Survival of animal and human associated Listeria monocytogenes in drinking water and biofilms

Land slugs are occasionally observed as contaminants in groundwater wells and drinking water treatment plants including storage tanks. Slugs may feed on carrion and feces, and they are potential vectors of pathogens such as Listeria monocytogenes . We isolated L. monocytogenes from the pest slug Arion vulgaris and examined the persistence and survival of human-and slug-derived L. monocytogenes in groundwater-based drinking water and bio ﬁ lms. L. monocytogenes survival was evaluated using cultivation and cultivation-independent techniques. L. monocytogenes remained culturable for 35 – 47 days in drinking water with ﬁ rst-order decay rates between 0.314 and 0.457 h (cid:1) 1 ( T 99 (cid:3) 10 days). Attachment of L. monocytogenes to ﬁ lter sand delayed washout from drinking water ﬁ lters and increased persistence 2 – 3-fold. Indigenous bio ﬁ lms stimulated initial surface attachment 10 – 100-fold but L. monocytogenes declined more rapidly in drinking water bio ﬁ lms compared with virgin ﬁ lters not colonized by microorganisms. Grazing by protozoa likely attenuated L. monocytogenes survival in some drinking water bio ﬁ lms. A comparable survival pattern was observed for L. monocytogenes and the fecal indicator bacterium E. coli . The study suggests that live L. monocytogenes can persist for weeks as sessile organisms in groundwater-based drinking water supplies and may subsequently be released to the drinking water.


INTRODUCTION
A. vulgaris is active during periods with high humidity and during the night (Cameron ).A. vulgaris prefers to hide in dark and moist places, and seeks protection in moist and frost-protected places during winter (Cameron ).A. vulgaris can range in size from >10 cm to <1 mm and this and other land slugs can penetrate protective barriers less than a few mm (Figure 1).As a result, A. vulgaris can be observed in many anthropogenic habitats and sometimes as a contaminant in urban and rural water utilities.A. vulgaris has been shown to be a potential vector for L. monocytogenes and this pathogen has been detected in slug mucus and feces at concentrations of 10-1,205 CFUs per g slug biomass (Gismervik et al. ).
Because A. vulgaris is a potential vector for Listeria it is likely that this and other pathogens can be transmitted to drinking water if the water utility is compromised by slugs.
Listeria monocytogenes is a facultative anaerobic Grampositive, non-spore-forming psychrotrophic bacterium that is relatively resistant to adverse environmental conditions (Ivanek et  In the present study, we investigated the persistence of slug-and human-associated L. monocytogenes strains in municipal drinking water produced from groundwater.
Flow systems were used to examine the fate and survival

Isolation and growth of bacteria
The Listeria strains used in the drinking water experiments are listed in Table 1.Six Listeria strains were isolated from the pest slug A. vulgaris (Figure 1). A. vulgaris was collected in areas with drinking water abstraction and slug samples    incubated in the dark at 120 rpm on a rotary shaker at 10 C.

Enumeration of bacteria
Gas and liquid sampled from the microcosms were analyzed for 14 CO 2 .Gas samples of 1 mL were injected into scintillation vials containing an 8 mL CO 2 trap (ethylene glycol monomethyl ether and ethanolamine 7:1; Merck, Germany).Vials were shaken whereafter a 10 mL scintillation cocktail was added (Hionic Fluor; PerkinElmer ® , USA).The 14 CO 2 in the liquid samples was evacuated and trapped after acidification.
The radioactivity was quantified using a Packard 1600 Tri-Carb Liquid Scintillation Analyzer (Packard, NL).Counts were corrected for quench using external standards.

Data analysis and statistics
Comparisons of differences in survival between Listeria strains and between different detection methods were carried out using the nonparametric Mann-Whitney U test (Wilcoxon rank sum test) using KaleidaGraph 4.5.4 (Synergy Software, USA).Drinking water microcosms were subsequently spiked with L. monocytogenes from the pest slug (Arion vulgaris), a strain from poultry (DSM 12464), a strain from rabbit (DSM 20600), and a human clinical isolate (SS-ENK).

RESULTS AND DISCUSSION
One of the spiked L. monocytogenes strains (DSM 20600) lost culturability after only nine days in drinking water whereas the three other strains remained culturable for up to 35-47 days (Figure 3).L. monocytogenes DSM 20600 is a laboratory strain (type strain) originally isolated in 1926, and this bacterium may subsequently have lost some ability for survival under nutrient-poor conditions resulting in relatively poor survival in drinking water.The first-order decay rate for L. monocytogenes DSM2060 was 0.703 day À1 .In contrast, decay rates for L. monocytogenes SS-ENK, L. monocytogenes DSM12464 and L. monocytogenes slug1 were 0.457, 0.425 and 0.314 day À1 , respectively.The decay rates for the latter three L. monocytogenes strains correspond to T99 values of 10-15 days in drinking water (i.e., the time for a 99% decrease in CFU) (Figure 3).Turnover of 14 C-labelled L. monocytogenes and E. coli was three and ten times greater in microcosms with an indigenous microflora (filter material with biofilm and drinking water) compared with microcosms without native drinking water microorganisms (Figure 6).In experiments with E. coli, the turnover of 14 C-labelled bacterial cells was also Contamination of water resources and drinking water with small animals and animal waste may result in transmission of live pathogens (Levy ; Bitton ; Hynds et al. ; Stokdyk et al. ).Gastropods such as land slugs are occasionally observed as contaminants in groundwater wells and drinking water storage tanks and as unwelcome intruders at drinking water treatment plants.Slugs often feed on a variety of organic food items and are also known to ingest feces from animals and humans as part of their diet (coprophiles).Several potential pathogens have been observed in the intestinal tract and on the exterior surface of land slugs including Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Clostridium perfringens, Clostridium botulinum and Listeria monocytogenes (Elliot ; Sproston et al. ; Gismervik et al. , ).The land slug Arion vulgaris (formerly Arion lusitanicus) is considered a major pest in many regions of Europe and the population has increased significantly in the last 20 years (Pfenninger et al. ; Zemanova et al. ).
al. ; Gandhi & Chikindas ).L. monocytogenes is widespread in the environment including soil, water and feces, and it has many known animal reservoirs (Ivanek et al. ; Hellberg & Chu ).L. monocytogenes is a pathogen and the causative agent of listeriolosis and other illnesses in animals and humans.Listeriolosis is associated with a relatively high case-fatality rate in humans, and because the mortality rate can be high, many countries have a zero tolerance for this bacterium in relation to food and water (Gião & Keevil ; Pandove et al. ).L. monocytogenes has multiple environmental transmission routes and the prevention of infection of animals and humans is complex and challenging (Ivanek et al. ).Interestingly, the environmental occurrence and survival of L. monocytogenes may increase with future climate changes (Hellberg & Chu ).However, little is known about the persistence of L. monocytogenes in many types of drinking water and in drinking water biofilms.Indigenous drinking water biofilms have been implicated as environmental reservoirs or safe havens for intruding pathogens (Keevil ; Wingender & Flemming ; Bitton ).
of L. monocytogenes in drinking water sand filters with and without biofilms.Radiolabeling of Listeria cells was used to study cell turnover, and results for L. monocytogenes were compared with results for the traditional fecal pollution indicator E. coli.

Figure 2 |
Figure 2 | Drinking water filter system for studying survival and attachment of L. monocytogenes and E. coli to filter quartz sand with and without an indigenous drinking water biofilm.
Presence and survival of L. monocytogenes in drinking water Source water from five rural drinking water treatment plants was examined for the presence of Listeria spp.The water supplies use groundwater as source water and employ conventional water treatment consisting of aeration and sand filtration.Disinfection is not part of the operating procedures at these treatment plants.Presumptive Listeria was not detected in water volumes <0.01 L whereas analysis of 1-4 L resulted in detection of presumptive Listeria in four out of five rural water supplies (0.3-90 CFU/L).This initial screening was not intended to be exhaustive but it indicated that low concentrations of Listeria were detectable and this suggested a potential for occurrence of Listeria in rural water supplies.It was not possible to establish the origin of the Listeria but it is known that rural groundwater is sometimes prone to contamination with live invertebrates and waste from wildlife and farm animals (Levy ; Bitton ; Hynds et al. ; Stokdyk et al. ).Survival of L. monocytogenes in drinking water was examined by spiking human and animal strains into drinking water microcosms at 10 C (Figure 3).The drinking water used in the microcosms was municipal drinking water from groundwater and 10 C is a typical temperature in such systems.The standard operating procedure in this municipality advocates distribution of water to consumers without water treatment or disinfection (non-treated pristine groundwater).The drinking water contained no detectable coliform bacteria or intestinal enterococci (<1 CFU/ 100 mL), and the heterotrophic plate count was <10 CFU/ mL. Background concentrations of L. monocytogenes were not detected in the microcosms (<1 CFU/1,000 mL).

Figure 3
Figure3| (a) Survival of different L. monocytogenes strains from animals and humans in drinking water at 10 C and (b) the time estimated for a 99% decrease in CFU (T99).See Table1 for Figure 5 | Recovery of L. monocytogenes DSM 12464 from the surface of filter material in drinking water filters without biofilm (white bars) and filter material with an established drinking water biofilm (black bars) using (a) cultivation-based enumeration and (b) FISH-based enumeration.Data represent means of triplicate samples ± standard deviation.

Figure 4 |
Figure 4 | Concentrations of (a) L. monocytogenes DSM 12464 and (b) E. coli ATCC 2522 in the effluent from drinking water filters without biofilm (open symbols) and filter material with an established drinking water biofilm (closed symbols).The theoretical washout curve is shown as a hatched line.Data represent means of triplicate experiments ± standard deviation.Some error bars are contained within the symbols.

Figure 6
Figure 6 | (a) Fate of 14 C-labelled L. monocytogenes DSM 12464 and (b) E. coli ATCC 25922 inoculated into drinking water microcosms measured as production of 14 CO2.(a) L. mono- cytogenes in filter sterilized drinking water with sterile filter material (open circles), L. monocytogenes in drinking water with indigenous microflora and sterile filter material (filled circles), L. monocytogenes in drinking water with indigenous microflora and biofilm-covered filter material (filled squares).(b) E. coli in filter sterilized drinking water with sterile filter material (open circles), E. coli in drinking water with indigenous microflora and sterile filter material (filled circles), E. coli in drinking water with indigenous microflora and biofilm-covered filter material (filled squares).Data represent means of triplicate experiments.

16 dH) abstracted directly from chalk aquifers. Limited water treatment is employed by this municipality before dis-
followed by distribution without disinfection.Aeration followed by filtration in open or closed sand filters without subsequent disinfection represent the standard operating procedure for the vast majority of municipal and private waterworks in Denmark.For microcosm experiments, municipal drinking water was obtained from Aalborg Municipality (Denmark).The source water was non-disinfected hard groundwater (12-

Table 1 |
Origin of the Listeria strains used in drinking water experiments Difco, USA) and ciprofloxacin at a final concentration of 1.0 μg mL À1 (Sigma-Aldrich, Germany).
Culturable Listeria in environmental samples were analyzed in triplicates by membrane filtration of 1-4 L groundwater using 0.45 μm mixed cellulose ester filters (Millipore, USA).The filters were transferred to sterile pads (Millipore, USA) soaked with Fraser Broth (Sigma-Aldrich), and resuscitated for 18 h at 30 C followed by a transfer to Brilliance Listeria agar with selective and differential supplements as described above.Listeria plates were incubated at 36 C and examined after 48 h.L. monocytogenes using a modified version of the method described by Besnard et al. ().Samples were filtered onto black polycarbonate membrane filters (0.2 μm pore size; Poretics Products, USA).Filters were placed on pads soaked with Brain Heart Infusion Broth containing 1.0% yeast extract (mately 5 × 10 5 cells mL À1 ) or 14 C-labelled E. coli (approximately 1 × 10 6 cells mL À1 ).The total amount of radioactivity was approximately 1.0 × 10 4 Bq per microcosm with 14 C-labelled L. monocytogenes and 1.7 × 10 4 Bq per microcosm inoculated with 14 C-labelled E. coli.Microcosms were Downloaded from http://iwaponline.com/ws/article-pdf/21/7/3515/960330/ws021073515.pdf by guest