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International Journal of Hygiene and Environmental Health 214 (2011) 417– 423

Contents lists available at ScienceDirect

International Journal of Hygiene andEnvironmental Health

j o ur nal homepage: www.elsev ier .de / i jheh

iofilms in drinking water and their role as reservoir for pathogens

ost Wingender, Hans-Curt Flemming ∗

iofilm Centre, University of Duisburg-Essen, Universitätsstraße 5, D-45141 Essen, Germany

r t i c l e i n f o

rticle history:eceived 15 March 2011eceived in revised form 20 May 2011ccepted 24 May 2011

eywords:iofilmsathogensygienic risk

a b s t r a c t

Most microorganisms on Earth live in various aggregates which are generally termed “biofilms”. They areubiquitous and represent the most successful form of life. They are the active agent in biofiltration andthe carriers of the self-cleaning potential in soils, sediments and water. They are also common on surfacesin 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.) opportunisticbacteria of environmental origin (e.g., Legionella spp., Pseudomonas aeruginosa), enteric viruses (e.g., aden-oviruses, rotaviruses, noroviruses) and parasitic protozoa (e.g., Cryptosporidium parvum). These organismscan 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. Thereare indications that at least a part of the biofilm populations of pathogenic bacteria persists in a viable butnon-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 forhumans if left unnoticed.

iofilms

The life of microorganisms in the environment is much differentrom that in laboratories. In biofilms, the organisms form assem-lages which are irreversibly associated with a surface and enclosed

n a matrix of extracellular polymeric substances (EPS) of their ownrigin which form matrix (Donlan, 2002; Hall-Stoodley et al., 2004).iofilms are mostly known on solid surfaces, although they occur

n a vast range of manifestations. All of them share common fea-ures and take substantial ecological benefits from these structures.mong those is the formation of stable, synergistic microcon-ortia, the EPS; containing extracellular enzymes which turn theatrix into an external digestion system; facilitated horizontal

ene transfer and intense intercellular communication. These fea-ures have recently been reviewed (Flemming, 2008; Flemmingnd Wingender, 2010). For long time, biofilms were considerediterally as a side issue and they experienced little awareness,lthough they were a common sight all the time. Their relevance

or environmental processes as well as in medicine and publicygiene has gained attention only in the past few decades. Sincehen, sophisticated methods have been introduced into biofilm

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

438-4639/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.ijheh.2011.05.009

© 2011 Elsevier GmbH. All rights reserved.

research such as fluorescence microscopy and confocal laser scan-ning microscopy (CLSM), micro-electrodes, advanced chemicalanalysis, and, most powerful, molecular biology (see Flemming,2008). All this has allowed investigating biofilm biology in muchgreater detail (Stewart and Franklin, 2008) and, thus, taking viewsof the life of microorganisms in the real world. From a point ofview 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 communitieswith division of labour, intense communication and many aspectsof multicellular life (Keller and Surette, 2006) – without beinga multicellular organism. This is certainly a new understandingof 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-Stoodleyet al., 2004; Shirtliff and Leid, 2009) or chronic wounds (Bjarnsholtet al., 2010), as well for hygiene and for the 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 ofsoil, 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.,Wuertz et al., 2003) and they are the drivers of biological waste dis-posal (e.g., Evans, 2005). They perform the composting processes

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nd, it is the thermophilic organisms which generate sufficient heato inactivate pathogens from compost raw materials (Diaz et al.,007).

This review, however, focuses on the role of biofilms as a habi-at for pathogens and other hygienically relevant microorganisms,ighlighting in particular the role of biofilms in drinking waterystems. For much further detail see Wingender (2011).

iofilms and health risks

On all surfaces in contact with non-sterile water, biofilmsevelop (Flemming, 2011). Pathogens, even present below detec-ion limit in water, can accidentally attach to biofilms which thenan act as their environmental reservoir and represent a poten-ial source of water contamination. Detachment from biofilms canccur by continuous erosion, but it has to be taken into accounthat erosion does not occur on a constant base. Also, patches ofiofilms can be detached, leading to locally high cell densities in theater phase (“clouds”). It has to be emphasized that bacterial num-

ers from the water phase do not indicate the quantity of biofilmsor their location. If human hosts are susceptible and exposed toontaminated water, a health risk is present (Fig. 1).

Infection can occur by ingestion of contaminated water, inhala-ion of aerosols containing pathogens or contact of skin, mucous

embranes, eyes and ears (WHO, 2006). Metabolic products suchs hydrogen sulfide and nitrite or endotoxins also belong to thempact of biofilms to the hygienic quality of water. Its astetheticuality can be impaired by discoloration, turbidity and malodours.

n some cases, biofilms support the trophic food chain, leading toccurrence and growth of protozoa and eventually invertebratenimals.

Particularly critical are the water systems of hospitals and otherealth-care facilities, where biofilm-born pathogens can consider-bly contribute to water-associated nosocomial infections (Exnert al., 2005). Biofilms can represent the source of pathogens at con-inuous exposure of patients, care-givers and all surfaces which

ay come into contact with contaminated water (Ortolano et al.,005).

In fact, about 95% of the bacterial numbers in a drinking waterystem are located at the surfaces while only 5% are found in theater phase and detected by sampling as commonly used for qual-

ty control (Flemming et al., 2002). The strategy of water supplierso limit microbial growth and, thus, biofilm formation is based onutrient depletion as a goal of water treatment. This results instable drinking water” which does not show elevated microbialumbers on the way to the consumer due to regrowth. Biofilms areresent on all inner surfaces and represent local accumulations ofells, but they occur usually thin and patchy. Fig. 2 (left) shows typ-cal biofilms on steel, as visualized by epifluorescence microscopy,ig. 2 (right) shows a scanning electron micrograph of a micro-olony formed on steel, both after exposure for 14 days to domesticrinking water.

After several weeks to months, a plateau phase of biofilm for-ation on inert materials employed in drinking water surfaces is

eached, which strongly varies. The total cell numbers range inhe order of 104 to 108 cells/cm2, while the numbers of culturableeterotrophic plate count (HPC) bacteria in established biofilmsan vary between approximately 101 to 106 colony-forming unitscfu)/cm2(Wingender and Flemming, 2004; Långmark et al., 2005).he proportion of culturable bacteria typically represents only aery small fraction of the total cell numbers and can be several

rders of magnitude lower, usually below 10 cfu mL−1. In an olig-trophic system, their proportion of the total cell number usuallyanges between 0.001 to a few percent of the total cell counts; lowulturability is considered to be characteristic for bacteria in drink-

iene and Environmental Health 214 (2011) 417– 423

ing water biofilms (Kalmbach et al., 1997; Martiny et al., 2003;Wingender and Flemming, 2004). Nutrient availability, hydraulicconditions, water temperature, the type and concentration of dis-infectant residues (Norton and LeChevallier, 2000) will influencebiofilm growth. Protozoa have been reported to control drinkingwater biofilms by grazing (Pedersen, 1990). The autochthonousmicroflora of biofilms in drinking water systems predominantlyconsists of environmental microorganisms without any relevancefor human health. These natural populations usually develop andconstitute the biofilms, and are commonly non-pathogenic.

At elevated nutrient levels, stronger biofilm formation isobserved. One source of nutrients can be the water phase. However,biodegradable compounds from synthetic polymers, e.g., plasti-cizers, antioxidants, etc. can also serve as nutrients when suchmaterials 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 noterase the problem. The pattern of the coliform occurrences indi-cated contamination originating from biofilms. After inspectionof internal surfaces of the systems, no significant biofilm growthwas observed on pipe surfaces, but in a number of cases, visiblebiofilms were detected on rubber-coated valves which harboredthe same coliform species as those found in the drinking watersamples. The rubber-coated valves seemed to act as point sourcesfor the contamination of water. It is usually low molecular weightadditives 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 favourable growth conditions. Scanning electron microscopy(Fig. 3, left) reveals massive biofilm formation with large cells(Fig. 3, right), indicating good nutrient conditions in an otherwisenutrient-poor drinking water. The problem could only be solvedby exchange of the coated valves by material which did not sup-port microbial growth. This example demonstrates the pivotal roleof materials. In German public drinking water systems, only suchmaterials are permitted which do not support microbial growth.This is certified by a standardized procedure (Anonymous, 2007) inwhich the materials have to pass the test.

Hygienically relevant microorganisms in drinking watersystems

Two categories of hygienically relevant microorganisms can bedistinguished:

(i) Microorganisms with pathogenic properties which have beenshown to be associated with water-related illness and out-breaks, and

(ii) Bacteria which are primarily used as index and indicator organ-isms in water analysis, indicating the presence of pathogenicorganisms of faecal origin (index organisms) or indicating theeffectiveness of water treatment processes as well as integrityof 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 usuallyfaecally 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 bythese organisms.

J. Wingender, H.-C. Flemming / International Journal of Hygiene and Environmental Health 214 (2011) 417– 423 419

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Opportunistic pathogens are frequently natural aquatic organ-sms, and thus adapted to oligotrophic environmental conditions

hich are typical of many drinking water systems (Feuerpfeil et al.,009). A number of the pathogenic microorganisms have beenecognized as emerging pathogens (Nel and Markotter, 2004; Nelnd Weyer, 2004; Nel et al., 2004), either as newly discoveredathogens (e.g., Campylobacter spp., H. pylori, Legionella spp., Cryp-osporidium spp.) or as new variants of already known species (e.g.,nterohaemorrhagic E. coli O157:H7) (Szewzyk et al., 2000).

The organisms may attach to surfaces as primary colonizersnd actively establish biofilms alone or in combination with othericroorganisms. However, they also can become integrated in pre-

xisting biofilms as secondary colonizers (see Fig. 1).

Heterotrophic bacteria, free-living protozoa and fungi can

ultiply if they have adapted to the oligotrophic conditions charac-eristic of many artificial water systems. Given suitable laboratoryonditions, all relevant water-related pathogenic bacterial species

ig. 2. (Left): Drinking water biofilm on steel surface after 14 days of exposure to drinkicrocolony on steel after 14 days of exposure to drinking water.

ms and as sources of contamination and infection in drinking water systems (from

have actually been shown to be able to adhere to solid surfacesand/or to form monospecies biofilms, indicating their potential asbiofilm organisms. However, enteric viruses and parasitic protozoaare obligate parasites and dependent on multiplication in animalor human hosts. Such organisms can only be expected to attach toand persist in biofilms without being able to proliferate.

Bacterial pathogens of faecal origin

Important waterborne bacterial pathogens which can infectthe gastrointestinal tract of humans and warm-blooded ani-mals and are excreted with the faeces into the environmentinclude Salmonella enterica (e.g., serovar Typhi, Paratyphi and

Typhimurium), Shigella spp., Vibrio cholerae, pathogenic E. colivariants (e.g., enterotoxigenic E. coli, enterohaemorrhagic E. coliO157:H7), Yersinia enterocolitica, Campylobacter spp. and Heli-cobacter pylori. These pathogens have in common that they are

ing water (magnification: 1000×) (from Donlan, 2002, with permission). (Right):

420 J. Wingender, H.-C. Flemming / International Journal of Hygiene and Environmental Health 214 (2011) 417– 423

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ig. 3. (Left) Scanning electron micrograph of a biofilm grown on synthetic rubberacteria, indicating favourable nutrient conditions in an otherwise oligotrophic dri

ransmitted by ingestion of faecally contaminated water and pri-arily cause gastrointestinal (diarrhoeal) diseases. All of them may

ave the potential to become components of microbial communi-ies in biofilms (Wingender, 2011).

aecal index and indicator organisms

Important index/indicator organisms include coliform bacte-ia (total coliforms, E. coli) and faecal streptococci/enterococciPayment et al., 2003). According to the WHO (2006), E. coli is thearameter of choice for monitoring drinking water quality. Col-

forms other than E. coli may also indicate the presence of faecalollution, but they could also originate from a non-faecal source.owever, their presence indicates an undesirable contaminationf water systems due to treatment deficiencies or lack of waterystem integrity. Enterococci are used as an additional parame-er of faecal pollution. Long-term survival and regrowth of thendex/indicator bacteria in biofilms may contribute to the contam-nation of water distribution systems by these organisms in thebsence of known contamination events (LeChevallier et al., 1987).rom a public health perspective, this phenomenon is of impor-ance since contamination of drinking water with coliforms fromiofilms in distribution systems can interfere with their functiono indicate faecal or other undesirable exogenous contaminationsnd mask true failures in water treatment and maintenance of theetwork. In addition, some index/indicator organisms can also beelevant as pathogens in water-related diseases.

nvironmental biofilm bacteria with pathogenic properties

Quite a few opportunistic bacterial pathogens naturally occurn aquatic and soil environments and are able to persist and

row in biofilms of drinking water systems. These bacteria includeeromonas spp., some coliforms (Citrobacter spp., Enterobacterpp., Klebsiella pneumoniae), Legionella spp., Mycobacterium spp.nd Pseudomonas aeruginosa. The infective doses of clinically rel-

rinking water system. (Right) Magnification of left image, note the large size of thewater (Kilb et al., 2003, with permission).

evant strains of these organisms are relatively high (106–108)for healthy individuals and are mostly harmless for them (Rusinet al., 1997). However, their infectious doses are lower and espe-cially critical for the increasing proportion of sensitive humanpopulations. This includes infants, the very elderly, hospitalizedindividuals, immunocompromised persons and those with otherunderlying diseases and under medical treatment. Depending onthe organism, the route of transmission leading to a water-relateddisease is ingestion, inhalation of aerosols or exposure to skin (e.g.,through wounds), ears and eyes. Currently, Legionella pneumophilaand some other Legionella species, Pseudomonas aeruginosa andnon-tuberculous mycobacteria are regarded as the most relevantopportunistic bacterial pathogens linked to water-related diseases.

Enteric viruses

Enteric viruses involved in water-related diseases cause acutegastrointestinal illness (e.g., noroviruses, rotaviruses) and can alsoaffect other organs like the liver (hepatitis A and E viruses) or thecentral nervous system (poliovirus). These viruses are excretedin 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 entericviruses in biofilms of water distribution systems. However, thereare some indications from field studies and laboratory experimentsthat pathogenic viruses may become incorporated into drinkingwater biofilms, persist there and can be released again to representa risk of infection (for review, see Skraber et al., 2005). Recently,enteroviruses and noroviruses were found by RT-PCR to be presentin wastewater biofilms which had grown for one month to morethan two years on polyethylene carriers in a moving-bed biofilmreactor of a wastewater treatment plant (Skraber et al., 2009). The

viruses could be detected in the biofilms also at a time when theirconcentrations were below the detection limit in wastewater, sug-gesting the ability of these viruses to persist in the biofilms. Virusesin biofilms seem to be protected against disinfectants such as chlo-

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ine as opposed to viruses in the water phase (Quignon et al., 1997). public health risk is given when virus-containing biofilm patchesre detached and released into the water phase.

ntestinal protozoan parasites and free-living protozoa

Important protozoan parasites which have been involved inaterborne outbreaks of gastrointestinal disease due to the

ontamination of DWDS and swimming pool water systemsnclude Cryptosporidium spp. (mainly Cryptosporidium parvum andryptosporidium hominis) and Giardia lamblia. They are obligate par-sites, multiply within human or animal hosts and are excreted inhe faeces in a fully infective form as oocysts (Cryptosporidium spp.)r cysts (G. lamblia). These transmissible stages can remain viableutside their hosts in aqueous environments for weeks to monthsnd are highly resistant to chlorine and chloramine (Fraise et al.,008). A need for a more thorough understanding of the fate ofocysts, especially their interaction with biofilms, was mentioneds essential for risk assessment of waterborne disease (Angles et al.,007). Biofilms seem to represent a potentially significant, long-erm reservoir of Cryptosporidium oocysts and Giardia cysts thatan be released back into the surrounding water. This explainshe appearance of oocysts in water distribution systems long after

contamination event. Thus, biofilms were suggested to be theeason for ongoing recoveries of oocysts from a drinking wateristribution system, following a waterborne cryptosporidiosis out-reak in England (Howe et al., 2002).

ree-living protozoa

Some free-living protozoa like Naegleria and Acanthamoebapecies are opportunistic pathogens and have been implicatedn water-related disease. Free-living protozoa are common mem-ers of biofilm communities in drinking water systems includingand filters and activated-carbon filters of water treatment plants,rinking water systems, plumbing systems and cooling towersPedersen, 1990; Hoffmann and Michel, 2003; Thomas et al.,008). For example, in different German distribution systems,ree-living amoebae, including hygienically relevant thermophiliccanthamoeba species, were detected in biofilms recovered fromipe surfaces at densities between 2 and over 300 amoebae/cm2

Hoffmann and Michel, 2003).

ungi

Fungi are supposed to be common constituents of water dis-ribution systems. Doggett (2000) report densities of filamentousungi ranged from 4.0 to 25.2 cfu cm−2, whereas yeast densitiesanged from 0 to 8.9 cfu cm−2 .Observations by scanning electronicroscopy further suggested that spores, not hyphae or vegeta-

ive cells, comprised the primary source of viable propagules. Fungiere isolated from water of municipal water distribution networks

nd from hospital plumbing systems (Anaissie et al., 2003; Warrist al., 2003; Hageskal et al., 2006). Even filamentous fungi have beeneported as biofilm formers (Harding et al., 2009). The observationsuggest that biofilms of drinking water distribution networks andospital plumbing systems can occasionally be a reservoir of fungiith pathogenic properties.

lgae

Algae belong to the most abundant biofilm forming organisms

n Earth (Cooksey and Wigglesworth-Cooksey, 2000). In surfaceaters, algae occurring both in planktonic form and as biofilms may

ontain species which form toxins such as microcystin (Leflaivend Ten-Hage, 2007) and represent a serious threat to human

iene and Environmental Health 214 (2011) 417– 423 421

health. Recently, a highly sensitive amperometric immunosensorfor microcystin detection in algae and their biofilms has beenreported (Campas and Marty, 2007). In drinking water distributionsystems and installations, however, algae do not occur due to lackof light.

The problem of detection

Traditionally, pathogenic bacteria in water are detected andquantified by cultural methods. However, they may make a transi-tion into a viable but non-culturable (VBNC) state. Bacteria in theVBNC state do not grow on conventional microbiological mediaon which they would normally develop into colonies, but are stillalive and are characterized by low levels of metabolic activity(Oliver, 2010). The conversion to the VBNC state is supposed tobe a response to adverse environmental conditions such as lack ofnutrients, 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 uponresuscitation under favourable conditions. Oliver (2010) provideda list of pathogens known to enter the VBNC state, in which allrelevant water-associated bacterial pathogens are included, e.g.,Pseudomonas aeruginosa, Legionella pneumophila, Salmonella typhi,or Vibrio vulnificus. Investigations of the VBNC state are usually per-formed with planktonic cells, so it is largely unknown. However, itcan be hypothesized that the VBNC state can also be induced bybiofilm environments.

To circumvent the shortcomings of non-culturability of biofilmorganisms, culture-independent methods are increasingly used inorder to characterize the composition and diversity of microbialbiofilm communities, and to identify pathogens in 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 fluorescence in situhybridization (FISH) or peptide nucleic acid FISH (PNA-FISH)with ribosomal RNA as a target for group- or species-specificfluorescent olignucleotide probes (Malic et al., 2009).

iii) Polymerase chain reaction (PCR) targeted at specific DNAsequences, alone or in combination with denaturating gradi-ent gel electrophoresis, cloning and sequencing of 16S rRNAgenes (Schwartz et al., 2003).

(iv) Enzymatic activity, e.g., esterases by cleaving of fluoresceindiacetate (Battin, 1997) or redox activity as visualized by CTC(Schaule et al., 1993).

Some hygienically relevant bacterial species have been detectedin 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 statein Pseudomonas aeruginosa and Legionella pneumophila by lowcopper concentrations (Dwidjosiswojo et al., in this volume).Significantly higher cell numbers were frequently detected byculture-independent methods compared to colony counts deter-mined on culture media, indicating that bacterial pathogens arepresent and may persist in a VBNC state in drinking water biofilms.However, this biofilm-associated non-culturable state and thepotential 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 theycan regain their virulence and are able to initiate infection whenthey revert to the culturable state under favourable environmen-tal conditions (Oliver, 2010; Dwidjosiswojo et al., in this volume).

422 J. Wingender, H.-C. Flemming / International Journal of Hygiene and Environmental Health 214 (2011) 417– 423

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hus, VBNC cells represent an infectious potential when present iniofilms of drinking water systems.

onclusions

Under epidemiological and ecological aspects, biofilms can beegarded as temporary or long-term reservoirs and habitats forathogens, whose biofilm mode of existence may even representart of their natural life cycle (Wingender, 2011). Thus, based onhe knowledge of the biology and ecology of the single pathogenpecies and their behaviour in biofilms as summarized in thiseview, specific modes of persistence can be attributed to the dif-erent types of pathogens after their attachment to or incorporationnto biofilms of drinking water systems (Fig. 4).

Environmental microorganisms like legionellae, mycobacteriar P. aeruginosa adapted to oligotrophic aquatic conditions can per-ist over long time periods in biofilms and possibly even multiplyn these environments. A transitory persistence for a few days to aew weeks seems to be possible for bacteria of faecal origin, whilenteric viruses and the (oo)cysts of parasitic protozoa are largelyliminated by washout from the biofilms. All these organisms canersist in biofilms or are released also at a time when they areot normally circulating in the water phase, so that their detec-ion can lead to false assumptions as to the origin of the pathogensnd impair risk assessment.

Biofilms and pathogens do interact. One aspect of this is thebservation that the life cycle within the biofilm can lead to ahange in the properties of some bacterial pathogens such ashe increase in biocide resistance or enhanced infectivity of L.neumophila and mycobacteria triggered by their passage in amoe-ae within biofilms. For some pathogens, it has been shown thatiofilms cells are more resistant to antimicrobial agents includingisinfectants used in practice for water treatment. Cells releasedrom biofilms still retain at least part of the enhanced resis-ance acquired during their passage in amoebae within biofilmsompared to planktonically grown cells as has been shown foregionellae and mycobacteria (Steed and Falkinham, 2006). Thus,urvival of cells released from biofilms into the water is enhancednd adherence to other surface locations downstream in the waterystem and initiation of biofilm formation is probable.

The introduction of culture-independent methods for the analy-is of water-related bacterial pathogens revealed that in many caseshe organisms in biofilms lose culturability, entering a VBNC state,nd thus, represent only a fraction of those which are detected by

oduction into established biofilms in drinking water systems (modified after Batté

culture-independent methods. The human health significance ofnon-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 ofthose VBNC organisms and to define the factors relevant in drinkingwater 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 ofpathogens is an important factor that has to be included in riskassessment applied to water-related pathogens. This knowledgecontributes to a basis for the proper operation and maintenance ofwater systems in order to ensure the provision of microbiologicallysafe drinking water and other types of water. Aim is to minimizethe disease burden of the human population potentially emanatingfrom man-made water systems.

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