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8/20/2019 264 Wingender Flemming Pathogens in Biofilms
Please cite this article in press as: Wingender, J., Flemming, H.-C., Biofilms in drinking water and their role as reservoir for pathogens. Int. J. Hyg.
Environ. Health (2011), doi:10.1016/j.ijheh.2011.05.009
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International Journal of Hygiene and Environmental Health xxx (2011) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Hygiene andEnvironmental Health
journal homepage: www.elsevier .de/ i jheh
Biofilms in drinking water and their role as reservoir for pathogens
Jost Wingender, Hans-Curt Flemming∗
Biofilm Centre, University of Duisburg-Essen,Universitätsstraße 5, D-45141 Essen, Germany
a r t i c l e i n f o
Article history:
Received 15 March 2011
Received in revised form 20 May 2011
Accepted 24 May 2011
Keywords:
Biofilms
Pathogens
Hygienic risk
a b s t r a c t
Most microorganisms on Earth live in various aggregates which are generally termed “biofilms”. They are
ubiquitous and represent the most successful form of life. They are the active agent in biofiltration and
the carriers of the self-cleaning potential in soils, sediments and water. They are also common on surfaces
in technical systems where they sometimes cause biofouling. In recent years it has become evident thatbiofilms in drinking water distribution networks can become transient or long-term habitats for hygieni-
cally relevant microorganisms. Important categories of these organisms include faecal indicator bacteria
Please cite this article in press as: Wingender, J., Flemming, H.-C., Biofilms in drinking water and their role as reservoir for pathogens. Int. J. Hyg.
Environ. Health (2011), doi:10.1016/j.ijheh.2011.05.009
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and,it is the thermophilic organisms which generatesufficient heat
to inactivate pathogens from compost raw materials (Diaz et al.,
2007).
This review, however, focuses on the role of biofilms as a habi-
tat for pathogens and other hygienically relevant microorganisms,
highlighting in particular the role of biofilms in drinking water
systems. For much further detail see Wingender (2011).
Biofilms andhealth risks
On all surfaces in contact with non-sterile water, biofilms
develop (Flemming, 2011). Pathogens, even present below detec-
tion limit in water, can accidentally attach to biofilms which then
can act as their environmental reservoir and represent a poten-
tial source of water contamination. Detachment from biofilms can
occur by continuous erosion, but it has to be taken into account
that erosion does not occur on a constant base. Also, patches of
biofilms can be detached, leading to locally high cell densitiesin the
waterphase(“clouds”).It has to be emphasized that bacterialnum-
bers from the water phase do not indicate the quantity of biofilms
nor their location. If human hosts are susceptible and exposed to
contaminated water, a health risk is present (Fig. 1).
Infection can occur by ingestion of contaminated water, inhala-tion of aerosols containing pathogens or contact of skin, mucous
membranes, eyes and ears (WHO, 2006). Metabolic products such
as hydrogen sulfide and nitrite or endotoxins also belong to the
impact of biofilms to the hygienic quality of water. Its astethetic
quality can be impaired by discoloration, turbidity and malodours.
In some cases, biofilms support the trophic food chain, leading to
occurrence and growth of protozoa and eventually invertebrate
animals.
Particularly critical are the water systems of hospitals andother
health-care facilities, where biofilm-born pathogens can consider-
ably contribute to water-associated nosocomial infections (Exner
et al., 2005). Biofilms can representthe source of pathogens at con-
tinuous exposure of patients, care-givers and all surfaces which
may come into contact with contaminated water (Ortolano et al.,2005).
In fact, about 95% of the bacterial numbers in a drinking water
system are located at the surfaces while only 5% are found in the
water phase anddetected by sampling as commonly used for qual-
ity control (Flemming et al., 2002). The strategy of water suppliers
to limit microbial growth and, thus, biofilm formation is based on
nutrient depletion as a goal of water treatment. This results in
“stable drinking water” which does not show elevated microbial
numbers on the wayto the consumer due to regrowth. Biofilms are
present on all inner surfaces and represent local accumulations of
cells,but they occurusually thin andpatchy. Fig.2 (left)shows typ-
ical biofilms on steel, as visualized by epifluorescence microscopy,
Fig. 2 (right) shows a scanning electron micrograph of a micro-
colony formedon steel, both after exposure for 14 days to domesticdrinking water.
After several weeks to months, a plateau phase of biofilm for-
mation on inert materials employed in drinking water surfaces is
reached, which strongly varies. The total cell numbers range in
the order of 104 to 108 cells/cm2, while the numbers of culturable
heterotrophic plate count (HPC) bacteria in established biofilms
can vary between approximately 101 to 106 colony-forming units
(cfu)/cm2(Wingender and Flemming, 2004; Långmark et al., 2005).
The proportion of culturable bacteria typically represents only a
very small fraction of the total cell numbers and can be several
orders of magnitude lower, usually below 10cfumL −1. In an olig-
otrophic system, their proportion of the total cell number usually
ranges between 0.001 to a few percent of the total cell counts; low
culturability is considered to be characteristic for bacteria in drink-
ing water biofilms (Kalmbach et al., 1997; Martiny et al., 2003;
Wingender and Flemming, 2004). Nutrient availability, hydraulic
conditions, water temperature, the type and concentration of dis-
infectant residues (Norton and LeChevallier, 2000) will influence
biofilm growth. Protozoa have been reported to control drinking
water biofilms by grazing (Pedersen, 1990). The autochthonous
microflora of biofilms in drinking water systems predominantly
consists of environmental microorganisms without any relevance
for human health. These natural populations usually develop and
constitute the biofilms, and are commonly non-pathogenic.
At elevated nutrient levels, stronger biofilm formation is
observed.One source of nutrients canbe the water phase. However,
biodegradable compounds from synthetic polymers, e.g., plasti-
cizers, antioxidants, etc. can also serve as nutrients when such
materials are employed in drinking water systems (Keevil, 2002;
Rogers et al., 1994). A case history may illustrate this (Kilb et al.,
2003): in water samples from drinking water distribution systems,
coliform bacteria (predominantly Citrobacter species) were repeat-
edly detected. Disinfection and flushing of the systems did not
erase the problem. The pattern of the coliform occurrences indi-
cated contamination originating from biofilms. After inspection
of internal surfaces of the systems, no significant biofilm growth
was observed on pipe surfaces, but in a number of cases, visible
biofilms were detected on rubber-coated valves which harboredthe same coliform species as those found in the drinking water
samples. The rubber-coated valves seemed to act as point sources
for the contamination of water. It is usually low molecular weight
additives of the polymers which can be utilized by the microor-
ganisms. Total biofilm cell counts varied from 106 to 108 cells/cm2
with HPC bacteria constituting up to 73% of total cell counts, indi-
Please cite this article in press as: Wingender, J., Flemming, H.-C., Biofilms in drinking water and their role as reservoir for pathogens. Int. J. Hyg.
Environ. Health (2011), doi:10.1016/j.ijheh.2011.05.009
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Fig. 1. Role of biofilms as environmental reservoirs of hygienically relevant microorganisms and as sources of contamination and infectionin drinking water systems (from
Wingender, 2011, with permission).
Opportunistic pathogens are frequently natural aquatic organ-
isms, and thus adapted to oligotrophic environmental conditions
whichare typical of many drinking water systems (Feuerpfeil et al.,
2009). A number of the pathogenic microorganisms have been
recognized as emerging pathogens (Nel and Markotter, 2004; Nel
and Weyer, 2004; Nel et al., 2004), either as newly discoveredpathogens (e.g., Campylobacter spp., H. pylori, Legionella spp., Cryp-
tosporidium spp.) or as new variants of already known species (e.g.,
enterohaemorrhagicE. coli O157:H7) (Szewzyk et al., 2000).
The organisms may attach to surfaces as primary colonizers
and actively establish biofilms alone or in combination with other
microorganisms. However, they also can become integrated in pre-
existing biofilms as secondary colonizers (see Fig. 1).
Heterotrophic bacteria, free-living protozoa and fungi can
multiplyif theyhave adapted to the oligotrophicconditions charac-
teristic of many artificial water systems. Given suitable laboratory
conditions, all relevant water-related pathogenic bacterial species
have actually been shown to be able to adhere to solid surfaces
and/or to form monospecies biofilms, indicating their potential as
biofilm organisms. However, enteric viruses and parasitic protozoa
are obligate parasites and dependent on multiplication in animal
or human hosts. Such organisms can only be expected to attach to
and persist in biofilms without being able to proliferate.
Bacterial pathogens of faecal origin
Important waterborne bacterial pathogens which can infect
the gastrointestinal tract of humans and warm-blooded ani-
mals and are excreted with the faeces into the environment
include Salmonella enterica (e.g., serovar Typhi, Paratyphi and
Typhimurium), Shigella spp., Vibrio cholerae, pathogenic E. coli
variants (e.g., enterotoxigenic E. coli, enterohaemorrhagic E. coli
O157:H7), Yersinia enterocolitica, Campylobacter spp. and Heli-
cobacter pylori. These pathogens have in common that they are
Fig. 2. (Left): Drinking water biofilm on steel surface after 14 days of exposure to drinking water (magnification: 1000×) (from Donlan, 2002, with permission). (Right):
Microcolony on steel after 14 days of exposure to drinking water.
Please cite this article in press as: Wingender, J., Flemming, H.-C., Biofilms in drinking water and their role as reservoir for pathogens. Int. J. Hyg.
Environ. Health (2011), doi:10.1016/j.ijheh.2011.05.009
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Fig. 3. (Left) Scanning electron micrograph of a biofilm grown on synthetic rubber in a drinking water system. (Right) Magnification of left image, note thelarge size of the
bacteria, indicating favourable nutrient conditions in an otherwise oligotrophic drinking water (Kilb et al., 2003, with permission).
transmitted by ingestion of faecally contaminated water and pri-
marily cause gastrointestinal (diarrhoeal) diseases. All of themmay
have the potential to become components of microbial communi-
ties in biofilms (Wingender, 2011).
Faecal index and indicator organisms
Important index/indicator organisms include coliform bacte-ria (total coliforms, E. coli) and faecal streptococci/enterococci
(Payment et al., 2003). According to the WHO (2006), E. coli is the
parameter of choice for monitoring drinking water quality. Col-
iforms other than E. coli may also indicate the presence of faecal
pollution, but they could also originate from a non-faecal source.
However, their presence indicates an undesirable contamination
of water systems due to treatment deficiencies or lack of water
system integrity. Enterococci are used as an additional parame-
ter of faecal pollution. Long-term survival and regrowth of the
index/indicator bacteria in biofilms may contribute to the contam-
ination of water distribution systems by these organisms in the
absence of known contamination events (LeChevallier et al., 1987).
From a public health perspective, this phenomenon is of impor-
tance since contamination of drinking water with coliforms from
biofilms in distribution systems can interfere with their function
to indicate faecal or other undesirable exogenous contaminations
and mask true failures in water treatment and maintenance of the
network. In addition, some index/indicator organisms can also be
relevant as pathogens in water-related diseases.
Environmental biofilmbacteria with pathogenic properties
Quite a few opportunistic bacterial pathogens naturally occur
in aquatic and soil environments and are able to persist and
grow in biofilms of drinking water systems. These bacteria include
Aeromonas spp., some coliforms (Citrobacter spp., Enterobacter
Please cite this article in press as: Wingender, J., Flemming, H.-C., Biofilms in drinking water and their role as reservoir for pathogens. Int. J. Hyg.
Environ. Health (2011), doi:10.1016/j.ijheh.2011.05.009
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rine as opposed to viruses in the water phase (Quignon et al., 1997).
A public health risk is given when virus-containing biofilm patches
are detached and released into the water phase.
Intestinal protozoan parasites and free-living protozoa
Important protozoan parasites which have been involved in
waterborne outbreaks of gastrointestinal disease due to the
contamination of DWDS and swimming pool water systems
include Cryptosporidium spp. (mainly Cryptosporidiumparvum and
Cryptosporidiumhominis)andGiardia lamblia. They areobligate par-
asites, multiply within human or animal hosts and are excreted in
the faecesin a fully infectiveform as oocysts (Cryptosporidiumspp.)
or cysts (G. lamblia). These transmissible stages can remain viable
outside their hosts in aqueous environments for weeks to months
and are highly resistant to chlorine and chloramine (Fraise et al.,
2008). A need for a more thorough understanding of the fate of
oocysts, especially their interaction with biofilms, was mentioned
as essentialfor risk assessment of waterborne disease (Angleset al.,
2007). Biofilms seem to represent a potentially significant, long-
term reservoir of Cryptosporidium oocysts and Giardia cysts that
can be released back into the surrounding water. This explains
the appearance of oocysts in water distribution systems long after
a contamination event. Thus, biofilms were suggested to be thereason for ongoing recoveries of oocysts from a drinking water
distribution system, following a waterborne cryptosporidiosis out-
break in England (Howe et al., 2002).
Free-living protozoa
Some free-living protozoa like Naegleria and Acanthamoeba
species are opportunistic pathogens and have been implicated
in water-related disease. Free-living protozoa are common mem-
bers of biofilm communities in drinking water systems including
sand filters and activated-carbon filters of water treatment plants,
drinking water systems, plumbing systems and cooling towers
(Pedersen, 1990; Hoffmann and Michel, 2003; Thomas et al.,
2008). For example, in different German distribution systems,free-living amoebae, including hygienically relevant thermophilic
Acanthamoeba species, were detected in biofilms recovered from
pipe surfaces at densities between 2 and over 300 amoebae/cm2
(Hoffmann and Michel, 2003).
Fungi
Fungi are supposed to be common constituents of water dis-
tribution systems. Doggett (2000) report densities of filamentous
fungi ranged from 4.0 to 25.2cfucm−2, whereas yeast densities
ranged from 0 to 8.9cfucm−2 .Observations by scanning electron
microscopy further suggested that spores, not hyphae or vegeta-
tive cells, comprised the primary source of viable propagules. Fungi
were isolated from water of municipal water distribution networksand from hospital plumbing systems (Anaissie et al., 2003; Warris
Please cite this article in press as: Wingender, J., Flemming, H.-C., Biofilms in drinking water and their role as reservoir for pathogens. Int. J. Hyg.
Environ. Health (2011), doi:10.1016/j.ijheh.2011.05.009
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Fig. 4. Fate of pathogenic microorganisms and faecal index/indicator organisms after introduction into established biofilms in drinking water systems (modified after Batté
et al., 2003 in Wingender, 2011, with permission).
Thus, VBNC cells represent an infectious potential when present in
biofilms of drinking water systems.
Conclusions
Under epidemiological and ecological aspects, biofilms can be
regarded as temporary or long-term reservoirs and habitats for
pathogens, whose biofilm mode of existence may even represent
part of their natural life cycle (Wingender, 2011). Thus, based on
the knowledge of the biology and ecology of the single pathogen
species and their behaviour in biofilms as summarized in this
review, specific modes of persistence can be attributed to the dif-
ferent typesof pathogens aftertheir attachment to or incorporation
into biofilms of drinking water systems (Fig. 4).
Environmental microorganisms like legionellae, mycobacteria
or P. aeruginosa adapted to oligotrophic aquatic conditions can per-sist over long time periods in biofilms and possibly even multiply
in these environments. A transitory persistence for a few days to a
few weeks seems to be possible for bacteria of faecal origin, while
enteric viruses and the (oo)cysts of parasitic protozoa are largely
eliminated by washout from the biofilms. All these organisms can
persist in biofilms or are released also at a time when they are
not normally circulating in the water phase, so that their detec-
tion can lead to false assumptions as to the origin of the pathogens
and impair risk assessment.
Biofilms and pathogens do interact. One aspect of this is the
observation that the life cycle within the biofilm can lead to a
change in the properties of some bacterial pathogens such as
the increase in biocide resistance or enhanced infectivity of L.
pneumophilaandmycobacteria triggered by their passage in amoe-bae within biofilms. For some pathogens, it has been shown that
biofilms cells are more resistant to antimicrobial agents including
disinfectants used in practice for water treatment. Cells released
from biofilms still retain at least part of the enhanced resis-
tance acquired during their passage in amoebae within biofilms
compared to planktonically grown cells as has been shown for
legionellae and mycobacteria (Steed and Falkinham, 2006). Thus,
survival of cells released from biofilms into the water is enhanced
and adherence to other surface locations downstream in the water
system and initiation of biofilm formation is probable.
The introductionof culture-independent methods for the analy-
sis of water-relatedbacterial pathogens revealed thatin manycases
the organisms in biofilms lose culturability, entering a VBNC state,
and thus, represent only a fraction of those which are detected by
culture-independent methods. The human health significance of
non-culturable pathogens is unclear, but there are indications thatVBNC bacteria such as legionellae are still able to cause infections.
More research is needed to evaluate the pathogenic potential of
thoseVBNC organisms andto define the factors relevant in drinking
water systems which trigger the VBNC state and induce resuscita-
tion to the culturable and infectious state (Wingender, 2011).
It has become clear that the biofilm mode of existence of
pathogens is an important factor that has to be included in risk
assessment applied to water-related pathogens. This knowledge
contributes to a basis for the proper operation and maintenance of
water systems in order to ensure the provisionof microbiologically
safe drinking water and other types of water. Aim is to minimize
the disease burden of the human population potentially emanating
from man-made water systems.
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