ARTICLE

Manual faucets induce more biofilms than electronic faucetsRiika Mäkinen, Ilkka T. Miettinen, Tarja Pitkänen, Jaana Kusnetsov, Anna Pursiainen, Sara Kovanen, Kalle Riihinen,and Minna M. Keinänen-Toivola

Abstract: Electronic faucets (types E1 and E2) and manual (M) faucets were studied for microbial quality, i.e., biomass andpathogenic microbes of biofilms in the faucet aerator, the water, and the outer surface of faucet in a hospital in Finland.Heterotrophic plate count content reflecting culturable microbial biomass and adenosine triphosphate content representingviable microbial biomass were smaller in the biofilms of E1-type electronic faucets than E2-type electronic faucets or M faucets.The likely explanation is the mixing point of cold and hot water (E1 and M: in the faucet; E2: in a separate box 50 cm before theactual faucet part). The highest amounts of Legionella (serogroups 2–15 of Legionella pneumophila) in a water sample (5000 cfu/L) andin biofilm samples (May–June 2008 sampling: 240 cfu/mL; November 2008: 1100 cfu/mL) were found in one E1-type faucet, whichwas lacking a back pressure valve due to faulty installation. This study reveals that certain types of electronic faucets seem topromote hospital hygiene, as they were associated with less microbial growth in biofilms in the faucet aerator, than some othertypes of electronic faucets or manual faucets, likely owing to the mixing point of cold and hot water. However, the faucet typehad no direct effect on the presence of Legionella spp. Also correct installation is crucial.

Key words: biofilm, drinking water, faucet, hospital, Legionella spp.

Résumé : Dans un hôpital en Finlande, on a étudié des robinets électroniques (types E1 et E2) et manuels (M) du point de vue dela qualité microbienne, autrement dit la biomasse et la teneur en microbes pathogènes de biofilms retrouvés dans l'aérateur durobinet, l'eau, et la surface externe du robinet. Le dénombrement des bactéries hétérotrophes reflétant la biomassemicrobiennecultivable, de même que la teneur en adénosine triphosphate représentant la biomasse microbienne viable, étaient inférieursdans les biofilms du robinet électronique E1 par rapport au robinet électronique E2 ou au robinetmanuel M. L'explication la plusvraisemblable serait que le point de mélange de l'eau chaude et de l'eau froide (dans le robinet pour E1 et M; dans un boîtierséparé, 50 cm avant le robinet comme tel, pour E2). On a retrouvé les plus grandes quantités de Legionella (sérogroupes 2–15 deLegionella pneumophila) dans un échantillon d'eau (5000 ufc/L) et dans des échantillons de biofilms (prélèvement demai–juin 2008:240 ufc/mL, novembre 2008: 1100 ufc/mL) dans un robinet de type E1, auquel il manquait une soupape de contre-pression a caused'une mauvaise installation. Cette étude révèle que certains types de robinets électroniques semblent favoriser l'hygiène enmilieu hospitalier puisqu'ils sont associés a un développement de microbes moins important dans les biofilms de l'aérateur qued'autres types de robinets électroniques oumanuels, une différence vraisemblablement attribuable au point demélange de l'eauchaude et froide. Cependant, le type de robinet n'aurait aucune incidence directe sur la présence de Legionella spp. De plus, uneinstallation adéquate est primordiale. [Traduit par la Rédaction]

Mots-clés : biofilms, eau potable, robinet, hôpital, Legionella spp.

IntroductionNosocomial infections in hospitals are among the most signifi-

cant infection problems in the developed countries. In Americanhospitals alone, health-care-associated infections account for anestimated 1.7million infections and 99 000 associated deaths eachyear (Klevens et al. 2007). The most important migration routes ofnosocomial infections are contact, droplet, and airborne infection(Vincent 2003).

The quality of hospital tap water influences human health, as ithas been the source of nosocomial infections (CDC 2003). Thepresence of and exposure to Legionella pneumophila has caused ep-idemics in hospitals in Finland and elsewhere (Perola et al. 2002;Stout et al. 2007). It is known that Legionella spp. grow in hot watersystems, have an optimum growth range of 20–45 °C, and spreadvia aerosols formed during bathing or showering. Legionella spp.,like other heterotrophicmicrobes, mostly grow in biofilms on thesurfaces of materials. Biofilms can harbour pathogens, as theyprovide an ecological niche for an abundant microbial commu-

nity. Biofilms are rapidly formed (Bachmann and Edyvean 2006;Zacheus et al. 2000) and represent a longer history of microbes ina water distribution system, while water quality can differ fromday to day or from season to season.

Faucets can be manual, i.e., use requires touch, or electronicsensor operated, i.e., touchless. In electronic faucets, water flowstarts when the sensor is activated, normally by movement. Fau-cets inwater distribution systems are known to pose a potential riskfor nosocomial infections, as they can harbor pathogenic microbes,such as Pseudomonas aeruginosa and Legionella spp. (CDC 2003; Halabiet al. 2001; Hargreaves et al. 2001). On the other hand, electronic, i.e.,touchless, faucets can promote hand hygiene and therefore preventnosocomial infections.

The aim of this study was (i) to study the presence of fecalindicator bacteria, pathogenic bacteria, andmicrobial biomass onthe surfaces of faucets, in the drinking water, and in the biofilms,and (ii) to compare manual and electronic faucets with respect tomicrobiological hygiene in a university hospital in Finland.

Received 21 February 2013. Revision received 22 April 2013. Accepted 23 April 2013.

R. Mäkinen and M.M. Keinänen-Toivola. Prizztech Ltd., WANDER Nordic Water and Materials Institute, Sinkokatu 11, FI-26100 Rauma, Finland; Satakunta University of Applied Sciences,Energy and Construction, Sinkokatu 11, FI-26100 Rauma, Finland.I.T. Miettinen, T. Pitkänen, J. Kusnetsov, A. Pursiainen, and S. Kovanen. National Institute for Health and Welfare (THL), Water and Health Unit, P.O. Box 95, FI-70701 Kuopio, Finland.K. Riihinen. Quantifire Ltd., Innopoli 2, Tekniikantie 14, FI-02150 Espoo, Finland.

Corresponding author: Riika Mäkinen (e-mail: riika.makinen@wander.fi).

407

Can. J. Microbiol. 59: 407–412 (2013) dx.doi.org/10.1139/cjm-2013-0131 Published at www.nrcresearchpress.com/cjm on 24 April 2013.

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Materials and methods

Study siteThe study site was a university hospital in Finland receiving its

drinking water from the municipal water works. The water orig-inated from 2 water treatment plants whose raw water wasmainly bank-filtrated water. In one water treatment plant, ironand manganese were removed by biological rapid sand filtration,and the water produced in the other plant was purified in thewater works by traditional chemical coagulation, including thepre-oxidation of iron and manganese with KMnO4 and aeration.The waters (2/3 from one treatment plant and 1/3 from the othertreatment plant) were mixed in the water works and finally disin-fected with chlorine (0.3 mg/L). The content of organic matter(total organic carbon (TOC)) was on average 1.9 mg/L. The distribu-tion systemconsistedmainly of cast iron andhighdensity polyeth-ylene pipeline materials. The retention time of water from waterworks to the hospital was approximately 12 to 24 h. The hospitalwas a large building complex with 8 hot water circuits varying inlength from 100 to 600 m. The main materials inside the hospitalwere copper and acid-proof steel.Water consumption in thehospitalwas a total of 120 000 m3 in 2007. During the 1990s there were 2microbiological outbreaks due to Sphingomonas paucimobilis andL. pneumophila, and their sources highly likely originated in the coldand hot water system of the hospital (Perola 2006). As far as we areaware, there were no nosocomial infections in the studied samplingareas during the sampling time.

Studied faucetsA total of 12 faucets were studied of which 4 were electronic

faucets type Oras Electra 6250G (hereafter E1-type faucets), 4 wereelectronic faucets type Oras Electra 6204 (hereafter E2-type fau-cets), and 4 were manual faucets type Oras Safira 282 (hereafter Mfaucets) (Table 1). Oras Electra 6250G (E1-type faucets) is a battery-operated touchless basin faucet with a temperature adjustmenthandle (factory default). Oras Electra 6204 (E2-type faucet) is atouchless basin faucet with a mixer valve. Oras Safira 282 (M fau-cet) is amanual single-lever basin faucet. The structural differencebetween the 2 types of electronic faucets was the place where themixing of hot and cold water took place. In M and E1-type faucets,mixing of hot and cold occurred in the faucet part. In E2-typefaucets, the mixing happened in a separate box 50 cm before theactual faucet part. The interior and the faucet aerator of E1- andE2-type faucets were plastic (mainly polyoxymethylene (POM)).The original faucet aerator of M faucets was stainless steel (AISI304). The outer surfaces of studied faucets were chrome plated.

The studied faucets were selected from as similar operatingenvironments as possible, i.e., from the same or similar wards. Inaddition, faucets operated by patients were considered to bemorerelevant for hospital hygiene than faucets in nurses' rest areas orpublic areas (Table 1). Faucets were specifiedwith A, B, C, andD forpresenting the results. E1-type faucets had been installed 2 yearsago in the hospital water system; E2-type and M faucets had been

operating for 10 years. None of the faucet aerators had been re-placed during their operating history, i.e., the faucet aerators wereoriginal and the same age as the faucets. In addition, the faucets hadnot been under maintenance or cleaning from inside during theiroperational history. The outer surfaces of all the faucets were wipedcleanwithaneutralwashingdetergent (pH6–8) everydayduring themorning, and if needed, the cleaningwas repeatedduring theday. Inaddition, all the faucets were cleaned with a chlorine-based deter-gent once a week. The faucets that were cleaned were not used forwashing the cleaning cloths or getting the cleaning water.

SamplingSampling took place at 0700 h. The last cleaning, which in-

cluded wiping the surfaces of the faucets, had been carried out onthe previous day, so sampling time presented the worst possiblecircumstances with respect to surface hygiene and water quality.The surface sample was taken by wiping the entire outer surfaceof a faucet with a sterile cotton stick that had been dipped intoRinger solution. The used end of the cotton stick was cut into asample bottle containing 25 mL of Ringer solution. When takingthe surface sample extra caution was needed with the motionsensors of the electronic faucets to prevent unnecessary wateroutburst, i.e., splatter or spillage. A water sample that had stag-nated overnight inside the faucet (i.e., 200 mL) was taken directlyfrom a faucet without water flow or any disinfection of the faucet.The water sample from the M faucets was taken so that the leverof the faucet was in the centre position and the faucet was openedto the same flow volume as the electronic faucets. The biofilmsample was taken from a faucet aerator. The aerator with its fix-tures was removed with sterile equipment, and the aerator with-out fixtures was placed into a sample bottle containing 25 mL ofRinger solution. The removed aerator was replaced with a newplastic aerator (mainly POM). The surface and biofilm sampleswere removed by sonicating the fixtures for 30 min with eluentsbeing cultured in the microbiological laboratory.

Surface, water, and biofilm samples for microbiological analy-ses were collected from all 12 faucets on 3 adjacent weeks: 21 May2008 (E2-type faucets), 27 May 2008 (E1-type faucets), and 6 June2008 (M faucets). Additional water and biofilm samples weretaken from the same 12 faucets after 25 ± 1 week, on 19 November2008 (E2-type faucets and 2 E1-type faucets) and 20 November 2008(M faucets and 2 E1-type faucets).

Water samples for analyses of chemical water quality, totalcarbon (TC), and TOCwere taken only on the first sampling periodinMay–June 2008. Similarly, a sample from thewater entering thehospital water system was taken from a fire hose in a mainte-nance tunnel at the first sampling period. Chemical water qualitywas analyzed from one sample of each of the 3 faucet types (n = 3).TC and TOC were analyzed from each faucet (n = 12).

Table 1. Faucets investigated in the study.

Faucet Faucet type Model Location and number of faucetsAge(years)

Mixing place of hotand cold water

Unitsinvestigatedin the study

E1 Electronic, i.e.,touchless

Oras Electra6250G

Operational rooms inotolaryngological*polyclinic (2 faucets) and surgicalpolyclinic (2 faucets)

2 In the faucet 4

E2 Electronic, i.e., touchless Oras Electra6204

Surgical ward, isolationrooms (4 faucets)

10 50 cm beforethe faucet

4

M Manual Oras Safira282

The same surgical ward as above,patient rooms (4 faucets)

10 In the faucet 4

*Otolaryngology is the study of ear, nose, and throat.

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Microbiological analysesThe microbiological analyses were made for the following mi-

crobial groups: Escherichia coli, Clostridium difficile, P. aeruginosa,Klebsiella sp., coliform bacteria, intestinal enterococci, Legionellaspp., heterotrophic plate count (HPC, R2A-method) analyzed withculture-based methods, and adenosine triphosphate (ATP) bio-luminescence method. All the microbiological analyses wereconducted with all the samples, i.e., surface, water, and biofilm,except for C. difficile and P. aeruginosa, which were not analyzedfrom water samples, and Legionella spp., which were not analyzedfrom surface samples in the May–June 2008 sampling. The sam-ples taken in November 2008 were analyzed for HPC, ATP, andLegionella spp. (only biofilm).

The counts of coliform bacteria and E. coliwere analyzed accord-ing to the membrane filtration method (SFS standard 3016 (SFS2001)) using Les Endo Agar medium (Merck, Darmstadt, Germany)and chromogenic Chromocult Coliform Agar (Merck) at 36 ± 2 °Cfor 21 ± 3 h. The presence of Klebsiella sp. was tested as part ofanalyses of coliform bacteria. The identification of species wasdone with API 20E and API 20NE tests (Biomerieux, Marcy l'Etoile,France).

Intestinal enterococci were analyzed according to the interna-tional standard method ISO 7899-2 (ISO 2000) using Slanetz andBartley agar medium (Oxoid, Basingstoke, UK).

Clostridium difficile was cultured from heat-treated water sam-ples following the pretreatment principles of the internationalstandard ISO 6461-2 (ISO 1986) and by using cycloserine–cefoxitin–egg yolk agar medium (Brazier 1993) at 37 °C for 3 days underanaerobic conditions.

Pseudomonas aeruginosa was analyzed according to the interna-tional standard method ISO 16266 (ISO 2006) using CN agar me-dium (Oxoid).

Culturing for Legionellawas based on the international standardmethod ISO 11731 (ISO 1998) and the Standard Operation Proce-dure of National Public Health Institute SOP MB131, in force since28 February 2003. With the water samples, the lowest theoreticalconcentration that was possible to detect was 50 cfu/L and forbiofilm samples it was 1 cfu/mL of the biofilm eluent volume.Water samples were concentrated by filtration and biofilm sam-ples by centrifugation. Acid wash and heat were the pretreatmentmethods. The inoculated glycine vancomycin polymyxin cy-cloheximide and buffered charcoal yeast extract media plateswere incubated at 36 °C up to 10 days.

HPC was analyzed with a spread plating method on R2A me-dium (Difco) for 7 days at 22 °C (APHA–AWWA–WEF 2012).

The content of ATP was measured with a Bio Orbit 1251 lumi-nometer (Turku, Finland) and by using ATP Biomass kit HS 266-311(BioThema AB, Handen, Sweden). The measurement is based onintensity of the light and is proportional to the amount of ATP andis measured before and after addition of ATP standard, whichcontains known amount of ATP. The ATP content of surface andbiofilm samples were measured with 3 duplicates, whereas waterand Ringer solution were measured without duplicates.

Microbial communities (total amount of 16S rRNA gene copies,Alpha-, Beta-, and Gamma-proteobacteria, Legionella spp.) were ana-lyzed by molecular biology technique of quantitative polymerasechain reaction (qPCR) based on 16S rRNA gene from biofilm sam-ples taken in November 2008. The amount of 16S rRNA gene cop-ies were measured with ABI SDS 7000 (Applied Biosystems, UK).The qPCR system is based on the detection and quantification offluorescent reporter. This signal increases in direct proportion tothe amount of amplified PCR product in the reaction (Morrisonet al. 1998). The fluorescent reporter used in the reactions wasSYBR Green I (Roche Diagnostics, Germany). A DNA extractionprior to qPCR was a method by QuantiFire Ltd. and primers in theqPCR reaction were a QuantiFire Ltd. design (primer informationavailable on request).

Analyses for chemical quality of waterTC and total non-purgeable organic carbon (TOC) were analyzed

with a high temperature combustion method (680 °C) with a Shi-madzu TOC 5000/5050 analyzer (Kyoto, Japan) (Lehtola et al. 2006).The analyses for chemical quality of water also included pH, alka-linity, electronic conductivity, total hardness, total nitrogen, totalphosphorous, iron, and manganese (data not shown). Neverthe-less, it can be stated that overall the chemical quality of colddrinking water was very similar in the studied faucets, i.e., thedistribution network had no effects on the chemical quality ofdrinking water. In addition, drinking water fulfilled national leg-islation in Finland, which is based on the Drinking Water Direc-tive (98/83/EC) of the European Union.

Statistical methodsAnalysis of variance (2-way ANOVA) followed by a Tukey's mul-

tiple comparison test (significance level of � = 0.05) was used todetect differences with sampling time and faucet types in HPCand ATP. In addition, 1-way ANOVA was used for total amount of16S rRNA gene copies; Alpha-, Beta-, and Gamma-proteobacteria; andLegionella spp. in biofilm samples taken in November 2008. Allstatistical analyses were performed using the Statistical Packagefor the Social Sciences (SPSS) forWindows version 16.0.1 (SPSS Inc.,Chicago, Illinois, USA).

Results and discussion

BiofilmIn biofilms collected from the faucet aerators, the faucet type

had an effect on HPCs, as expressed by viable microbial biomass(P < 0.001) (Fig. 1A). HPCs were lower for E1-type faucets (8.1 ×104 cfu/mL, n = 8) than for E2-type faucets (1.9 × 106 cfu/mL, n = 8,P < 0.01) or M faucets (3.3 × 106 cfu/mL, n = 8, P < 0.001). Similarly,the faucet type influenced the microbial biomass in the biofilmsbased on the ATP content (P< 0.01) (Fig. 1B). The ATP concentrationwas lower for electronic E1-type faucets (0.1 pmol/mL, n = 8) thanfor electronic E2-type faucets (2.2 pmol/mL, n = 8, P < 0.01) or Mfaucets (2.3 pmol/mL, n = 8, P < 0.01). The qPCR of total 16S rRNAgenes, representing bacterial biomass of biofilms taken inNovem-ber 2008 (age 25 ± 1 weeks), similarly showed that there was lessbacterial biomass in E1-type faucets (under detection limit, i.e.,

Fig. 1. (A) Heterotrophic plate count (HPC) representing culturablemicrobial biomass (R2A) and (B) adenosine triphosphate (ATP)content representing viable microbial biomass of biofilm samples(faucet aerators). E1-type, electronic faucet, water mixing point inthe faucet; E2-type, electronic faucet, water mixing point 50 cmbefore the faucet; M, manual faucet, water mixing point in thefaucet. n = 8 for each faucet type. **, P < 0.01; ***, P < 0.001.

0

1000000

2000000

3000000

4000000

5000000

6000000

E2-type faucets M faucetsE1-type faucets

***

M faucetsE2-type faucets

HP

C (c

fu/m

L)

E1-type faucets

**

0

1

2

3

4

5

6

****

ATP

(pm

ol/m

L)

A

B

Mäkinen et al. 409

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2.20 × 103 total 16S rRNA gene copies/mL) than in E2-type faucets(7.56 × 106 ± 1.49 × 107 total 16S rRNA gene copies/mL) or M faucets(1.91 × 106 ± 1.55 × 106 total 16S rRNA gene copies/mL). However,this difference was not statistically significant owing to a largevariation in numeric values between E2-type faucets and M fau-cets. Neither Alpha- nor Beta-proteobacteria were found in biofilmsof E1-type faucets (detection limits 2.20 × 103 and 4.00 × 102 16SrRNA genes/mL, respectively). The numbers of Alphaproteobacteriain E2-type and M faucets were 2.94 × 106 ± 5.64 × 106 rRNA and3.86 × 105 ± 1.95 × 105 gene copies/mL, respectively, representingthe largest proportion of the microbial population. In Betaproteo-bacteria, there was a statistically significant difference (P < 0.05)between E1-type (under detection limit) and E2-type faucets (5.13 ×103 ± 3.55 × 103 16S rRNA genes/mL). In one M faucet, M-B, thenumber of Betaproteobacteria was 3.89 × 103 ± 1.95 × 103 16S rRNAgenes/mL. Gammaproteobacteria were detected with positive signalonly in faucet E2-D (detection limit 4.00 × 102 16S rRNA genes/mL).

The critical structural difference between the studied faucetswas the place where hot and cold water was mixed, i.e., in theE1-type and M faucets, mixing was achieved within the faucet,whereas in the E2-type faucets, mixing took place 50 cm beforethe faucet. The E2-type and M faucets had been installed in thehospital water system 10 years previously, whereas the E1-typefaucets had been in the hospital water system for 2 years. As well,the materials present in the faucet aerators were different (E1:POM; E2: POM; M: AISI 304) in the May–June 2008 sampling butwere the same (E1, E2, M: POM) in the November 2008 sampling.However, based on statistical testing, the differences in the HPCsand the ATP content did not originate from variations in the fau-cet age or the aerator material in faucet. Biofilms are rapidlyformed in drinking water systems; they are known to reach steady-state growth in a time frame of 3weeks to 4months (Bachmann andEdyvean 2006; Zacheus et al. 2000). Future studies should be concen-trated on determining the technical reason for these results. Gener-ally, less microbial biomass in biofilms, as in E1-type faucets, isfavorable, since a dense biofilm can more effectively harbor patho-gens suchas Legionella spp.Also the locationof the faucet in thewatersystem of the hospital might partly explain the results, since theE2-type and M faucets were located in the same ward either in pa-tient rooms (M) or in isolation areas (E2), but the E1-type faucetswerelocated in the surgical area of 2neighboringpolyclinics.However, nodifferences inwater qualitywere detectedbetween theward (patientroom or isolation area) and the polyclinics. Biofilms represent a lon-ger history of microbes in a water distribution system, while waterquality can differ from day to day or from season to season.

This and a previous study (Wang et al. 2009) showed a largevariation in microbial quantity and an uneven distribution offlora among the faucets. The present observations support theconcept that tap water faucets become contaminated within thehealth care environment at the point of use (Trautmann et al.2005; Exner et al. 2005; Wang et al. 2009).

Legionella spp. was found in the May–June 2008 sampling fromthe biofilm of one E1-type faucet, E1-D, which was missing a backpressure valve caused by faulty installation (Table 2). (Themissingback pressure valve was installed immediately after the fault wasdiscovered/confirmed, in June.) In the November 2008 sampling,Legionella spp. was found with culture techniques in 3 faucets of2 faucetmodels. However, the highest concentrationwas detectedin the same faucet E1-D as in the May–June 2008 sampling. Inaddition, based on qPCR analyses of 16S rRNA of Legionella spp.from samples taken in November 2008, it was detected only insample E2-D, but no value for concentration could be providedbecause the number of cells was so low. The hospital personnelwere informed about the Legionella results immediately after theMay–June 2008 sampling, and a new back pressure valve was in-stalled. The Legionella strains detected belonged to serogroups 2–15of L. pneumophila. In a larger study, the mean Legionella concentra-

tions in biofilms of faucets with aerators, of faucets with a laminarflow device, and of control faucets without an aerator or laminarflow device have been similar to each other (520, 650, and530 cfu/L, respectively, n = 102) (Huang and Lin 2007). Thus, it ispostulated that the other environmental factors like water tem-perature, pipeline material, and microbial population have gen-erally a greater impact on the Legionella concentrations in biofilmthan does the presence or absence of an aerator. This highlightsthe importance of correct installations.

Coliform bacteria (detected only in faucet E1-B at �40 cfu/1 mL,and in faucet M-B at 2 cfu/2 mL), Klebsiella sp. (present only infaucet E1-B), and intestinal enterococci (detected only in faucetE2-D at 1 cfu/2mL) were very rarely detected in biofilms. Escherichiacoli, C. difficile, and P. aeruginosa were never detected in any of thebiofilm samples (faucet aerators). These indicator bacteria can bepresent in Finnish drinking water distribution systems, but arevery rare, normally being related to contamination of a watersystem.

WaterLegionella spp. was found in 3 out of 12 water samples from all

3 faucet types (Table 2). The highest concentration (5000 cfu/L) ofLegionella was found in the E1-D faucet, which lacked the backpressure valve, i.e., the situation was similar to the biofilm sam-ple. The Legionella strains detected belonged to serogroups 2–15 ofL. pneumophila similar to the biofilms. In an earlier study, in thesame hospital, water samples from taps and showers have con-tained greater Legionella concentrations (mean 1.0 × 104 cfu/L) thanthe circulating hot water samples (mean 1800 cfu/L) (Kusnetsovet al. 2003). In the European guidelines, the action level concen-tration in cold and hot water for Legionella spp. in hotels is1000 cfu/L, thus concentrations over 1000 cfu/L should cause re-view of control methods and preventive actions (EWGLI 2011).However, in hospitals, even lower concentrations are recom-mended and thus more attention should be paid to the watersystems. Basically, even though the cold water was cold and thehot water hot, the temperatures of the system were within theoptimum growth range of Legionella spp. Achieving a high enoughtemperature is themost effectivemethod for preventing Legionellaspp. (WHO 2007). According to Finnish legislation, the tempera-ture of water systems in all points should be at least 55 °C after ashort period of flushing. With this temperature limit, more than90% of viable Legionella spp. will be destroyed within 10–20 min(WHO 2007). From another perspective, the legislation in Finland

Table 2. Legionella spp. concentrations in water and bio-film samples based on culture techniques.

Biofilm (cfu/mL) Water (cfu/L)

FaucetMay–June2008

November2008

May–June2008

E1-A <1 <1 <50E1-B <1 <1 <50E1-C <1 6 <50E1-D 240* 1100 5000*E2-A <1 <1 150E2-B <1 3 <50E2-C <1 <1 <50E2-D <1 <1 <50M-A <1 <1 <50M-B <1 <1 <50M-C <1 <1 50M-D <1 <1 <50

Note: E1, electronic faucet, water mixing point in the faucet;E2, electronic faucet, watermixing point 50 cm before the faucet;M, manual faucet, water mixing point in the faucet. A–D refer tothe 4 studied faucets of each type.

*Faucet E1-D was lacking a back pressure valve due to faultyinstallation.

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limits the maximum water temperature from faucets and show-ers to 65 °C to avoid scalding. It is clear that correct design, instal-lation, and maintenance of the whole water distribution systemare needed to protect human health against the risks posed byLegionella.

The ATP concentration (faucet type: P < 0.05) was higher in watersamples fromE2-type faucets thanM faucets (0.08 ± 0.04 pmol/mL vs0.04±0.00pmol/mL, respectively; E2:n=8,M:n=7,P<0.05). TheATPconcentration of water samples in the E1-type faucets was 0.05 ±0.01 pmol/mL.

HPCs were similar in all water samples (data not shown). Esche-richia coli, coliform bacteria, intestinal enterococci, and Klebsiellasp. were not detected in any of the water samples. Our resultsdepict the worst possible circumstances of water quality, as thewater had stagnated overnight.

SurfaceThe results from the surface samples indicate that the ATP con-

tent, representing viable microbial biomass, was lower for thesurfaces of the E1-type (0.01 ± 0.01 pmol/mL, n = 4) and E2-typefaucets (0.28 ± 0.51 pmol/mL, n = 4) than for the surfaces of the Mfaucets (0.73 ± 0.84 pmol/mL, n = 4). However, the result is notstatistically significant, as the standard deviation of the resultswas large. The HPC results representing culturable microbial bio-mass (1.4 ± 2.2 × 102 cfu/mL, n = 12) did not reveal any similar trend.Only a small number of heterotrophic microbes of surface sam-ples grew on the R2A plates at 22 °C. R2A plates for HPC areoriginally developed for microbes in drinking water systems fa-voring microbes growing in conditions with low concentrationsof nutrients. This might indicate that microbes, detected in highernumbers, by the ATP assay, on the surfaces of the manual faucetsthan the electronic faucets, were normal flora from human hands.This supports the hypothesis that the electronic, i.e., touchless, fau-cets support hand hygiene and possibly prevent nosocomial infec-tions. Covering manual faucet handles with paper towels duringhand washing or installing a touchless faucet has been shown to beprotective against Shigella sonnei (Mermel et al. 1997).

Coliform bacteria were detected in very small numbers in 3 out of4 surface samples from the E2-type electronic faucets (E2-A, 2 cfu/3mL;E2-B, 1 cfu/1mL; andE2-D, 2 cfu/3mL) and inone surface samplefrom the M faucets (M-A, 2 cfu/3 mL). The cell numbers of coliformbacteria were low, likely showing unusual touching of the E2-typefaucets. Intestinal enterococci were detected only on faucet E2-D(�450 cfu/2.6 mL). Escherichia coli, C. difficile, P. aeruginosa, or Klebsiellasp. were not detected in any of the surface samples. With respect tocleaning, the surface samples represented theworst possible circum-stances, as the cleaning was done just 1 day before sampling.

In conclusion, the E1-type electronic faucets promoted less mi-crobial biomass (based onHPC, ATP, and total 16S rRNA gene) thanthe E2-type electronic faucets or the manual faucets. The reasonfor this difference could be the mixing point of cold and hot waterbetween the faucets or an uneven distribution of flora among thefaucets due to point-of-use contamination. It was found that Legion-ella spp. were present and able to multiply in the water system, butthe faucet type had no direct effect on the presence of Legionella spp.

AcknowledgementsThe financial support by the Regional Council of Satakunta –

European Regional Development Fund (ERDF) (50%) and Oras Ltd.(50%) and the National Technology Agency of Finland (project3047/31/2011) is gratefully acknowledged. The personnel from thetechnical services of the hospital are especially thanked for theirassistance in the practical details and implementation of the sam-pling. Piia Airaksinen, Henni Knuutinen, Siru Meckelborg, andMarjo Tiittanen from National Institute for Health and Welfare(Kuopio, Finland) are gratefully acknowledged for their laboratoryassistance in the research. Dr. Ewen MacDonald is thanked forgrammatical advice on the article.

ReferencesAPHA–AWWA–WEF (American PublicHealthAssociation –AmericanWaterWorks

Association – Water Environment Federation). 2012. Standard methods for theexamination ofwater andwastewater. 22nd ed. APHA–AWWA–WEF,Washington,D.C., USA.

Bachmann, R.T., and Edyvean, R.G.J. 2006. Biofouling: an historic and contem-porary review of its causes, consequences and control in drinking waterdistribution systems. Biofilms, 2(1): 1–31. doi:10.1017/S1479050506001979.

Brazier, J.S. 1993. Role of the laboratory in investigations of Clostridium difficilediarrhea. Clin. Infect. Dis. 16(Suppl. 4): S228–S233. doi:10.1093/clinids/16.Supplement_4.S228. PMID:8324124.

CDC (Centers for Disease Control and Prevention). 2003. Guidelines for environ-mental infection control in health-care facilities. Recommendations fromCDC and the Healthcare Infection Control Practices Advisory Committee(HICPAC). U.S. Department of Health andHuman Services Centers for DiseaseControl and Prevention (CDC), Atlanta, Ga., USA. Available from http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5210a1.htm [accessed 11 February2013].

EWGLI (European Technical Guidelines for the Investigation). 2011. EuropeanTechnical Guidelines for the Investigation, Control and Prevention of TravelAssociated Legionnaires' Disease. September 2011, Version 1.1. Produced bymembers of the EuropeanWorking Group for Legionella Infections. Availablefrom http://www.escmid.org/fileadmin/src/media/PDFs/3Research_Projects/ESGLI/European_Guidelines_September_2011_v1_1.pdf [accessed 11 February2013].

Exner, M., Kramer, A., Lajoie, L., Gebel, J., Engelhart, S., and Hartemann, P. 2005.Prevention and control of health care-associated waterborne infections inhealth care facilities. Am. J. Infect. Control, 33(5 Suppl. 1): S26–S40. doi:10.1016/j.ajic.2005.04.002. PMID:15940114.

Halabi, M., Wiesholzer-Pittl, M., Schöberl, J., and Mittermayer, H. 2001. Non-touch fittings in hospitals: a possible source of Pseudomonas aeruginosa andLegionella spp. J. Hosp. Infect. 49: 117–121. doi:10.1053/jhin.2001.1060. PMID:11567556.

Hargreaves, J., Shireley, L., Hansen, S., Bren, V., Fillipi, G., Lacher, C.,Esslinger, V., and Watne, T. 2001. Bacterial contamination associated withelectronic faucets: a new risk for healthcare facilities. Infect. Control Hosp.Epidemiol. 22(4): 202–205. doi:10.1086/501889. PMID:11379709.

Huang, W.-K., and Lin, Y.E. 2007. A controlled study of Legionella concentrationsin water from faucets with aerators or laminar water flow devices. Infect.Control Hosp. Epidemiol. 28(6): 765–766. doi:10.1086/516797. PMID:17520562.

ISO (International Organization for Standardization). 1986. Water quality —detection and enumeration of the spores of sulfite-reducing anaerobes(clostridia) — Part 2: Method by membrane filtration. Standard 6461-2. ISO,Geneva, Switzerland.

ISO (International Organization for Standardization). 1998. Water quality —detection and enumeration of Legionella. Standard 11731. ISO, Geneva, Switzer-land.

ISO (International Organization for Standardization). 2000. Water quality —detection and enumerationof intestinal enterococci—Part 2:Membranefiltrationmethod. Standard 7899-2. ISO, Geneva, Switzerland.

ISO (International Organization for Standardization). 2006. Water quality —detection and enumeration of Pseudomonas aeruginosa — method by mem-brane filtration. Standard 16266. ISO, Geneva, Switzerland.

Klevens, R.M., Edwards, J.R., Richards, C.L., Jr., Horan, T.C., Gaynes, R.P.,Pollock, D.A., and Cardo, D.M. 2007. Estimating health care-associated infec-tions and deaths in U.S. hospitals, 2002. Public Health Rep. 122(2): 160–166.Available from http://www.cdc.gov/HAI/pdfs/hai/infections_deaths.pdf [ac-cessed 11 February 2013].

Kusnetsov, J., Torvinen, E., Perola, O., Nousiainen, T., and Katila, M.L. 2003.Colonization of hospital water systems by legionellae, mycobacteria andother heterotrophic bacteria potentially hazardous to risk group patients.APMIS, 111(5): 546–556. doi:10.1034/j.1600-0463.2003.1110503.x. PMID:12887506.

Lehtola, M.J., Laxander, M., Miettinen, I.T., Hirvonen, A., Vartiainen, T., andMartikainen, P.J. 2006. The effects of changing water flow velocity on theformation of biofilms and water quality in pilot distribution system consist-ing of copper or polyethylene pipes. Water Res. 40(11): 2151–2160. doi:10.1016/j.watres.2006.04.010. PMID:16725175.

Mermel, L.A., Josephson, S.L., Dempsey, J., Parenteau, S., Perry, C., andMagill, N.1997. Outbreak of Shigella sonnei in a clinical microbiology laboratory. J. Clin.Microbiol. 35(12): 3163–3165. Available from http://jcm.asm.org/cgi/reprint/35/12/3163 [accessed 11 February 2013]. PMID:9399513.

Morrison, T.B., Weis, J.J., and Wittwer, C.T. 1998. Quantification of low-copytranscripts by continuous SYBR Green I monitoring during amplification.BioTechniques, 24(6): 954–958, 960, 962. PMID:9631186.

Perola, O. 2006. Hospital water supply as a source of nosocomial infections.Ph.D. thesis, University of Kuopio, Kuopio, Finland. Available from http://urn.fi/URN:ISBN:951-27-0582-6 [accessed 11 February 2013].

Perola, O., Kauppinen, J., Kusnetsov, J., Heikkinen, J., Jokinen, C., andKatila, M.-L. 2002. Nosocomial Legionella pneumophila serogroup 5 outbreakassociated with persistent colonization of a hospital water system. APMIS,110(12): 863–868. doi:10.1034/j.1600-0463.2002.1101204.x. PMID:12645664.

Mäkinen et al. 411

Published by NRC Research Press

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

McM

aste

r U

nive

rsity

on

11/1

4/14

For

pers

onal

use

onl

y.

SFS (Finnish Standards Association). 2001. Water quality. Member filter tech-nique for enumeration of total coliform bacteria in water. Standard 3016.Finnish Standards Association (SFS).

Stout, J.E., Muder, R.R., Mietzner, S., Wagener, M.M., Perri, M.B., DeRoos, K.,Goodrich, D., Arnold, W., Williamson, T., Ruark, O., Treadway, C.,Eckstein, E.C., Marshall, D., Rafferty, M.E., Sarro, K., Page, J., Jenkins, R.,Oda, G., Shimoda, K.J., Zervos, M.J., Bittner, M., Camhi, S.L., Panwalker, A.P.,Donskey, C.J., Nguyen, M.-H., Holodniy, M., and Yu, V.L.; the Legionella StudyGroup 2007. Role of environmental surveillance in determining the risk ofhospital-acquired legionellosis: a national surveillance study with clinicalcorrelations. Infect. Control Hosp. Epidemiol. 28(7): 818–824. doi:10.1086/518754. PMID:17564984.

Trautmann, M., Lepper, P.M., and Haller, M. 2005. Ecology of Pseudomonas aerugi-nosa in the intensive care unit and the evolving role of water outlets as a

reservoir of the organism. Am. J. Infect. Control, 33(5 Suppl. 1): S41–S49.doi:10.1016/j.ajic.2005.03.006. PMID:15940115.

Vincent, J.-L. 2003. Nosocomial infections in adult intensive-care units. Lancet,361: 2068–2077. doi:10.1016/S0140-6736(03)13644-6. PMID:12814731.

Wang, J.L., Chen, M.L., Lin, Y.E., Chang, S.C., and Chen, Y.C. 2009. Associationbetween contaminated faucets and colonization or infection by nonferment-ing gram-negative bacteria in intensive care units in Taiwan. J. Clin. Micro-biol. 47(10): 3226–3230. doi:10.1128/JCM.00034-09. PMID:19587299.

WHO (World Health Organization). 2007. Legionella and prevention of legionel-losis.WorldHealthOrganization, Geneva, Switzerland. Available fromhttp://www.who.int/water_sanitation_health/emerging/legionella.pdf [accessed 11February 2013].

Zacheus, O.M., Iivanainen, E.K., Nissinen, T.K., Lehtola, M.J., andMartikainen, P.J. 2000. Bacterial biofilm formation on polyvinyl chloride,polyethylene and stainless steel exposed to ozonated water. Water Res. 34(1):63–70. doi:10.1016/S0043-1354(99)00113-X.

412 Can. J. Microbiol. Vol. 59, 2013

Published by NRC Research Press

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. J. M

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. Dow

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by

McM

aste

r U

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rsity

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11/1

4/14

For

pers

onal

use

onl

y.