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WATER AND HEALTH (T WADE, SECTION EDITOR)
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
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 [4–6]. 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 [7–9].
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) [10–12]. 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
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 [17–19]. Furthermore, conventional
methods of enteric pathogen detection are generally time-con-
suming, expensive, and often insensitive even in fresh feces
. 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
Limitations of the fecal indicator paradigm have long been
acknowledged [21–23]. 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 [24–26].
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 [28–30].
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 [25,31–35]. The FIB found to
correlate with health risks also vary widely by site . 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 [37–42].
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.
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
concern—particularly coliphages, viruses that infect E. coli
bacteria, and obligate anaerobes, thought to have host speci-
ficity and to derive exclusively from recent fecal
contamination—have frequently been identified as potential
alternative indicators expected to better represent risks to hu-
man health [44–46]. 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 [47–51]. 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—
E. coli, enterococci Hazard identification, regulatory
Fecal indicator bacteria—
E. coli, enterococci Hazard identification, regulatory
Fecal indicator viruses Coliphages, Bacteroides bacteriophages Assess risk from enteric viruses Late
Bacteroides HF183, HumM2, PMMoV,
Determine source of contamination Middle
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
ESBL E. coli, MAR E. coli, MRSA Assess environmental antimicrobial
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 antibiotic–resistant E. coli,
MRSA methicillin-resistant Staphylococcus aureus
312 Curr Envir Health Rpt (2020) 7:311–324
sources, notably human noroviruses, drive the global burden
of gastrointestinal illness, including from recreational water
exposures [2•,52–56]. Highly persistent and infectious proto-
zoan parasites are shed at high rates by livestock [2•,57–59],
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 [57,60–64]. 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 . 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
, with recently published MST validation studies conduct-
ed in Australia [67,68], Bangladesh [69,70], Costa Rica ,
India , Japan [73,74], Mozambique , New Zealand
, Nepal [77,78], Singapore , Thailand , 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
[84–89]. Human-associated E. coli markers, long-desired for
their direct correspondence to a common FIB used for regu-
latory purposes, have also been developed [90–92], though
they may lack the analytical sensitivity for effective use in
ambient waters . The identification of new markers is
increasingly supported by advances in sequencing technology
and bioinformatics [84,94–96], and next-generation sequenc-
ing (NGS)–based MST approaches continue to be refined
. Although highly dependent on fecal library composition
(the collection of metagenomic sequences from known fecal
sources that informs source identification algorithms)
[97–101], 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 [105–107]. 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•,71,74,78,108–110]. Nonetheless, Bacteroides
HF183 and its variants have arguably consolidated their role
as the default tool for human source tracking , featuring
consistently high concentrations in sewage globally [83•], fre-
quent detection in surface waters [61,93,110–112], 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
. 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
313Curr Envir Health Rpt (2020) 7:311–324
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 [125–127]. Furthermore, the obli-
gate anaerobes and host-specific viruses proposed as alterna-
tive indicators for MST are often not amenable to laboratory
culture . A range of alternative detection methods contin-
ue to be developed and are the subject of several recent com-
prehensive reviews [97,128–131], 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 .
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 , However, this analysis may
detect residual signals from organisms that are not viable or
infectious at the time of collection . 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-
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 . 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 [144–146]. 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 [61,114,128,150–152].
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 ,
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 [128,154–156]. 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,
157–161]. 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 [160,162–164].
Generalized data reporting guidelines notwithstanding , 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
314 Curr Envir Health Rpt (2020) 7:311–324
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 . 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 . 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 . 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 .
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 [17,27,177–179]. 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,
178,180–182]. Despite less frequent detection of alternative
indicators in recreational waters , 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 .
Upon introduction to the environment, microbial contami-
nants are subject to highly variable dispersal and decay process-
es . 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
. Many studies have since investigated the persistence of
different organisms under various conditions, often using seeded
mesocosms . 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
315Curr Envir Health Rpt (2020) 7:311–324
markers . 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 [184–189]. 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•,179,190–193]. Forensic applications are increasingly pur-
sued to assign responsibility for fecal pollution, largely enabled
by wider adoption of MST approaches and molecular detection
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 . Furthermore, clear
long-term indicator trends do not necessarily represent con-
comitant changes in pathogen hazards .
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 . 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 , 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]. Precipitation—perhaps 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,
195,202–204]; for many applications, providing recreational
guidance on the basis of recent precipitation may well present
the most reliable method for protecting public health .
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
316 Curr Envir Health Rpt (2020) 7:311–324
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
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