Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary

STOTEN-20325; No of Pages 11 Science of the Total Environment xxx (2016) xxx–xxx

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Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary Christelle Schang a, Anna Lintern a, Perran L.M. Cook b, Catherine Osborne c, Anand McKinley a, Jonathon Schmidt d, Rhys Coleman e, Graham Rooney e, Rebekah Henry a, Ana Deletic a, David McCarthy a,⁎ a

Environmental and Public Health Microbiology Laboratory (EPHM Lab), Department of Civil Engineering, Monash University, Wellington Road, Clayton 3800, Victoria, Australia School of Chemistry, Monash University, Wellington Rd, Clayton 3800, Victoria, Australia Jomar Life Research, Melbourne, Victoria, Australia d ALS Environmental, Dalmore Drive, Scoresby 3179, Victoria, Australia e Melbourne Water Corporation, La Trobe Street, Docklands 3008, Australia b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Thermophilic Campylobacter spp. can survive for 14 days in the water column of microtidal estuaries. • Campylobacter spp. can survive for up 21 days in estuarine bed and bank sediments. • Inactivation rates of E. coli and Campylobacter spp. are similar in estuarine water.

a r t i c l e

i n f o

Article history: Received 17 April 2016 Received in revised form 24 June 2016 Accepted 24 June 2016 Available online xxxx Editor: D. Barcelo Keywords: Thermophilic Campylobacter E. coli Estuary Faecal contamination Presence Survival

a b s t r a c t Urban estuaries throughout the world typically contain elevated levels of faecal contamination, the extent of which is generally assessed using faecal indicator organisms (FIO) such as Escherichia coli. This study assesses whether the bacterial FIO, E. coli is a suitable surrogate for Campylobacter spp., in estuaries. The presence and survival dynamics of culturable E. coli and Campylobacter spp. are compared in the water column, bank sediments and bed sediments of the Yarra River estuary (located in Melbourne, Australia). The presence of E. coli did not necessarily indicate detectable levels of Campylobacter spp. in the water column, bed and bank sediments, but the inactivation rates of the two bacteria were similar in the water column. A key finding of the study is that E. coli and Campylobacter spp. can survive for up to 14 days in the water column and up to 21 days in the bed and bank sediments of the estuary. Preliminary data presented in this study also suggests that the inactivation rates of the two bacteria may be similar in bed and bank sediments. This undermines previous hypotheses that Campylobacter spp. cannot survive outside of its host and indicates that public health risks can persist in aquatic systems for up to three weeks after the initial contamination event. © 2016 Published by Elsevier B.V.

⁎ Corresponding author at: Department of Civil Engineering, Monash University, Wellington Road, Clayton 3800, Victoria, Australia. E-mail address: [email protected] (D. McCarthy).

http://dx.doi.org/10.1016/j.scitotenv.2016.06.195 0048-9697/© 2016 Published by Elsevier B.V.

Please cite this article as: Schang, C., et al., Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.06.195

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C. Schang et al. / Science of the Total Environment xxx (2016) xxx–xxx

1. Introduction Urban estuaries provide multiple functions to the community, including flood and disease mitigation, improved aesthetics and recreational opportunities (Findlay and Taylor, 2006). However, they are also under increased stress due to climate change, population growth and urbanisation, all of which can result in poor water quality. Faecal microorganisms are the leading cause of pollution in coastal waters and estuaries (Burton and Pitt, 2001) and faecal contamination is traditionally measured using faecal indicator organisms (FIO). FIO levels have not been linked to human health outcomes in estuarine environments (NHMRC, 2008). It is therefore necessary to develop a greater understanding of the behaviour of both FIO and potential pathogens in estuarine environments before we can assume that FIO levels in estuarine environments are an accurate indicator of potential human health risks. One concern regarding the use of FIO for assessing human health risks is that the survival of FIO might not necessarily be representative of the survival of faecal-derived pathogens in aquatic systems (Pachepsky and Shelton, 2011). Extended survival of faecal microbes in aquatic sediments depends on a large number of parameters such as seasonality, proximity to sources, temperature, organic matter, nutrients, and O2 and CO2 concentrations (Pachepsky and Shelton, 2011). Although there is evidence suggesting that FIOs can persist in the bed and bank sediments of tidally influenced systems, where they are sheltered from predation and inactivation (Solo-Gabriele et al., 2000; Desmarais et al., 2002; Ishii et al., 2006; Fries et al., 2008), the survival of most pathogens in estuarine bed and bank sediments is not well understood. As such, there is some uncertainty as to whether pathogens have similar survival patterns as FIO in estuarine environments. The possibility that pathogens will survive, even for a short time, in bed and bank sediments of urban estuaries presents a potential risk to recreational users. The water-sediment interface is not a static system, meaning that the sediments and the pathogens within these sediments can be easily resuspended by people wading or swimming, currents, storms, high winds, boats and dredging (Gerba and McLeod, 1976). In estuaries, the movement of the salt wedge and the rising tides can also resuspend bed and bank sediments and this process has been shown to lead to higher concentration of faecal microorganisms within the water column (Solo-Gabriele et al., 2000). It has also been demonstrated that resuspension of bed sediments during simulated storm events can release microbes into the overlying water columns (Kay and McDonald, 1980; Wilkinson et al., 1995). Therefore, better understanding of the survival rates of water-borne pathogens in estuarine beds and banks is necessary, not only to assess the adequacy of using FIO as surrogates for pathogenic organisms in aquatic systems, but also to inform faecal contamination management strategies within estuaries and improve the modelling of microbes in these systems. Water-borne pathogens known to occur in intertidal sediments are thermophilic members of the genus Campylobacter (Obiri-Danso and Jones, 1999; Obiri-Danso et al., 2001). They are the etiological agents of Campylobacterioris and are spread into the environment through the release of faecal material which may be directly or indirectly deposited into waterways (St-Pierre et al., 2009). Many have already linked Campylobacter spp. to illness in users of recreational waters (Schönberg-Norio et al., 2004; Olivieri, 2007; Hughes et al., 2009; Hughes and Gorton, 2013). Indeed, Schönberg-Norio et al. (2004) linked swimming in natural waters to sporadic Campylobacter infection. As such, Campylobacter jejuni is one of the three reference pathogens used when testing or challenging alternate urban water treatment systems (NRMMC–EPHC–NHMRC, 2009) and also one of the five reference pathogens used when conducting a quantitative microbial risk assessment (QMRA) for animal-impacted waters (USEPA, 2010). Although it is clear that Campylobacter is a significant water-borne pathogen and can be present in estuaries, very little is known about the dynamics of this pathogenic bacterium in estuarine environments. According to a

recent literature review conducted by Sterk et al. (2013), there are no known studies of Campylobacter spp. survival in estuarine bed or bank sediments. Furthermore, Campylobacter spp. has been demonstrated to enter a viable but not culturable (VBNC) state under stress conditions adding further uncertainty to quantitative analysis of complex environments (Thomas et al., 2002). Due to the human health risks posed by Campylobacter, there is an urgent need to better understand the survival dynamics of these pathogens in estuaries. This paper discusses the presence and survival of the two microorganisms, Escherichia coli and Campylobacter spp. in all components of an estuarine system: the water column, the bank sediments and the bed sediments. By doing so, this study aims to address the use of the FIO, E. coli, for assessing the level of a reference pathogen genus, Campylobacter spp., present in an estuary. The Yarra River estuary (Victoria, Australia) is used as a case study, as it is well recognized for elevated faecal microbe contamination (Nguyen, 2005; Fyfe, 2006; Cauchi, 2008; Ker, 2011; Arup, 2014; Gillett, 2014). This study not only demonstrates the suitability of using E. coli as a surrogate for Campylobacter spp., but also increases our understanding of Campylobacter spp. survival dynamics, which will assist in the management of Campylobacter contamination in estuaries. 2. Materials and methods 2.1. Study area Six sampling sites, with varying characteristics, (Table 1) were located along the Yarra River estuary, which flows through the city of Melbourne, Australia (Fig. 1). Water column, bank sediment and bed sediment samples collected from these sites were used to explore the presence and survival of E. coli and Campylobacter spp. This paper focuses on using culture-based methods; whilst this will not capture VBNC bacteria, it will describe viable bacteria which is considered essential for future use in quantitative microbial risk assessments (QMRAs). 2.2. Presence and survival of E. coli and Campylobacter spp. in the water column 2.2.1. Presence Between October 2010 and June 2011, 19 water samples were collected during dry and wet weather from the estuary at CS (Site 6). All samples were analysed for E. coli and Campylobacter. E. coli was analysed using a 1:10 dilution with Colilert® 24 h (IDEXX) as per AS4726.212005-Method 21 (Standards Australia, 2005). Campylobacter was quantified as per AS 4276.19:2001.11 using an 11 tube MPN with the following volumes: 5 × 10 mL, 5 × 100 mL and 1 × 500 mL (Henry et al., 2015). The detection limit for this test was b1 MPN/L. All microbial analyses were performed by ALS Environmental, which is a NATA accredited laboratory located in Melbourne, Australia. Additional FIOs and other potential pathogens were also enumerated, however these results are not discussed in this paper and both the assay methods and results are provided in Appendix A of the Supplementary material. 2.2.2. Survival To compare the survival dynamics of E. coli and Campylobacter in the water column of the Yarra River estuary, water column samples were collected in June and July 2011 at CS (Site 6). On both occasions, 30 L of water was sampled, transported to the laboratory and immediately distributed in 23 × 1 L and 14 × 250 mL sterile containers. Bottles were arranged in a circle and each received alternating 100 mL and 25 mL increments for 10 rotations, as described in McCarthy et al. (2008). After distribution, all samples (i.e. 23 × 1 L and 14 × 250 mL) were placed in a chamber with a constant temperature of 11 °C (the temperature of the river at the day and time of sampling) and rotated (samples inverted) at 30 rpm for 14 days. Although viable but not culturable cells

Please cite this article as: Schang, C., et al., Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.06.195

C. Schang et al. / Science of the Total Environment xxx (2016) xxx–xxx

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Table 1 Site location and characteristics for each of the experiments conducted in this study. Site

Ref.

Site name

Site characteristics

Experiment for which sample utilised

Sample collection date

E. coli

Campylobacter spp.

1

BR

Bridge Road, Richmond

Bed sediment

June 2011 to September 2011

✓

✓

2

SKC

St Kevins College Boathouse, Hawthorn

Bank sediment (presence)

20/4/2011 2/5/2011 9/5/2011 18/5/2011

✓

18/5/2011 and 9/5/2011 only

3

GR

Grange Road, Toorak

Tidally influenced area, with rare intrusion of salt wedge, medium recreational use. Predominately anoxic in low flows Tidally influenced area, more dynamic salt wedge intrusion (no intrusion during this study period), high recreational use. Medium vegetation, grass and reeds scattered across site. Upstream of Gardiners Ck Tidally influenced area, with salt wedge intrusion, high recreational use. Vegetated site, contains reeds and grass

✓

18/5/2011 only

4

5

6

WCB

RS

CS

Wesley College Boathouse, Toorak

River Street, South Yarra

Church Street, South Yarra

Tidally influenced area, with salt wedge intrusion, high recreational use. Sandy bank, experiences foot traffic. Tidally influenced area, with salt wedge intrusion, high recreational use. Rock layer, 500 mm under surface sediment Tidally influenced area, with salt wedge intrusion, high recreational use. Downstream of major stormwater drain.

can grow at this temperature, this study focussed on culturable cells only, as cell viability was more important to capture than VBNC for future use of this data (e.g. QMRA studies). Samples were randomly taken from the chamber on days 0, 1, 2, 3, 7, 10 and 14 during the incubation period. On each of these sampling days, five bottles (three 1 L samples and two 250 mL samples) were randomly selected from the chamber without replacement. Samples selected from the chamber were kept on ice and delivered to ALS Environmental and the Monash Water Studies Centre (both laboratories with NATA accredited protocols), and were analysed immediately upon arrival. As such, all samples

Bank sediment (presence) Bank sediment (survival) Bank sediment (presence) Bank sediment (survival) Bank sediment (presence) Bank sediment (survival) Water column (presence) Water column (survival)

9/5/2011 only ✓ 9/5/2011 and 18/5/2011 only ✓

October 2010 to June 2011 June 2011 to July 2011

18/5/2011 only

18/5/2011 only

18/5/2011 and 2/5/2011 only 18/5/2011 only

✓

✓

✓

✓

were analysed b 30 min after removal from the chamber. The sample on day 0 (i.e. the first set) was analysed within 2 h of collection from the river. It should be noted that due to logistic constraints, samples taken on day 0 for the June 2011 could not be analysed for Campylobacter. The temperature of the samples was measured as soon as they were taken out of the chamber (prior to transport), and once again upon arrival at the laboratory to ensure that the temperature of the water did not change significantly during transport on ice. Water samples were analysed for E. coli and Campylobacter spp. using the same methods described previously for the water column presence study. E. coli levels

Fig. 1. Site locations along the Yarra River estuary in Melbourne, Australia. Sites characteristics are provided in Table 1. Insert (a) shows a map of Australia with the state of Victoria filled in black. Insert (b) shows the state of Victoria with the Yarra River catchment filled in black.

Please cite this article as: Schang, C., et al., Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.06.195

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were analysed in all five sample replicates but due to the complexity of analytical procedures and due to the volume of water required, Campylobacter was quantified in the three replicate 1 L samples. Extra 1 L control replicates were taken out of the chamber on days 0 and 14 and analysed for suspended solids and nutrients (FRP, ammonia and NOx) (APHA-AWWA-WEF, 2005) using a Lachat Quick-Chem 8500, HACH, USA, temperature, pH, electrical conductivity (EC) and dissolved oxygen (DO) (multiprobe, Horiba, Japan), to identify the extent of water quality change over the 14 day period. This experiment was repeated in July 2011. E. coli and Campylobacter decay rates in the water column were estimated based on the log concentrations and using the die-off rate model described by Chick (1908). E. coli were compared to Campylobacter concentrations detected in the water column over time using the Spearman Rank Correlation Coefficient (α = 0.05), to determine the similarities in the survival trends of these two bacteria (Spearman, 2010). The Spearman Rank Correlation Coefficient was used due to the non-normality of the data (p b 0.05 with a Shapiro-Wilk test). 2.3. Presence and survival of E. coli and Campylobacter in the bank sediments 2.3.1. Presence To compare E. coli and Campylobacter concentrations present in estuarine bank sediments, bank sediment samples were collected at SKC (Site 2), GR (Site 3), WCB (Site 4) and RS (Site 5) at low tide on four occasions in 2011: April 20th, May 2nd, 9th and 18th (Fig. 1). During each of these four occasions, five samples were taken from each site. These samples were taken by equally dividing the distance between the waterline (where sediments are inundated most of the time; LOW) and a constant point of reference (where sediments are only briefly inundated during high tides; HIGH) into four sections (Fig. 2). The top 5 cm of bank sediments (approximately 500 g) at these five locations were collected using disposable sterile utensils, placed in zip-lock bags and transported on ice to the laboratory at 4 °C for immediate analysis. 10 g was immediately taken from each sample and used to estimate moisture contents (AS1289 B1.1-1977) (Standards Australia, 1977). A

further 5 g from each sample was used for E. coli analysis. These 5 g were placed into 100 mL deionised water with 0.05% Tween 80 (Sigma-Aldrich, USA), inverted for 10 min at 10–15 rpm, and left to settle for 10 min. 1 mL of the supernatant was then taken and E. coli levels were enumerated using the Colilert® technique as for the water column and bed sediment samples (AS4726.21-2005-Method 21) (Standards Australia, 2005). Campylobacter in bank sediments was analysed using AS4276.19:2001.11 with the following modifications: 10 g of bulk soil was resuspended in 1 L of de-ionised water and this water was processed for an 11 tube MPN using following filtered volumes: 1 × 50 mL, 5 × 10 mL and 5 × 1 mL. The detection limit of the assay was b0.1/g wet weight. E. coli concentrations were enumerated for all samples collected on all four occasions: 20/4/2011, 2/5/2011, 9/5/2011, and 18/5/2011. Due to the complexity of the assay, Campylobacter analysis was conducted on a fewer number of samples: (1) LOW, MID, HIGH samples taken from all sites (SKC; Site 2, GR; Site 3, WCB; Site 4 and RS; Site 5. Fig. 1) on 18/05/11; (2) a MID sample from RS (Site 5) on 2/05/2011 and (3) a MID sample from SKC (Site 2) on 9/05/11. Summary statistics of E. coli and Campylobacter concentrations detected at the LOW, MID and HIGH positions (Fig. 2) for all four sites on all four test dates were calculated. The concentrations of E. coli and Campylobacter were compared to moisture content and rainfall totals in the 24 and 48 h prior to sampling using the Spearman Rank Correlation Coefficient (α = 0.05) (Spearman, 2010). Rainfall totals at the Melbourne Regional Office Gauge were obtained from the Bureau of Meteorology (www.bom.gov.au). 2.3.2. Survival To compare the survival trends of E. coli and Campylobacter in bank sediments, approximately 500 g of bulk sediments were taken from the banks of the Yarra River at SKC (Site 2), GR (Site 3), WCB (Site 4) and RS (Site 5) (Fig. 1) at the LOW, MID and HIGH positions (Fig. 2). These sediments were kept in sterile and transparent bags and placed outside in a sheltered area covered by a clear, plastic roof. This allowed the sediment to be exposed to the environmental conditions (e.g., irradiation, temperature variations) observed in the field. However, we expect some experimental biases due to the fact that the amount of sediment diminished over time, and that the sediments were not exposed to the tidal cycle or rainfall. Approximately 10 g samples were taken from these bags using a sterile spatula roughly every second day, for 27 days. This study was conducted four times: the first two using sediment sampled from WCB (Site 4) on 09/05/2011 and 18/05/ 2011 and the third and fourth times using sediment sampled from GR (Site 3) and RS (Site 5) on 09/05/2011 and 18/05/2011 respectively. All samples (from the three positions and four sites) were analysed for E. coli and moisture content (using similar methods described above in the microorganism presence in bank sediments study). Campylobacter survival was tested at RS (Site 5) for the LOW and HIGH positions for the 18/05/2011 run. The trends in concentrations of E. coli and Campylobacter at each position on the four occasions were compared visually by plotting their concentrations over time to assess whether the E. coli and Campylobacter inactivation rates were similar. 2.4. Presence and survival of E. coli and Campylobacter in the bed sediments

Fig. 2. Sampling set-up along the river bank. The arrows represent where the samples were taken along the river bank at low tide for presence analysis. LOW, MID and HIGH indicate locations where the samples for survival analysis were taken from (the same relative locations have been used at all sampling sites: Sites 2–5 from Fig. 1). Distances between water level and reference point for each sampling location on each of the four sampling dates provided in Table B3 in the Supplementary materials.

2.4.1. Presence The presence of E. coli and Campylobacter in bed sediments was investigated by collecting 15 bed sediment cores from BR (Site 1) in June 2011 and again in September 2011. In June 2011 the cores were collected near the bank of the river (water depth of approximately 1.5 m) and in September 2011, they were obtained near the middle of the river (water depth of approximately 3.5 m). Clean and sterile Perspex columns were inserted approximately 200 mm deep into the bed sediment, trapping also 50 to 100 mm of water on top of the sediment

Please cite this article as: Schang, C., et al., Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.06.195

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surface. Once the columns were removed, a clean and sterile stopper was placed at the top and bottom of each of the 15 columns to prevent disturbance and mixing of the sample during transport. Once the cores were transported to the laboratory (within 1 h of collection), a clean and sterile spatula was used to sample the TOP (0– 10 mm), MID (10–20 mm) and BOT sediment (90–100 mm) of three cores. These samples were immediately analysed for E. coli, Campylobacter levels and moisture content. As with the water analyses, E. coli in the bed sediments was enumerated using the Colilert® technique (AS 4726.21-2005-Method 21) (Standards Australia, 2005). 1 g of bulk sediment was directly introduced to the 100 mL Colilert® vessel with 1 mL of 0.5% Tween 80 (final concentration in the bottle 0.05%, Sigma-Aldrich, USA) and rotated for 10 min at 10–15 rpm, prior to enumeration. For Campylobacter analysis, 10 g of soil (wet weight), harvested by centrifugation of the sludge, were resuspended initially into 1 L (in the June 2011 experiment) or 100 mL (in the September 2011 experiment) of reagent grade water and 100 mL was filtered through a 0.45 μm filter as described in the AS 4276.19:2001.11. The concentrated solutions were then analysed using an 11 tube MPN method using the following volumes: 1 × 50 mL, 5 × 10 mL and 5 × 1 mL. This method had a detection limit of b 10 MPN/L or b 0.1/g wet weight. Moisture content analysis was conducted using 10 g of bulk soil (AS1289 B1.1-1977) (Standards Australia, 1977). Summary statistics (minimum, geometric mean, maximum) of E. coli and Campylobacter concentrations in the TOP, MID and BOT samples for the June and September 2011 tests were calculated and compared visually to assess the presence of these two microorganisms in the bed sediments.

2.4.2. Survival To monitor the survival of E. coli and Campylobacter in the bed sediment of the estuary, 15 cores were collected from BR (Site 1) in June 2011 and again in September 2011, using the core collection and transportation methods described previously. These cores were in addition to the 15 cores collected for the bed sediment presence study. After 1 h of collection, the 15 cores were fully submerged in a water bath containing 200 L of water. This water was collected from the Yarra River at BR (Site 1), approximately 0.5 to 1 m below the water surface, immediately after sampling. The temperature of the water bath was maintained at between 10 and 13°, to ensure that it would be equivalent to the temperature measured on the day of sampling. Oxygenation of the water was maintained using aerators. The water in each Perspex column was gently mixed using a clean magnetic stirrer placed just above the sediment to mimic river conditions. These cores remained in the water bath for 27 days in the June 2011 experiment and for 21 days in the September 2011 experiment. Three cores were randomly sampled on days 0, 6, 10, 14, 21 and 27 days. On each of these occasions, TOP (0–10 mm), MID (10– 20 mm) and BOT (90–100 mm) sediment samples were taken from the cores and analysed for E. coli, Campylobacter and moisture content. The methods used for the enumeration were the same as those used for the bed sediment presence study above. To assess the impact of the experimental set-up on the cores, dissolved oxygen (DO) microprofiles of a core was undertaken at the start and end of the September 2011 bed sediment survival test using a Firesting oxygen microsensor system connected to a MUX3 micro manipulator (Pyroscience, Germany). E. coli decay rates were calculated for the TOP, MID and BOT samples from the sediment cores. Due to the lack of consistent detection, Campylobacter decay rates could not be calculated. Temporal change in E. coli concentrations between the initial concentration and the final concentration was also assessed using the t-test for unequal variances (α = 0.05). E. coli concentrations detected in sediment cores over time were compared to moisture content using the Spearman Rank Correlation Coefficient (α = 0.05) (Spearman, 2010).

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3. Results and Discussion 3.1. Presence and survival of E. coli and Campylobacter in the water column 3.1.1. Presence E. coli was detected in all 19 samples taken at CS (Site 6) between October 2010 and June 2011. There was a high variability in concentrations (110–9300 MPN/100 mL) and a median of 280 MPN/100 mL. Campylobacter was detected in 16 out of 19 samples taken at the same location and concentrations were much lower and less variable (b 1 to 35 MPN/L) and a median of 2.1 MPN/L. However, there is no statistically significant positive correlation between E. coli and Campylobacter concentrations (Rs = 0.368, p = 0.196). This could suggest that E. coli and Campylobacter densities in estuarine systems are governed by different processes. Similar results have been found in other studies of the Yarra River estuary. For example, Henry et al. (2015) did not observe statistically significant positive correlations between E. coli and Campylobacter in the estuary. 3.1.2. Survival Both E. coli and Campylobacter levels decreased with time, but were still detectable after 14 days at CS (Site 6, Fig. 3). Over these 14 days, the water quality of the estuarine water generally did not change except for a slight increase in DO, and decrease in ammonia and FRP, likely due to the microbial activity occurring in the water column (Søndergaard et al., 2003; Ferguson and Eyre, 2004). E. coli inactivation rates (proportion lost per day) in June 2011 and July 2011 were 0.10 and 0.17 log/day respectively, which is comparable to those previously described in the literature. For example, Blaustein et al. (2013) found E. coli die-off rates of 0.1 to 0.3 log/day in estuarine water and seawater at 10 °C. The inactivation rates for Campylobacter in the water from CS (Site 6) were lower, at 0.08 and 0.09 log/day in June 2011 and July 2011, respectively. These die-off rates in the water from CS (Site 6) are slightly higher than those found in literature for riverine environments (e.g. 0.06 log/day; (Thomas et al., 1999)). It is important to note, that the die-off rates reflect only the culturable population within the sediments and do not account for the formation of VBNC which could account for a further 5– 20% of the viable population (Thomas et al., 2002). We hypothesize that this difference is because CS (Site 6) experiences saline conditions, which is known to reduce the survival duration of Campylobacter (Doyle and Roman, 1982). The results also demonstrate a strong positive correlation between Campylobacter and E. coli in the two experiments (Rs = 0.76, p = 3.1 × 10−8). Thus, whilst their initial concentrations may differ, the inactivation trends of Campylobacter and E. coli in the water column follow similar trends. A key finding of this test is that thermophilic Campylobacter spp. can survive without any new or additional nutrients in the water column for 14 days. This raises concerns about health risks for recreational users of this estuary as it indicates that potential pathogens are still viable in the water column up to 14 days after they enter. As predation was not controlled in these experiments, we believe that these die-off rates provide a realistic representation of the survival and die-off dynamics of thermophilic Campylobacter spp. in microtidal estuaries. 3.2. Presence and survival of E. coli and Campylobacter in the bank sediments 3.2.1. Presence High levels of E. coli were detected at all four sampling sites and on all sampling days (Table 2). The average E. coli initial concentration, across all sites and positions, was approximately 6500 MPN/g dw and concentrations varied by over two orders of magnitude both spatially (by site) and temporally (approximately 250 to 36,000 MPN/g dw; Table 2). Similar to E. coli, Campylobacter concentrations were also elevated at all four sites on 18/5/2011 (Table 2). The average concentration was 25 MPN/g dw, and there was a high variability between sites and

Please cite this article as: Schang, C., et al., Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.06.195

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C. Schang et al. / Science of the Total Environment xxx (2016) xxx–xxx

Fig. 3. E. coli (left) and Campylobacter (right) concentration in the water column collected at CS (Site 6) in June 2011 (top) and July 2011 (bottom). When results were below the detection limit, concentration was taken as equal to half the detection limit of the method.

in E. coli concentrations with distance from the waterline do not match those of Campylobacter concentrations (Table 2 and Appendix B). On one hand, this may be indicating that differing factors may be affecting the presence of the two microorganisms in bank sediments. However, it is also possible that this lack of correlation is due to the sampling

over time, with Campylobacter concentrations being below detection at some sites, but reaching up to 90 MPN/g dw (at RS; Site 5 on 02/05/ 2011; Table 2). The variability in E. coli and Campylobacter concentrations by site are similar; highest concentrations were at site SKC (Site 2), and the lowest were at site WCB (Site 4). However, the trends

Table 2 Summary statistics of the initial concentration of E. coli and Campylobacter and moisture content recorded across the bank section at each of the four sites (Wesley College Boathouse, River Street, Grange Road and St Kevins College) and for all four sampling days (20/04/2011, 02/05/2011, 09/05/2011 and 18/05/2011).

WCB

RS

GR

SKC

Test date

Bank length [m]

20/04/2011 02/05/2011 09/05/2011 18/05/2011 20/04/2011 02/05/2011 09/05/2011 18/05/2011 20/04/2011 02/05/2011 09/05/2011 18/05/2011 20/04/2011 02/05/2011 09/05/2011 18/05/2011

4 2.8 5.6 5.2 2.4 0.6 1.2 2.4 4 2.8 4 2.4 3.2 0.8 3 5

E. coli [MPN/g dry]

Campylobacter [MPN/g dry]

Moisture content [%]

Mean

Min

Max

St dev

Low

Mid

High

Mean

St dev

4520 432 1014 7913 11,771 4398 2707 19,175 5714 3284 2743 8350 6900 4459 2656 20,234

894 265 259 4390 10,388 3980 1472 8669 2257 510 426 1662 4699 2952 550 7936

10,681 614 2196 18,030 12,342 4795 3551 36,074 12,248 3905 8867 17,972 9257 7962 6051 32,305

3854 125 722 5746 818 369 860 10,506 4134 1345 3460 6545 1972 2127 2136 8986

– – – b2 – 90 – 15 – – – 21 – – – 86

– – – 10 – – – b3 – – – 2 – – b2 30

– – – 7 – – – b3 – – – 28 – – – 63

41 38 34 51 60 61 60 66 54 49 48 54 52 51 47 64

14 8 11 12 1 1 1 4 5 4 6 5 6 5 8 6

“–” indicates when the samples were not analysed. “b” indicates the results were less than the detection limit in MPN/g dry.

Please cite this article as: Schang, C., et al., Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.06.195

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methodology employed. Only one sample was taken from each location and position, and it is possible that the general trend in Campylobacter was not adequately captured in these single samples. Further studies involving multiple sampling from each location and position are required to better capture potential spatial variability and identify whether the E. coli and Campylobacter have similar trends across banks. There is a strong positive correlation between E. coli concentrations (at all positions and sites over time), and sediment moisture content (Rs = 0.65, p b 0.01; Supplementary material Appendix B). Soil moisture content is known to impact the growth of E. coli, which ceases to grow at water potential of −4.6 MPa and below this water potential, it can only survive for 2 days (Potts, 1994). This may explain why E. coli levels at WCB (Site 4) were lower than other sites (Table 2). In addition, the sediments at WCB (Site 4) was sandier than other sites and literature demonstrates that sand is less conducive to bacterial colonisation both because of its lower ability to retain moisture and fewer adsorption sites (Craig et al., 2004). However, it should be noted that E. coli levels may also be affected by the proximity to sources. For example SKC, which has the highest E. coli concentrations is proximal to a known source of E. coli (Gardiners Creek) (Jovanovic et al., 2015). The relationship between Campylobacter levels in bank sediments and sediment moisture content is less clear (Rs = 0.57, p = 0.085). Whilst there is a positive correlation between the two variables, this correlation is not statistically significant. On one hand, this could merely be a problem with statistical power and low sample numbers. On the other, it is possible that soil moisture is not a key factor in influencing Campylobacter levels in sediments. Instead, the proximity of bank sediments to sources of Campylobacter may also be a factor influencing Campylobacter levels in sediments. Highest concentrations (out of all sampled sites) were generally observed at SKC (Site 2), which is immediately downstream of Gardiners Creek (Fig. 1), a known source of Campylobacter (data not shown, Henry et al., 2015). WCB (Site 4) had the lowest Campylobacter concentrations, with all of its samples below 10 MPN/g dry. The reason for this is not clear, but it is possible that similar to E. coli, the sandy nature of the banks at this site could also be influencing Campylobacter colonisation due to its lower moisture retention and fewer adsorption sites. Temporal variability of E. coli concentrations in bank sediments are likely to be influenced by antecedent rainfall conditions. There were significant negative correlations between E. coli levels and rainfall totals from the previous 24 and 48 h (Rs = −0.64; p b 0.05; Supplementary materials Appendix B). These correlations were marginally stronger for samples taken near the waterline (Supplementary materials Appendix B). This relationship may be due to rain washing E. coli from the river banks into the main river channel, and if so, estuary banks could also be a source of E. coli. The factors underlying the temporal variability of Campylobacter could not be investigated as only samples collected on 18/05/2011 were assessed for Campylobacter at all four sites. 3.2.2. Survival Fig. 4 and Table 3 show that E. coli is able to survive in bank sediments that are not supplemented with moisture or extra nutrients, and exposed to natural temperatures and radiation. Some variability in survival rates were observed, with E. coli levels remaining constant for at least 5 to 7 days in bank sediments sampled on the 9th of May 2011 (Fig. 4a and b) whilst E. coli levels were maintained for over 20 days in the bank sediments taken on the 18th of May 2011 (Fig. 4c and d). These temporal differences in survival patterns (by site), could be due to the antecedent conditions of the bank sediment before collection. Samples where E. coli decayed faster had lower initial moisture contents (Table B3). The decay rates also appear to be affected by distance from the waterline, with sediments furthest from the waterline (5.6 m for WCB and 4 m for GR) maintaining higher E. coli concentrations during dieoff (Fig. 4a and b). It is possible that when E. coli are inundated less frequently, they develop desiccation resistance mechanisms which

7

enables them to survive longer in challenging conditions. SoloGabriele et al. (2000) hypothesised that in the outer fringe of the bank, where the drying conditions are the most extreme, E. coli are able to survive due to its capacity to outcompete its predators in relatively dry soils. This also suggests that the type of E. coli present in the outer fringe of the banks are strains that have become naturalised to the environment and hence may not be from recent contamination. This trend was not observed at RS on the 18th of May 2011, probably because the duration of the experiment was not sufficient to capture decay in any of the configurations, let alone any differences in decay patterns. Table 3 indicates that, Campylobacter is able to survive in bank sediments for over 27 days. In fact, Table 3 shows higher concentrations of Campylobacter after 27 days in some cores. In particular, Table 3 indicates that both bacteria were detected at high levels at 12 days. This undermines the hypotheses that reference pathogens such as thermophilic Campylobacter (in particular, C. jejuni and Campylobacter coli) are unable to survive outside their hosts for long periods of time (Jones, 2001). On most sampling days, the samples taken closer to the waterline have higher Campylobacter concentrations compared to those taken further from the waterline (Table 3; p = 0.07 in paired Student's t-test). Although the Campylobacter concentrations over time do not follow a similar trend to E. coli for the RS sediment collected on 18/05/2011 (Table 3), because both bacteria were found to survive in the bank sediments for at least 12 days it is possible that E. coli survival may be a surrogate for Campylobacter spp. However, further experimental work, with a larger number of samples taken from each position and location on each day is required to confirm these findings. 3.3. Presence and survival of E. coli and Campylobacter in the bed sediment 3.3.1. Presence E. coli concentrations had a maximum of 3000 MPN/g dw and Campylobacter a maximum of 17 MPN/g dw (Table 4). Both microorganisms were found in higher concentrations in the top 20 mm of the bed sediments and were often not detected below 90 mm. Desmarais et al. (2002) and Hathaway et al. (2011) also reported decreasing E. coli concentrations with sediment core depth. This is to be expected as the concentration of live microbes are generally low below 100 mm in terrestrial soils (Sait et al., 2002; Sangwan et al., 2005). It is possible that depths of 90–100 mm within the sediment cores are unfavourable for bacterial survival and growth of the bacteria that was deposited months or years previously where there may be less nutrients and organic matter (Pachepsky and Shelton, 2011). The September 2011 cores had lower concentrations for both E. coli and Campylobacter (Table 4). This may be due to spatial or temporal variability in E. coli and Campylobacter levels in the sediments, as the September 2011 cores were taken further away from the bank than the June 2011 cores. Both E. coli and Campylobacter had lower concentrations in the bottom of the cores, and also exhibited lower concentrations in September 2011. This suggests that spatial (vertically and horizontally) and temporal trends in E. coli levels in bed sediments could be reflective of overall trends in Campylobacter levels. However, Campylobacter was not always detected in the bed sediment cores even when E. coli was present. 3.3.2. Survival Although the initial E. coli concentrations differ between June and September 2011, both tests showed similar trends in E. coli concentrations over time (Fig. 5). In addition, whilst the trends in sediment samples from 0–10 mm and 10–20 mm were similar, the concentrations in the bottom layer (90–100 mm) were consistently below the detection limit. Bed sediments from the top 20 mm of the cores showed a slight increase in E. coli concentration within the first 7 days of incubation, followed by a gradual decrease. The initial increase is likely to be associated with settling of E. coli from the overlying water column onto the

Please cite this article as: Schang, C., et al., Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.06.195

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Fig. 4. E. coli survival in bank sediments of the Yarra River estuary following collection of samples on the 9th May 2011 at WCB (a) and GR (b) and the 18th May 2011 at WCB (c) and RS (d). The distance in the legend represents the sampling location from the waterline. For 9th May 2011, when no E. coli was detected with the Colilert® method after day 12 at WCB, the value was taken as equal to half the detection limit (0.5 MPN/g).

bed sediment. After N20 days of testing, E. coli concentrations in the bed sediment were still above the detection limit. The overall E. coli inactivation rates in the TOP layers were 0.01 log/ day (June 2011 cores) and 0.03 log/day (September 2011 cores). These were around ten times lower than the rates observed in the water column (Fig. 3), indicating that E. coli can survive longer in the sediment than in the water column. In the MID layer, the overall decay rates were similar to that of the TOP layers at 0.04 log/day (June 2011 cores) and 0.02 log/day (September 2011 cores). These results show that the E. coli levels remained quite constant, with minimal die-off over time in both layers throughout both testing periods (t-test p N 0.05), confirming what is generally described in the literature for tidally influenced systems (Desmarais et al., 2002; Craig et al., 2004; Fries et al., 2008; Pachepsky and Shelton, 2011).

Table 3 E. coli and Campylobacter concentration in bank sediments from River Street collected on 18th May 2011. ‘Low’ refers to samples taken closest to the waterline and ‘High’ refers to samples taken furthest from the waterline. Concentrations are based on one single measurement each day. Day

0 7 9 12 20 27 ‘NT’ - nontested.

E. coli [MPN/g dry]

Campylobacter [MPN/g dry]

Low

High

Low

High

20,850 442 NT 2940 3227 NT

12,879 4956 NT 2773 2709 NT

15 93 28 266 b0.9 21

b1.4 b1.2 b1.1 263 b0.9 78

Campylobacter was detected ten times during the June 2011 test and only three times during the September 2011 test (Table 4). Due to the lack of detection of Campylobacter at many instances during the experiments, it was difficult to identify a clear temporal trend in Campylobacter levels, and estimate inactivation rates at any of the sampling points. As such, future work should focus on repeating survival studies of Campylobacter in estuarine bed sediments using lower detection levels and further replicates. However, regardless of this inability to identify clear trends, the survival data demonstrates that there were detectable levels of Campylobacter in the top 20 mm of bed sediment, even after 27 days. In addition, on the last day (day 27) of both tests (day 27 of the June 2011 test, and day 13 of the September 2011 test) both E. coli and Campylobacter were detected. As such, both organisms are able to survive outside of their hosts for extended periods of time. This suggests that E. coli survival in estuarine bed sediments may be representative of that of Campylobacter, but further confirmation of this relationship is required. Campylobacter was detected at a lower frequency and at lower concentrations in the September 2011 study, which is to be expected because of the different seasons in which the tests were conducted. The June 2011 study was conducted during the Australian autumn and winter, where the outside temperature was rarely above 20 °C and the temperature of the overlying water column was below 15 °C – temperatures known to favour the survival of both E. coli (An et al., 2002) and Campylobacter (Obiri-Danso and Jones, 2000). Furthermore, it would be expected that the E. coli and Campylobacter concentrations would differ between the two seasons due to varying rainfall rates in winter and spring, and the resulting variations in the levels of bacteria entering the estuary (e.g., Jovanovic et al., 2015). Additionally, as the June 2011 and September 2011 cores were taken from two different locations in the river cross-section, the different survival patterns in the June and

Please cite this article as: Schang, C., et al., Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.06.195

C. Schang et al. / Science of the Total Environment xxx (2016) xxx–xxx

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Table 4 E. coli and Campylobacter concentrations in the bed sediment cores collected in June and September 2011 from Bridge Road (BR, Site 1). The concentration per gram of dry sediment was calculated using the detection limit value adjusted with the moisture content. Sampling day

Position in core

E. coli [MPN/g dry]

Campylobacter [MPN/g dry]

June 2011

Day 0

Day 6

Day 13

Day 21

Day 27

Top (0–10 mm) Mid (10–20 mm) Bot (90–100 mm) Top Mid Bot Top Mid Bot Top Mid Bot Top Mid Bot

September 2011

June 2011

September 2011

Core 1

Core 2

Core 3

Core 1

Core 2

Core 3

Core 1

Core 2

Core 3

Core 1

Core 2

Core 3

764

3424

1538

154

146

50

3.7

24.1

0.1

b0.3

b0.3

b0.2

993

1433

424

87

124

35

b2.4

b1.7

b1.6

b0.2

0.2

0.5

b261

b177

b229

b33

b16

21

b3

b1.9

b2.4

b0.2

b0.1

b0.2

4510 11,759 b364 1724 1352 21 3076 1453 b17 1231 2271 b9

10,810 34,938 b530 4150 4214 68 1984 20,686 7 326 1155 b8

2556 12,716 b226 6289 3561 b17 1845 773 b10 1181 1737 b13

324 105 b21 105 82 b18 31 14 b13 NT NT NT

122 115 b10 90 41 b12 27 22 b12 NT NT NT

205 118 b23 128 23 b14 14 34 b9 NT NT NT

0.4 b6.1 b3.8 b1.8 b1.1 b2.3 2.1 b0.2 b0.1 b0.1 0.2 b0.1

b1.4 b7.3 b5.7 0.8 0.6 b2.8 b0.2 b0.2 b0.1 b0.2 0.1 b0.1

b1.2 b5.3 b2.3 1.0 b2.1 b2.0 b0.2 b0.2 b0.1 b0.2 b0.2 b0.1

b0.2 b0.2 b0.2 b0.2 b0.2 b0.2 NT NT NT NT NT NT

b0.2 b0.2 b0.1 b0.3 b0.2 b0.1 NT NT NT NT NT NT

b0.3 b0.2 b0.3 0.4 b0.1 b0.2 NT NT NT NT NT NT

‘NT’ - nontested.

September 2011 experiments may be reflecting micro-spatial variation in Campylobacter. To identify the influence of the DO levels on E. coli and Campylobacter survival and presence, the DO micro-profile within the bed sediment was recorded on day 7 and day 21 in the September 2011 experiment. There was a sharp depletion of oxygen in the top 5–7 mm of the sediment core (Fig. 6) and below this depth, anoxic conditions were observed. These DO profiles however only partially explain the stratification seen along the depth of the cores (i.e. lower concentrations at 90–100 mm depth compared to 0–20 mm depth). With E. coli and Campylobacter present below 7 mm depth, this suggests that even in anoxic conditions, the environmental conditions were sufficient to enable survival (Pachepsky and Shelton, 2011). In fact, Campylobacter may prefer anoxic conditions to oxic conditions, as optimal growth occurs in microaerophilic conditions. 3.4. Uncertainties in the presence and survival studies There were some limitations in the experimental methodology, which may be affecting the presence and survival trends in E. coli and Campylobacter. Firstly, the presence and survival studies discussed above had a limited number of repeats and samples. For example, when sampling bank sediments, only one sample was taken from each

Fig. 5. Average E. coli concentration and standard deviation in the bed sediment samples collected from the Yarra River at Bridge Road in June 2011 (blue and red) and September 2011 (green and purple). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

location and position. Similarly, for the bed sediment study, one sample was taken from each position in each core. Given the uncertainties in the enumeration of Campylobacter (Henry et al., 2015), in future studies it would be advisable to employ a larger number of repeats and samples. Secondly, seasonality in the Campylobacter and E. coli presence and survival trends was not investigated in this study. The water column survival studies were conducted in June and July 2011 (winter), the bank sediment presence and survival studies were conducted in April and May 2011 (autumn), and the bed sediment presence and survival studies were conducted in June and September 2011 (winter and spring). As such, further work is required to identify if presence and survival trends differ seasonally. For example, whilst it was identified that E. coli and Campylobacter inactivation rates are similar in the water column during winter months (June and July 2011), it is possible that they may differ in the summer months, as the temperature of the Yarra River can vary up to 12 degrees between the summer and winter (Bruce et al., 2014). Finally, the Campylobacter enumeration methods employed had high detection limits. Detection limits are generally high and variable when culturing bacteria from environmental samples, especially when techniques used to culture microbes from drinking water are used (Hugenholtz et al., 1998). This may be contributing to the lack of

Fig. 6. Dissolved oxygen (DO) depth micro-profile within the bed sediment cores collected from the Yarra River at Bridge Road (Site 1), in September 2011 on day 7 (blue) and day 21 (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Schang, C., et al., Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.06.195

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consistency in Campylobacter spp. concentrations identified, particularly in the bed sediments presence and survival experiments. Furthermore, owing to the higher number of tubes used to estimate the Most Probable Number of E. coli (97 vs. 11 for Campylobacter spp.), these results inherently have lower uncertainty (Russek and Colwell, 1983; McBride et al., 2003), which may explain the more consistent results for E. coli. We recommend that this work be repeated with lower detection limits, and a higher number of multiple tubes, of Campylobacter to better assess the presence and survival trends between E. coli and Campylobacter.

4. Conclusions The objectives of this study were to first compare the presence and survival patterns of E. coli and Campylobacter in the water column, bed and bank sediments of an urban estuary and, secondly, to identify whether E. coli was a suitable indicator for Campylobacter. We found that in the water column, Campylobacter is not always detected even when E. coli is present. Despite this discrepancy, E. coli survival rates in the water column over a 14-day period were comparable to that of Campylobacter, which suggests that E. coli survival rates in the water column could be used as an indicator or a proxy of Campylobacter survival. Spatial trends in E. coli levels in sediments across the cross section of the estuary banks did not correlate to those of Campylobacter. As such, it is possible that the presence of the two microorganisms is being influenced by different factors such as naturalisation and previous exposure. In the bed sediments E. coli and Campylobacter exhibited similar vertical profiles (a decreasing trend) down through the cores as well as similar spatio-temporal variability. However, the presence of E. coli did not always indicate detectable levels of Campylobacter in the cores. The extended survival of both bacteria in the bed and bank sediments suggests that E. coli survival rates might be similar to that of Campylobacter. We identified that both bacteria were able to survive for at least 21 days (in June 2011) and 13 days (in September 2011) in the bed sediments. However, the data did not provide conclusive evidence as to whether E. coli presence and survival is representative of Campylobacter in estuarine sediments. Further experiments, involving a larger number of samples, taken in different seasons, and a Campylobacter enumeration method with lower detection limits are required to confirm these findings. The critical importance of accurately and easily identifying Campylobacter in estuaries was highlighted in this study by the finding that this microorganism can survive outside of its host in estuarine environments for at least two weeks in the water column, and for at least three weeks in bed and bank sediments. The extended survival of Campylobacter in sediments is particularly of concern, as these sediments could act as sources of pathogen contamination when they are resuspended by tidal movements, high flows or anthropogenic activities in the river (e.g. boating). As such, further work is required to identify the external environmental influences and factors (e.g. wetting and drying cycles of bank sediments, proximity to sources, and exposure to temperature variations) that affect Campylobacter presence and survival, so that we can develop alternative strategies for monitoring aquatic systems for this pathogen for the protection of public health.

Acknowledgements We would like to acknowledge the contributions made by the Australian Research Council and Melbourne Water through the Linkage Program (LP120100718) for this work. The authors also acknowledge the contribution of the Monash University Water Studies Centre, Keralee Brown, Vera Eate, Peter Kolotelo and Richard Williamson in the collection and laboratory analysis of samples and Peter Bach for his help with producing Fig. 1.

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Please cite this article as: Schang, C., et al., Presence and survival of culturable Campylobacter spp. and Escherichia coli in a temperate urban estuary, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.06.195