8/20/2019 264 Wingender Flemming Pathogens in Biofilms

http://slidepdf.com/reader/full/264-wingender-flemming-pathogens-in-biofilms 1/8

8/20/2019 264 Wingender Flemming Pathogens in Biofilms

http://slidepdf.com/reader/full/264-wingender-flemming-pathogens-in-biofilms 2/8

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

ARTICLE IN PRESSGModel

IJHEH-12471; No.of Pages7

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

(e.g., Escherichia coli), obligate bacterial pathogens of faecal origin (e.g., Campylobacter spp.) opportunistic

bacteria of environmental origin (e.g., Legionella spp.,Pseudomonasaeruginosa), enteric viruses (e.g., aden-

oviruses, rotaviruses, noroviruses) and parasitic protozoa (e.g.,Cryptosporidium parvum). These organisms

can attach to preexisting biofilms, where they become integrated and survive for days to weeks or even

longer, depending on the biology and ecology of the organism and the environmental conditions. There

are indications that at least a part of the biofilm populations of pathogenic bacteria persists in a viable but

non-culturable (VBNC) state and remains unnoticed by the methods appointed to their detection. Thus,

biofilms in drinking water systems can serve as an environmental reservoir for pathogenic microorgan-

isms and represent a potential source of  water contamination, resulting in a potential health risk for

humans if left unnoticed.

© 2011 Elsevier GmbH. All rights reserved.

Biofilms

Thelife of microorganisms in theenvironment is much different

from that in laboratories. In biofilms, the organisms form assem-

blages which are irreversibly associatedwitha surface andenclosed

in a matrix of extracellular polymericsubstances (EPS)of their own

origin which formmatrix (Donlan,2002; Hall-Stoodley et al.,2004).

Biofilms are mostly known on solid surfaces, although they occur

in a vast range of manifestations. All of them share common fea-

turesand take substantial ecological benefits fromthese structures.

Among those is the formation of stable, synergistic microcon-

sortia, the EPS; containing extracellular enzymes which turn the

matrix into an external digestion system; facilitated horizontal

gene transfer and intense intercellular communication. These fea-tures have recently been reviewed (Flemming, 2008; Flemming

and Wingender, 2010). For long time, biofilms were considered

literally as a side issue and they experienced little awareness,

although they were a common sight all the time. Their relevance

for environmental processes as well as in medicine and public

hygiene has gained attention only in the past few decades. Since

then, sophisticated methods have been introduced into biofilm

∗ Corresponding author.

E-mail address: hc.flemming@uni-due.de (H.-C. Flemming).

research such as fluorescence microscopy and confocal laser scan-

ning microscopy (CLSM), micro-electrodes, advanced chemical

analysis, and, most powerful, molecular biology (see Flemming,

2008). All this has allowed investigating biofilm biology in much

greater detail (Stewart and Franklin, 2008) and, thus, taking views

of the life of microorganisms in the real world. From a point of 

view of life science, the most exciting aspect is that microorgan-

isms today cannot be simply viewed as independent individuals,

competing as much as they can, but as complex communities

with division of labour, intense communication and many aspects

of multicellular life (Keller and Surette, 2006) – without being

a multicellular organism. This is certainly a new understanding

of microbiology with big consequences. In medicine, it is impor-

tant for the understanding of implant-related infections which aremainly caused by biofilms (Costerton et al., 1987; Hall-Stoodley

et al., 2004; Shirtliff and Leid, 2009) or chronic wounds (Bjarnsholt

et al., 2010), as well forhygieneand forthe understanding of micro-

bial problems in technical processes (Flemming, 2011).

It should not be overlooked that biofilms have very benefi-

cial aspects. They are the carriers of the self-cleaning potential of 

soil, sediment and water by mineralizing organic matter. They are

employed for biological purification of drinking water in biofilters

(e.g., Gimbel et al., 2006), of biological waste water treatment (e.g.,

Wuertzet al., 2003) and they arethe drivers of biological waste dis-

posal (e.g., Evans, 2005). They perform the composting processes

1438-4639/$ – seefrontmatter © 2011 Elsevier GmbH. All rights reserved.

doi:10.1016/j.ijheh.2011.05.009

8/20/2019 264 Wingender Flemming Pathogens in Biofilms

http://slidepdf.com/reader/full/264-wingender-flemming-pathogens-in-biofilms 3/8

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

ARTICLE IN PRESSGModel

IJHEH-12471; No.of Pages7

2   J. Wingender, H.-C. Flemming / International Journal of Hygiene andEnvironmental Health xxx (2011) xxx–xxx

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-

cating favourablegrowthconditions. Scanningelectron microscopy

(Fig. 3, left) reveals massive biofilm formation with large cells

(Fig. 3, right), indicating good nutrient conditions in an otherwise

nutrient-poor drinking water. The problem could only be solved

by exchange of the coated valves by material which did not sup-

port microbial growth. This example demonstrates the pivotal role

of materials. In German public drinking water systems, only such

materials are permitted which do not support microbial growth.This is certified by a standardized procedure (Anonymous, 2007) in

which the materials have to pass the test.

Hygienically relevantmicroorganisms in drinkingwater

systems

Two categories of hygienically relevant microorganisms can be

distinguished:

(i) Microorganisms with pathogenic properties which have been

shown to be associated with water-related illness and out-

breaks, and

(ii) Bacteria whichare primarilyused as indexand indicatororgan-

isms in water analysis, indicating the presence of pathogenic

organisms of faecal origin (index organisms) or indicating the

effectiveness of water treatment processes as well as integrity

of water distribution systems (indicator organisms) (WHO,

2006).

Obligate water-related pathogens, i.e., those which cause dis-

ease in humans independent of their health status are usually

faecally derived. Others are opportunistic pathogens which cause

disease in sensitive human subgroups such as the elderly, children,

immunocompromised individuals, patients with preexisting dis-

ease or other predisposing conditions which facilitate infection by

these organisms.

8/20/2019 264 Wingender Flemming Pathogens in Biofilms

http://slidepdf.com/reader/full/264-wingender-flemming-pathogens-in-biofilms 4/8

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

ARTICLE IN PRESSGModel

IJHEH-12471; No.of Pages7

 J. Wingender, H.-C. Flemming / International Journal of Hygiene and Environmental Health xxx (2011) xxx–xxx 3

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.

8/20/2019 264 Wingender Flemming Pathogens in Biofilms

http://slidepdf.com/reader/full/264-wingender-flemming-pathogens-in-biofilms 5/8

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

ARTICLE IN PRESSGModel

IJHEH-12471; No.of Pages7

4   J. Wingender, H.-C. Flemming / International Journal of Hygiene andEnvironmental Health xxx (2011) xxx–xxx

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 

spp., Klebsiella pneumoniae), Legionella spp., Mycobacterium spp.

and Pseudomonas aeruginosa. The infective doses of clinically rel-

evant strains of these organisms are relatively high (106–108)

for healthy individuals and are mostly harmless for them (Rusin

et al., 1997). However, their infectious doses are lower and espe-

cially critical for the increasing proportion of sensitive human

populations. This includes infants, the very elderly, hospitalized

individuals, immunocompromised persons and those with other

underlying diseases and under medical treatment. Depending on

the organism, the route of transmission leading to a water-related

disease is ingestion, inhalation of aerosols or exposure to skin (e.g.,

through wounds), ears and eyes. Currently, Legionella pneumophila

and some other Legionella species, Pseudomonas aeruginosa and

non-tuberculous mycobacteria are regarded as the most relevant

opportunistic bacterial pathogens linked to water-related diseases.

Enteric viruses

Enteric viruses involved in water-related diseases cause acute

gastrointestinal illness (e.g., noroviruses, rotaviruses) and can also

affect other organs like the liver (hepatitis A and E viruses) or the

central nervous system (poliovirus). These viruses are excreted

in the faeces of infected humans and are transmitted predomi-

nantly by ingestion. In contrast to bacterial pathogens, relativelylittle information exists on the occurrence and survival of enteric

viruses in biofilms of water distribution systems. However, there

are some indications from field studies and laboratory experiments

that pathogenic viruses may become incorporated into drinking

water biofilms, persist there and canbe released again to represent

a risk of infection (for review, see Skraber et al., 2005). Recently,

enteroviruses and noroviruses were found by RT-PCR to be present

in wastewater biofilms which had grown for one month to more

than two years on polyethylene carriers in a moving-bed biofilm

reactor of a wastewater treatment plant (Skraber et al., 2009). The

viruses could be detected in the biofilms also at a time when their

concentrations were below the detection limit in wastewater, sug-

gesting the ability of these viruses to persist in the biofilms. Viruses

in biofilms seem to be protected against disinfectants such as chlo-

8/20/2019 264 Wingender Flemming Pathogens in Biofilms

http://slidepdf.com/reader/full/264-wingender-flemming-pathogens-in-biofilms 6/8

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

ARTICLE IN PRESSGModel

IJHEH-12471; No.of Pages7

 J. Wingender, H.-C. Flemming / International Journal of Hygiene and Environmental Health xxx (2011) xxx–xxx 5

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

etal.,2003;Hageskal etal., 2006). Evenfilamentous fungi havebeen

reported as biofilm formers (Harding et al., 2009). The observations

suggest that biofilms of drinking water distribution networks and

hospital plumbing systems can occasionally be a reservoir of fungi

with pathogenic properties.

 Algae

Algae belong to the most abundant biofilm forming organisms

on Earth (Cooksey and Wigglesworth-Cooksey, 2000). In surface

waters, algae occurringboth in planktonic formand as biofilms may

contain species which form toxins such as microcystin (Leflaive

and Ten-Hage, 2007) and represent a serious threat to human

health. Recently, a highly sensitive amperometric immunosensor

for microcystin detection in algae and their biofilms has been

reported (Campas and Marty, 2007). In drinking water distribution

systems and installations, however, algae do not occur due to lack

of light.

 The problem of detection

Traditionally, pathogenic bacteria in water are detected and

quantified by cultural methods. However, they may make a transi-

tion into a viable but non-culturable (VBNC) state. Bacteria in the

VBNC state do not grow on conventional microbiological media

on which they would normally develop into colonies, but are still

alive and are characterized by low levels of metabolic activity

(Oliver, 2010). The conversion to the VBNC state is supposed to

be a response to adverse environmental conditions such as lack of 

nutrients, unfavourable water temperature, the presence of disin-

fectants or toxic metal ions such as copper (Dwidjosiswojo et al.,

in this volume). VBNC bacteria can become culturable again upon

resuscitation under favourable conditions. Oliver (2010) provided

a list of pathogens known to enter the VBNC state, in which all

relevant water-associated bacterial pathogens are included, e.g.,

Pseudomonas aeruginosa, Legionella pneumophila, Salmonella typhi,orVibrio vulnificus. Investigationsof the VBNC state areusually per-

formed with planktonic cells, so it is largely unknown. However, it

can be hypothesized that the VBNC state can also be induced by

biofilm environments.

To circumvent the shortcomings of non-culturability of biofilm

organisms, culture-independent methods are increasingly used in

order to characterize the composition and diversity of microbial

biofilm communities,and to identifypathogensin biofilms of drink-

ing water systems. Of relevance are:

(i) Immunological (antibody-based) techniques (Hausner et al.,

2000).

(ii) Nucleic acid-based methods,which include fluorescencein situ

hybridization (FISH) or peptide nucleic acid FISH (PNA-FISH)

with ribosomal RNA as a target for group- or species-specific

fluorescent olignucleotide probes (Malic et al., 2009).

(iii) Polymerase chain reaction (PCR) targeted at specific DNA

sequences, alone or in combination with denaturating gradi-

ent gel electrophoresis, cloning and sequencing of 16S rRNA

genes (Schwartz et al., 2003).

(iv) Enzymatic activity, e.g., esterases by cleaving of fluorescein

diacetate (Battin, 1997) or redox activity as visualized by CTC

(Schaule et al., 1993).

Some hygienically relevant bacterialspecies have beendetected

in real or experimental drinking water biofilms, using culture-

independent methods, alone or in combination with conventionalculture methods. An example is the induction of the VBNC state

in Pseudomonas aeruginosa and Legionella pneumophila by low

copper concentrations (Dwidjosiswojo et al., in this volume).

Significantly higher cell numbers were frequently detected by

culture-independent methods compared to colony counts deter-

mined on culture media, indicating that bacterial pathogens are

present andmay persist in a VBNC state in drinking water biofilms.

However, this biofilm-associated non-culturable state and the

potential of resuscitation of these bacteria has not yet been char-

acterized in detail. From a health perspective, the relevance of 

pathogens in the VBNC state may be underestimated, since they

can regain their virulence and are able to initiate infection when

they revert to the culturable state under favourable environmen-

tal conditions (Oliver, 2010; Dwidjosiswojo et al., in this volume).

8/20/2019 264 Wingender Flemming Pathogens in Biofilms

http://slidepdf.com/reader/full/264-wingender-flemming-pathogens-in-biofilms 7/8

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

ARTICLE IN PRESSGModel

IJHEH-12471; No.of Pages7

6   J. Wingender, H.-C. Flemming / International Journal of Hygiene andEnvironmental Health xxx (2011) xxx–xxx

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.

References

Anaissie, E.J., Stratton, S.L., Dignani, M.C., Lee, C., Summerbell, R.C., Rex, J.H., Mon-son, T.P., Walsh, T.J., 2003. Pathogenic molds (including  Aspergillus species) inhospital water distribution systems: a 3-year prospective study and clinicalimplicationsfor patientswith hematologic malignancies.Blood101, 2542–2546.

Anonymous, 2007. Technische Regel W 270. Vermehrung von Mikroorganismenauf Werkstoffen für den Trinkwasserbereich – Prüfung und Bewertung.DVGW,Bonn.

Angles, M.L.,Chandy,J.P., Cox,P.T., Fisher, I.H., Warnecke,M.R., 2007.Implications of biofilm-associated waterborne Cryptosporidium oocysts for thewater industry.Trends Parasitol. 23, 352–356.

Battin, T., 1997. Assessment of fluoresceindiacetatehydrolysis as a measure of totalesterase activity in natural stream sediment biofilms. Sci. Total Environ. 189,51–60.

Bjarnsholt, T., Moser, C., Jensen, P.R., Høiby, N. (Eds.), 2010. Biofilm Infections.Springer International, Heidelberg, New York.

Campas, M., Marty, J.-L., 2007. Highly sensitive amperometric immunosensors formicrocystin detection in algae. Biosens. Bioelectron. 22, 1034–1040.

Cooksey, K.E., Wigglesworth-Cooksey, B., 2000. Diatoms in biofilms. In: Bitton, G.(Ed.), Environmental Microbiology. , pp. 1051–1063.

Costerton, J.W., Cheng, K.-J., Geesey, G.G., Ladd, T.I., Nickel, J.C., Dasgupta, M., Mar-rie, T.J., 1987. Bacterial biofilms in nature and disease. Ann. Rev. Microbiol. 41,435–464.

Diaz,L.F., de Bertholdi, M., Bidlingmaier, W. (Eds.), 2007.CompostScience and Tech-nology (waste management). Oxford, Butterworth Heinemann.

Doggett, M.S., 2000. Characterization of fungal biofilms within a municipal waterdistribution system. Appl. Environ. Microbiol. 66, 1249–1251.

Donlan, R.M., 2002. Biofilms: microbial life on surfaces.Emerg. Inf. Dis. 8, 881–890.Dwidjosiswojo, Z., Richards, J., Moritz, M.M., Dopp, E., Flemming, H.-C., Wingen-

der, J. Influence of copper ions on the viability and cytotoxicity of Pseudomonasaeruginosa under conditions relevant to drinking water. Int. J. Hyg. Environ.Health, in this volume.

Evans, G. (Ed.), 2005. Biowaste and Biological Waste Treatment. Earthscan, London.

8/20/2019 264 Wingender Flemming Pathogens in Biofilms

http://slidepdf.com/reader/full/264-wingender-flemming-pathogens-in-biofilms 8/8