This study was based on the hypothesis that groundwater-derived biofilms may provide a reservoir for coliform or pathogenic bacteria as has been observed in drinking water distribution systems. Escherichia coli, labelled with green fluorescent protein, was found to colonize all layers of mixed-population biofilms developed in association with indigenous groundwater micro-organisms in a laboratory-scale reactor. Biofilm-associated E. coli was removed at a slower rate from the reactor flasks than planktonic E. coli under a continuous flow regime. During flow-through of groundwater, planktonic E. coli removal was slower in flasks containing coverslips for enhanced biofilm development compared to a control flask without coverslips. Conversely, during flow-through of treated effluent, planktonic E. coli removal was faster in flasks with coverslips compared to without. Removal of attached E. coli was also fastest in the coverslip-containing flasks with effluent flow-through. This suggests that an increase in available nutrients may reduce E. coli survival potential due to either enhanced competition for nutrients or enhanced antagonism by the indigenous microbial population. Under identical conditions, GFP-labelled Pseudomonas aeruginosa was found to persist in the biofilms for longer than E. coli, most notably when exposed to flow-through of treated effluent. However, prolonged persistence of P. aeruginosa in the effluent could not be attributed to an association with the biofilms. This study has shown that under certain conditions the presence of mixed-population biofilms may limit the survival potential of enteric bacterial pathogens introduced into groundwater.
BanningN,
TozeS.,
MeeB. J.
2002; Escherichia coli survival in groundwater and effluent measured using a combination of propidium iodide and the green fluorescent protein. J Appl Microbiol 93:69–76
BlockJ. C.
1992; Biofilms in drinking water distribution systems. In Biofilms – Science and Technology pp 469–485 Edited by
BottT. R.,
MeloL.,
FletcherM.,
CapedevilleB.
Dordrecht: Kluwer;
BloembergG. V,
O'TooleG. A,
LutenbergB. J. J.,
KolterR.
1997; Green fluorescent protein as a marker for Pseudomonas spp. Appl Environ Microbiol 63:4543–4551
BuswellC. M,
HerlihyY. M,
LawrenceL. M,
McGuigganJ. T. M,
MarshP. D,
KeevilW.,
LeachS. A.
1998; Extended survival and persistence of Campylobacter spp. in water and aquatic biofilms and their detection by immunofluorescent-antibody and -rRNA staining. Appl Environ Microbiol 64:733–741
BuswellC. M,
HerlihyY. M,
KeevilC. W,
MarchP. D.,
LeachS. A.
1999; Carbon load in aquatic ecosystems affects the diversity and biomass of water biofilm consortia and the persistence of the pathogen Campylobacter jejuni within them. J Appl Microbiol 85:161S–167S
BuswellC. M,
NichollH. S.,
WalkerJ. T.
2001; Use of continuous culture bioreactors for the study of pathogens such as Campylobacter jejuni and Escherichia coli O157 in biofilms. Methods Enzymol 337:70–78
CamperA. K,
LeChevallierM. W,
BroadawayS. C.,
McFetersG. A.
1985; Growth and persistence of pathogens on granular activated carbon filters. Appl Environ Microbiol 50:1378–1382
CamperA. K,
JonesW. L.,
HayesJ. T.
1996; Effect of growth conditions and substratum composition on the persistence of coliforms in mixed-population biofilms. Appl Environ Microbiol 62:4014–4018
ColbourneJ. S,
PrattD. J,
SmithM. G,
Fisher-HochS. P.,
HarperD.
1984; Water fittings as sources of Legionella pneumophila in a hospital plumbing system. Lancet i:210–213
DowdS. E.,
PillaiS. D.
1997; Survival and transport of selected bacterial pathogens and indicator viruses under sandy aquifer conditions. J Environ Sci Health 32:2245–2258
GerbaC. P.,
GoyalS. M.
1985; Pathogen removal from wastewater during groundwater recharge. In Artificial Recharge of Groundwater pp 283–317 Edited by
AsanoT.
Boston, MA: Butterworth;
GilbertP.,
BrownM. R. W.
1995; Mechanisms of the protection of bacterial biofilms from antimicrobial agents. In Microbial Biofilms pp 118–130 Edited by
Lappin-ScottH. M.,
CostertonJ. W.
Cambridge: Cambridge University Press;
LowderM,
UngeA,
MarahaN,
JanssonJ. K,
SwiggettJ.,
OliverJ. D.
2000; Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506. Appl Environ Microbiol 66:3160–3165
MackayW. G,
GribbonL. T,
BarerM. R.,
ReidD. C.
1999; Biofilms in drinking water systems: a possible reservoir for Helicobacter pylori
. J Appl Microbiol 85:52S–59S
MarraoG,
VerissimoA,
BowkerR. G.,
daCostaM. S.
1993; Biofilms as major sources of Legionella spp. in hydrothermal areas and their dispersion into stream water. FEMS Microbiol Ecol 12:25–33
MombaM. N. B,
CloeteT. E,
VenterS. N.,
KfirR.
1999; Examination of the behaviour of Escherichia coli in biofilms established in laboratory-scale units receiving chlorinated and chloraminated water. Water Res 33:2937–2940
MurgaR,
ForsterT. S,
BrownE,
PrucklerJ. M,
FieldsB. S.,
DonlanR. M.
2001; Role of biofilms in the survival of Legionella pneumophila in a model potable-water system. Microbiology 147:3121–3126
MurgelG. A,
LionL. W,
AchesonC,
ShulerM. L,
EmersonD.,
GhiorseW. C.
1991; Experimental apparatus for selection of adherent microorganisms under stringent growth conditions. Appl Environ Microbiol 57:1987–1996
OlofssonA.-C,
ZitaA.,
HermanssonM.
1998; Floc stability and adhesion of green-fluorescent-protein-marked bacteria to flocs in activated sludge. Microbiology 144:519–528
PavelicP.,
DillonP. J.
1997Review of international experience in injecting natural and reclaimed waters into aquifers for storage and reuseCentre for Groundwater Studies Report No. 74 South Australia;
PavelicP,
DillonP. J,
BarryK. E.,
HerczegA. L.
1998; Well clogging effects determined from mass balances and hydraulic response at a stormwater ASR site. In Artificial Recharge of Groundwater: Proceedings of the Third International Symposium on Artificial Recharge of Groundwater pp 61–67 Edited by
PetersJ. H.
Amsterdam: Balkema;
Rinck-PfeifferS.
2000Physical and biochemical clogging processes arising from aquifer storage and recovery (ASR) with treated wastewater PhD thesis Flinders University of South Australia;
RobinsonP. J,
WalkerJ. T,
KeevilC. W.,
ColeJ.
1995; Reporter genes and fluorescent probes for studying the colonisation of biofilms in a drinking water supply line by enteric bacteria. FEMS Microbiol Lett 129:183–188
RogersJ,
DowsettA. B,
DennisP. J,
LeeJ. V.,
KeevilC. W.
1994; Influence of plumbing materials on biofilm formation and growth of Legionella pneumophila in potable water systems. Appl Environ Microbiol 60:1842–1851
ScottK. P,
MercerD. K,
GloverL. A.,
FlintH. J.
1998; The green fluorescent protein as a visible marker for lactic acid bacteria in complex ecosystems. FEMS Microbiol Ecol 26:219–230
VessR. W,
AndersonR. L,
CarrJ. H,
BondW. W.,
FaveroM. S.
1993; The colonization of solid PVC surfaces and the acquisition of resistance to germicides by water micro-organisms. J Appl Bacteriol 74:215–221
WalkerJ. T,
MackernessC. W,
RogersJ.,
KeevilC. W.
1995; Heterogeneous mosaic biofilm – a haven for waterborne pathogens. In Microbial Biofilms pp 196–204 Edited by
Lappin-ScottH. M.,
CostertonJ. W.
Cambridge: Cambridge University Press;