health effects previously reported in these trials. Our findings suggest that the basic sanitation interventions implemented in these studies did not contain human waste and did not adequately reduce exposure to enteropathogens in the environment. Funding: Bill and Melinda Gates Foundation and the UK Foreign and Commonwealth Development Office.
Twenty-two wastewater discharging emergency outfalls located within Kuwait Bay have the ability to pollute marine life. The quality of wastewater discharged to the sea has a direct and significant impact on the ecosystem including marine organisms, and an indirect effect on human health. The current study aims to evaluate the quality of wastewater discharged at selected sites of Kuwait Bay and to compare the obtained results with the standards of the Environmental Public Authority (EPA) for discharging treated wastewater to Kuwait Bay. Five locations were selected near emergency outfalls, and onsite field measurements for water quality were carried out, including temperature, pH, Electrical Conductivity (EC), and Dissolved Oxygen (DO). Furthermore, 15 mixed water samples (wastewater and seawater) were collected throughout November 2021 and January 2022 during low and high tides and within 1 m away from wastewater discharging outfalls. These samples were analyzed for nutrients, heavy metals, and bacteria parameters. The field results indicated the presence of slight alkalinity (pH 7.01–8.0), freshwater to saline water type (EC, 1.27 ms/cm–65.07ms/cm), oxidized environment (DO, 0.91 mg/l–5.28 mg/l). The laboratory results of water samples revealed that the concentrations of nutrients (total nitrogen, 2.0–35 mg/l) and all targeted heavy metals were detected in mixed samples for all sites in concentrations of microgram per liter and within EPA acceptable limits. On the other hand, phosphate concentration (4.2–13.6 mg/l) and the counts of microbial indicators (Fecal coliform, E. coli, and Fecal Streptococci) were high and above EPA standards, which shows the grave biological pollution that occurred at these sites. The variation in the contamination levels at the selected locations indicates the need for periodic monitoring of the quality of mixed water near wastewater emergency outlets and searching for the appropriate solutions to reduce the contaminated wastewater discharged directly into the beaches.
In this study, we assessed six Oklahoma streams for Enterococcus sediment and water concentrations along with water quality, sediment, hydrologic and geographical factors. We also conducted a microcosm experiment from two stream sediments to evaluate Enterococcus survivability under stable laboratory conditions. Stream sites exhibited common relationships between Enterococcus and other environmental factors, including significant correlations to antecedent dry period, Escherichia coli, impervious area, dissolved oxygen, and turbidity. These correlations were found for Enterococcus in both water and sediment. Specifically for Enterococcus in sediment, concentrations were also significantly correlated to turbidity and sediment percent organic matter, but not to hydrological conditions. Conversely, concentrations of Enterococcus in water exhibited significant moderate correlations to precipitation, antecedent dry period, drainage area, impervious area, and discharge, as well as streambed particle size. High variability between geographical attributes and stream conditions increased uncertainties and relationships between Enterococcus concentrations in the stream among most factors. However, when grouping sites by similar watershed and sediment characteristics, strong significant relationships for water-quality parameters and Enterococcus concentrations in water and sediment were observed. The microcosm study indicated that sediment Enterococcus concentrations for two streams with contrasting sediment properties were stable, except for a considerable increase between day 0 and day 1, with no decay shown for a 31 day period. Collectively, our field and laboratory results revealed that Enterococcus can survive for extended periods under both dynamic and stable sediment and water conditions, and that environmental factors can be used to characterize freshwater streams and rivers for Enterococcus concentrations in freshwater streams and rivers.
Contaminated irrigation water is among many potential vehicles of human pathogens to food plants, constituting significant public health risks especially for the fresh produce category. This review discusses some available guidelines or regulations for microbiological safety of irrigation water, and provides a summary of some common methods used for characterizing microbial contamination. The goal of such exploration is to understand some of the considerations that influence formulation of water testing guidelines, describe priority microbial parameters particularly with respect to food safety risks, and attempt to determine what methods are most suitable for their screening. Furthermore, the review discusses factors that influence the potential for microbiologically polluted irrigation water to pose substantial risks of pathogenic contamination to produce items. Some of these factors include type of water source exploited, irrigation methods, other agro ecosystem features/practices, as well as pathogen traits such as die-off rates. Additionally, the review examines factors such as food safety knowledge, other farmer attitudes or inclinations, level of social exposure and financial circumstances that influence adherence to water testing guidelines and other safe water application practices. A thorough understanding of relevant risk metrics for the application and management of irrigation water is necessary for the development of water testing criteria. To determine sampling and analytical approach for water testing, factors such as agricultural practices (which differ among farms and regionally), as well as environmental factors that modulate how water quality may affect the microbiological safety of produce should be considered. Research and technological advancements that can improve testing approach and the determination of target levels for hazard characterization or description for the many different pollution contexts as well as farmer adherence to testing requirements, are desirable.
Fecal contamination of fresh produce from human and animal sources is a public health concern due to the risk of foodborne illnesses. The current standard laboratory procedures for microbiological analyses usually require an enrichment step that involves several hours. Molecular techniques such as polymerase chain reaction (PCR) have been used to directly detect pathogens from the samples, however, due to the low quantity of pathogen present and small volumes used for PCR, enrichment is usually required. Additionally, the need for specialized equipment and experienced workers hinders the use of these molecular techniques for field testing. Here, we developed a rapid risk-assessment assay for fecal contamination by targeting Bacteroidales using loop-mediated isothermal amplification (LAMP). The assay allows for naked-eye observation of reactions with as few as ∼8 copies of Bacteroidales per cm² of the surface in the field. We evaluated this assay with complex field samples as well as on-site field studies. Our on-field studies demonstrated that the Bacteroidales LAMP assay enables us to easily and quickly (<50 min) assess the risk of fecal contamination from animal operations, with a concordance of 85.3% when compared to lab-based qPCR. These results were obtained without expensive equipment (when compared to standard laboratory procedures). . These assays could be used to determine site-specific risk and help the decision-making process of fresh produce growers.
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Characterizing fecal contamination exposure from drinking water can introduce exposure measurement errors, i.e., differences between the observed and true exposure. These errors can mask the true relationship between fecal contamination exposure and waterborne diseases. We present a framework to quantify the impact of measurement errors on exposure–outcome health effect estimates introduced by variability in measured drinking water fecal contamination levels and household versus community sampling strategies. We matched fecal indicator bacteria (FIB) data for >37,000 drinking water samples to children aged 0–72 months from 19 studies in low- and middle-income countries and took two complementary analytical approaches. We found that household-level exposure assessments may attenuate effect estimates of FIB concentrations in drinking water on diarrhea, and single water samples may attenuate health effect estimates of FIB concentrations on linear growth. To understand the health effects of fecal contamination exposure, measurement error frameworks can be used to estimate more biologically relevant exposures.
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Current microbiological water safety testing methods are not feasible in many settings because of laboratory, cost, and other constraints, particularly in low-income countries where water quality monitoring is most needed to protect public health. We evaluated two promising E. coli methods that may have potential in at-scale global water quality monitoring: a modified membrane filtration test followed by incubation on pre-prepared plates with dehydrated culture medium (CompactDryTM), and 10 and 100 ml presence–absence tests using the open-source Aquatest medium (AT). We compared results to membrane filtration followed by incubation on MI agar as the standard test. We tested 315 samples in triplicate of drinking water in Bangalore, India, where E. coli counts by the standard method ranged from non-detect in 100 ml samples to TNTC (>200). Results suggest high sensitivity and specificity for E. coli detection of candidate tests compared with the standard method: sensitivity and specificity of the 100 ml AT test was 97% and 96% when incubated for 24 h at standard temperature and 97% and 97% when incubated 48 h at ambient temperatures (mean: 27 °C). Sensitivity and specificity of the CompactDryTM test was >99 and 97% when incubated for 24 h at standard temperature and >99 and 97% when incubated 48 h at ambient temperatures. Good agreement between these candidate tests compared with the reference method suggests they are suitable for E. coli monitoring to indicate water safety.
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Identifying the origin of fecal contamination can support more effective interventions to interrupt enteric pathogen transmission. Microbial source tracking (MST) assays may help to identify environmental routes of pathogen transmission although these assays have performed poorly in highly contaminated domestic settings, highlighting the importance of both diagnostic validation and understanding the context-specific ecological, physical, and sociodemographic factors driving the spread of fecal contamination. We assessed fecal contamination of compounds (clusters of 2–10 households that share sanitation facilities) in low-income neighborhoods of urban Maputo, Mozambique, using a set of MST assays that were validated with animal stool and latrine sludge from study compounds. We sampled five environmental compartments involved in fecal microbe transmission and exposure: compound water source, household stored water and food preparation surfaces, and soil from the entrance to the compound latrine and the entrances to each household. Each sample was analyzed by culture for the general fecal indicator Escherichia coli (cEC) and by real-time PCR for the E. coli molecular marker EC23S857, human-associated markers HF183/BacR287 and Mnif, and GFD, an avian-associated marker. We collected 366 samples from 94 households in 58 compounds. At least one microbial target (indicator organism or marker gene) was detected in 96% of samples (353/366), with both E. coli targets present in the majority of samples (78%). Human targets were frequently detected in soils (59%) and occasionally in stored water (17%) but seldom in source water or on food surfaces. The avian target GFD was rarely detected in any sample type but was most common in soils (4%). To identify risk factors of fecal contamination, we estimated associations with sociodemographic, meteorological, and physical sample characteristics for each microbial target and sample type combination using Bayesian censored regression for target concentration responses and Bayesian logistic regression for target detection status. Associations with risk factors were generally weak and often differed in direction between different targets and sample types, though relationships were somewhat more consistent for physical sample characteristics. Wet soils were associated with elevated concentrations of cEC and EC23S857 and odds of detecting HF183. Water storage container characteristics that expose the contents to potential contact with hands and other objects were weakly associated with human target detection. Our results describe a setting impacted by pervasive domestic fecal contamination, including from human sources, that was largely disconnected from the observed variation in socioeconomic and sanitary conditions. This pattern suggests that in such highly contaminated settings, transformational changes to the community environment may be required before meaningful impacts on fecal contamination can be realized.
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Human contact with fecally contaminated waters often raises public health concern. The infection potential closely relates to the fecal source type and the aging persistence of waterborne pathogens. In this study, the health risk of contracting gastroenteritis from exposure to aging fecal contamination was predicted using source-associated markers. Microbial decay characteristics in typical summer seawater were incorporated into a pathogen dose estimation model for a constant fecal input. Results show that the median illness probability commensurate with the health benchmark of 36/1,000 corresponded to the marker concentrations of ~7.8, ~6.6, ~3.7 and ~3.5 log10 gene copies/100 mL for seagulls, cattle, raw sewage and treated effluent, respectively. The error in risk estimates due to neglecting microbial decay was linearly correlated to the decay differences between markers and pathogens. Specifically, the health risk associated with non-human sources, which is primarily contributed by bacterial and parasitic pathogens, can be substantially overestimated, while that for virus-dominated human sources was insignificantly affected by the differential decay. Additionally, seagulls dominated Enterococcus concentration in waters with a mixture of above-mentioned sources, although they posed limited health risk. This study provides an approach to understanding the influence of fecal aging on health risk estimation.
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Levels of fecal indicator bacteria (FIB) provide a surrogate measure of the microbial quality of water used for a wide range of applications. Despite the common use of these measures, a significant limitation is a delay in results due to the time required for cultivation and enumeration of FIB. Testing requires at least 18-24 h, and therefore, FIB cannot be used to identify current or real-time microbial water quality. An approach of nowcasting or empirical modelling approaches that incorporate water quality, environmental, and weather variables to predict FIB levels in real-time has been developed with some success. However, FIB levels are dependent on a complex interaction of numerous variables, which can be challenging to model with ordinary linear regression or classification methods most commonly applied. In this study, novel use of Bayesian Belief Networks (BBNs) that allow for a probabilistic representation of complex variable interactions is investigated for real-time modelling of FIB levels surface waters. In particular, the integration of both water quality measures and current/historical weather for prediction of fecal coliforms and Escherichia coli levels is achieved using BBNs. For 4-bin classification of fecal coliform levels, BBNs increased prediction accuracy by 25%-54% compared to other previously used techniques including logistic regression, Naïve Bayes, and random forests. Binary prediction of E. coli levels exceeding a threshold of 20 CFU/100 mL was also significantly improved using BBNs with prediction accuracies >90% for all monitoring sites. Advantages of the BBN approach are also demonstrated identifying the ability to make predictions from incomplete monitoring data as well as probabilistic inference of variable importance in FIB levels. In particular, the results indicate that water quality surrogates such as conductivity are essential to real-time prediction of FIB. The results and models described in this work can be readily utilized to provide accurate and real-time assessments of FIB levels in surface waters utilizing commonly monitored parameters.
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During June 2019, an outbreak of campylobacteriosis occurred in Askøy, an island northwest of Bergen, Norway. According to the publicly available records, over 2000 residents fell ill and 76 were hospitalised, and two deaths were suspected to be associated with Campylobacter infection. By investigating the epidemic pattern and scope, an old caved drinking water holding pool was identified that had been faecally contaminated as indicated by the presence of Escherichia coli (E. coli). Furthermore, Campylobacter bacteria were found at several points in the water distribution system. In the escalated water health crisis, tracking down the infectious source became pivotal for the local municipality in order to take prompt and appropriate action to control the epidemic. A major task was to identify the primary faecal pollution source, which could further assist in tracking down the epidemic origin. Water from the affected pool was analysed using quantitative microbial source tracking (QMST) applying host-specific Bacteroidales 16S rRNA genetic markers. In addition, Campylobacter jejuni, Enterococcus faecalis, Clostridium perfringens and Shiga toxin-producing E. coli were detected. The QMST outcomes revealed that non-human (zoogenic) sources accounted predominantly for faecal pollution. More precisely, 69% of the faecal water contamination originated from horses.
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Nucleic acid based techniques, such as quantitative PCR (qPCR) and next generation sequencing (NGS), provide new insights into microbial water quality, but considerable uncertainty remains around their correct interpretation. We demonstrate, for different water sources in informal settlements in the Kathmandu Valley, Nepal, significant Spearman rank correlations between conventional and molecular microbiology methods that indicate faecal contamination. At family and genera level, 16S rRNA amplicon sequencing results obtained with the low-cost, portable next generation sequencer MinION from Oxford Nanopore Technologies had significant Spearman rank correlations with Illumina MiSeq sequencing results. However, method validation by amplicon sequencing of a MOCK microbial community revealed the need to ascertain MinION sequencing results for putative pathogens at species level with complementary qPCR assays. Vibrio cholerae hazards were poorly associated with plate count faecal coliforms, but flagged up by the MinION screening method, and confirmed by a qPCR assay. Plate counting methods remain important to assess viability of faecal coliforms in disinfected water sources. We outline a systematic approach for data collection and interpretation of such complementary results. In the Kathmandu Valley, there is high variability of water quality from different sources, including for treated water samples, illustrating the importance of disinfection at the point of use.
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Sanitary surveys are used in low- and middle-income countries to assess water, sanitation, and hygiene conditions, but have rarely been compared with direct measures of environmental fecal contamination. We conducted a cross-sectional assessment of sanitary conditions and E. coli counts in soils and on surfaces of compounds (household clusters) in low-income neighborhoods of Maputo, Mozambique. We adapted the World Bank’s Urban Sanitation Status Index to implement a sanitary survey tool specifically for compounds: a Localized Sanitation Status Index (LSSI) ranging from zero (poor sanitary conditions) to one (better sanitary conditions) calculated from 20 variables that characterized local sanitary conditions. We measured the variation in the LSSI with E. coli counts in soil (nine locations/compound) and surface swabs (seven locations/compound) in 80 compounds to assess reliability. Multivariable regression indicated that a ten-percentage point increase in LSSI was associated with 0.05 (95% CI: 0.00, 0.11) log10 fewer E. coli/dry gram in courtyard soil. Overall, the LSSI may be associated with fecal contamination in compound soil; however, the differences detected may not be meaningful in terms of public health hazards.
Microbial pollution of recreational waters poses a significant public health risk which, unless mitigated, will continue to increase with population growth. Water managers must implement strategies to accurately discriminate and source human from animal faecal contamination in complex urbanised environments. Our case-study used a new combination of chemical (i.e. ammonia) and microbial (i.e. Escherichia coli, Bacteroides spp.) faecal monitoring tools in a targeted multi-tiered approach to quickly identify pollution hot-spots and track high-risk subterranean stormwater drains in real-time. We successfully located three point sources of human faecal pollution (both episodic and constant pollution streams) within 11 catchments in a total monitoring time of four months. Alternative approaches for obtaining such fine-scale accuracy are typically labour intensive and require expensive equipment.
Fecal microorganisms can enter water bodies in diverse ways, including runoff, sewage discharge, and direct fecal deposition. Once in water, the microorganisms experience conditions that are very different from intestinal habitats. The transition from host to aquatic environment may lead to rapid inactivation, some degree of persistence, or growth. Microorganisms may remain planktonic, be deposited in sediment, wash up on beaches, or attach to aquatic vegetation. Each of these habitats offers a panoply of different stressors or advantages, including UV light exposure, temperature fluctuations, salinity, nutrient availability, and biotic interactions with the indigenous microbiota (e.g., predation and/or competition). The host sources of fecal microorganisms are likewise numerous, including wildlife, pets, livestock, and humans. Most of these microorganisms are unlikely to affect human health, but certain taxa can cause waterborne disease. Others signal increased probability of pathogen presence, e.g., the fecal indicator bacteria Escherichia coli and enterococci and bacteriophages, or act as fecal source identifiers (microbial source tracking markers). The effects of environmental factors on decay are frequently inconsistent across microbial species, fecal sources, and measurement strategies (e.g., culture versus molecular). Therefore, broad generalizations about the fate of fecal microorganisms in aquatic environments are problematic, compromising efforts to predict microbial decay and health risk from contamination events. This review summarizes the recent literature on decay of fecal microorganisms in aquatic environments, recognizes defensible generalizations, and identifies knowledge gaps that may provide particularly fruitful avenues for obtaining a better understanding of the fates of these organisms in aquatic environments.