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Investigating the decay rates of Escherichia coli relative to Vibrio parahemolyticus and Salmonella Typhi in tropical coastal waters
w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 6 1 e1 5 7 0
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Investigating the decay rates of Escherichia coli relative to Vibrio parahemolyticus and Salmonella Typhi in tropical coastal waters Choon Weng Lee a,*, Angie Yee Fang Ng a, Chui Wei Bong a, Kumaran Narayanan b, Edmund Ui Hang Sim c, Ching Ching Ng a a
Laboratory of Microbial Ecology, Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia School of Science, Monash University, Sunway Campus, Selangor, Malaysia c Department of Molecular Biology, Faculty of Resource Science and Technology, University Malaysia Sarawak, Malaysia b
article info
abstract
Article history:
Using the size fractionation method, we measured the decay rates of Escherichia coli, Salmo-
Received 24 June 2010
nella Typhi and Vibrio parahaemolyticus in the coastal waters of Peninsular Malaysia. The size
Received in revised form
fractions were total or unfiltered, <250 mm, <20 mm, <2 mm, <0.7 mm, <0.2 mm and <0.02 mm.
18 November 2010
We also carried out abiotic (inorganic nutrients) and biotic (bacterial abundance, production
Accepted 19 November 2010
and protistan bacterivory) measurements at Port Dickson, Klang and Kuantan. Klang had
Available online 27 November 2010
highest nutrient concentrations whereas both bacterial production and protistan bacterivory rates were highest at Kuantan. We observed signs of protistebacteria coupling via the
Keywords:
following correlations: Protistan bacterivoryBacterial Production: r ¼ 0.773, df ¼ 11, p < 0.01;
Bacterial decay rate
ProtistBacteria: r ¼ 0.586, df ¼ 12, p < 0.05. However none of the bacterial decay rates were
Size fractionation
correlated with the biotic variables measured. E. coli and Salmonella decay rates were
Top-down control
generally higher in the larger fraction (>0.7 mm) than in the smaller fraction (<0.7 mm) sug-
Straits of Malacca
gesting the more important role played by protists. E. coli and Salmonella also decreased in the
South China sea
<0.02 mm fraction and suggested that these non-halophilic bacteria did not survive well in seawater. In contrast, Vibrio grew well in seawater. There was usually an increase in Vibrio after one day incubation. Our results confirmed that decay or loss rates of E. coli did not match that of Vibrio, and also did not correlate with Salmonella decay rates. However E. coli showed persistence where its decay rates were generally lower than Salmonella. ª 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Coastal waters account for less than 10% of the ocean area. However they are highly productive and account for 25% of primary production in the ocean (Berger et al., 1989). Coastal waters are increasingly exploited by humans for food, recreation, transport and other needs, and at present most are in various stages of degradation (Alongi, 1998). There is also an increasing public health threat from pathogens (Hazen and
Toranzos, 1990; Moe, 1997). Disposal of inadequately treated waste is considered faecal pollution and a main source of bacterial pathogens in the sea (Solo-Gabriele et al., 2000). For faecal pollution studies, the concept of bacterial indicator is standard (Wolf, 1972). A fundamental assumption to this concept is the parity in the survival of indicator and enteric pathogens over a wide range of aquatic environments (Bonde, 1977). It is however acknowledged that these indicators are inadequate to predict the presence of pathogenic
* Corresponding author. E-mail address: [email protected] (C.W. Lee). 0043-1354/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2010.11.025
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microorganisms (Rhodes and Kator, 1988; Kay et al., 1994; Borrego and Figueras, 1997). At present, the coliform group of microorganisms or microorganisms found in the intestines of all warm blooded animals especially Escherichia coli is used as a standard indicator of faecal contamination in many countries (Hazen and Toranzos, 1990) including Malaysia (Department of Environment, 2008). The value of E. coli as an indicator microorganism is significantly enhanced by the ease with which it can be detected and cultured (Wolf, 1972) when compared with other bacterial pathogens. In order to use E. coli as an indicator bacterium in seawater, knowledge of its survival must be acquired as it is not a halophilic microorganism. Loss rates of indicator microorganisms pose an interesting problem especially in coastal waters as many factors affect the survival times of E. coli for example temperature, light, salinity, predation, nutrients and pollutants (Fujioka et al., 1981; Munro et al., 1989; Presser et al., 1998; Solo-Gabriele et al., 2000; Rozen and Belkin, 2001; Sinton et al., 2002). Since the 1950s, Carlucci and Pramer (1959; 1960a; 1960b) have studied the survival of bacteria including E. coli in seawater. Numerous studies have shown that decay rates of E. coli do not reflect Vibrio cholerae in estuarine waters and also Salmonella spp. (Colwell et al., 1981; Rhodes and Kator, 1988). However to the best of our knowledge, there is no study that compares the relative loss rates of E. coli with both halophilic and non-halophilic bacterial pathogens. In this study, we investigated whether the decay rates for E. coli were similar to a non-halophilic bacterial pathogen (i.e. Salmonella Typhi) and a halophilic bacterial pathogen (i.e. Vibrio parahaemolyticus)? Our results could help properly evaluate the risk posed by such bacteria either to the health of bathers in recreational waters or to the safety of fisheries or marine aquaculture.
2.
Materials and methods
2.1.
Sampling
We sampled at three stations located on the east (Kuantan: 03 48.40 N 103 20.60 E) and west (Klang: 03 00.10 N 101 23.40 E and Port Dickson: 02 29.50 N 101 50.30 E) of Peninsular Malaysia from April until October 2006 (Fig. 1). The stations at Kuantan and Klang were located in estuaries whereas the station at Port Dickson was sandy coast. Surface seawater samples were collected during high tide, using an acid-cleaned bucket, and in-situ measurements of temperature (0.1 C) and salinity (0.1 ppt) were carried out using a salinometer (YSI-30, US). Seawater samples were then kept in a cooler box for no more than 4 h until processing in the laboratory. In the laboratory, seawater samples for dissolved nutrient analyses were filtered through pre-combusted (450 C for 5 h) Whatman GF/F filters, and stored at 20 C until analysis.
2.2.
Environmental conditions
Dissolved inorganic nitrogen (nitrate (NO3), nitrite (NO2), ammonium (NH4)), and phosphate (PO4) concentrations were measured using a spectrophotometer (Parsons et al., 1984). All nutrient measurements above were carried out in triplicates.
Fig. 1 e Map showing the location of the sampling sites, east (Kuantan) and west (Klang and Port Dickson) of Peninsular Malaysia.
Coefficient of variation (CV) for NH4, NO2 and PO4 analyses were <5%, and <10% for NO3 analysis. Bacteria and protist were determined using the direct count method by an epifluorescence microscope (Olympus BX60, Japan) with a U-MWU filter cassette (excitor 330385 nm, dichroic mirror 400 nm, barrier 420 nm). For protist, 10 ml sample was filtered onto a black 0.8 mm pore size Isopore filter (Millipore, Ireland), and then stained with the fluorochrome primulin (40 mg ml1 final concentration) for 5 min (Bloem et al., 1986) whereas for bacteria, 2 ml sample was filtered onto a black 0.2 m pore size Isopore filter, and then stained with 40 6diamidino-2-phenylindole (DAPI, 1 mg ml1 final concentration) for 10 min (Kepner and Pratt, 1994). Slides were kept frozen for < 3 days before enumeration. A minimum of 10 microscope fields or 500 cells were counted for bacteria and for protist, at least 30 microscope fields were observed.
2.3.
Bacterial decay rates
For bacterial decay experiments, seawater samples were sizefractionated as total or unfiltered, <250 mm (through a 250 mm stainless steel mesh), <20 mm (20 mm pore size nylon mesh), <2 mm (2.0 mm polycarbonate membrane filter), <0.7 mm (GF/F filter), <0.2 mm (0.2 mm polycarbonate membrane filter) and <0.02 mm (0.02 mm Whatman Anodisc). The size fractions were inoculated separately with 1% (v/v) fresh cultures of E. coli, Salmonella Typhi (hereafter referred to as Salmonella) and V. parahaemolyticus (hereafter referred to as Vibrio), and then incubated at 30 C for about three days. Both E. coli and Salmonella inocula were prepared on nutrient broth whereas
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for Vibrio, 3% NaCl (final concentration) was added to the nutrient broth. From the inoculated size fractions, the initial E. coli, Salmonella and Vibrio concentration was determined as colony forming unit (cfu) ml1 via spread plating. The number of cfus was observed one day after spread plating on MacConkey agar (for E. coli and Salmonella) and Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar with 3% NaCl (for Vibrio). A portion of the sample was also removed every day to determine the change in E. coli, Salmonella and Vibrio concentration. Bacterial decay rate was modeled by the standard differential equation: dN/dt ¼ kN (Chick, 1908) where k ¼ decay constant, N is the cfu ml1, and t ¼ time of incubation. Integrating the differential equation gave the following ln N ¼ kt þ C. Hence for decay rate assessment, we transformed the cfu data by natural logarithm and plotted the ln cfu ml1 against incubation time. Linear regression analysis was then used to find the best-fit slope i.e. decay rate. We also measured separately, the concurrent bacterial production and protistan bacterivory rates by measuring bacterial growth rate in both <0.7 mm and <20 mm fractions after 12 h incubation. Bacterial growth rate in each fraction was calculated as the increase in natural logarithmic bacterial abundance over time. The bacterial growth rate in the <0.7 mm fraction was assumed without grazing (m0.7) whereas growth rate in the <20 mm fraction (m20) was the product of both growth and grazing. Bacterial production (BP) was then estimated by the following equation: BP ¼ Bacterial abundance m0.7 (Lee et al., 2009a) whereas bacterivory rate was estimated by Bacterial abundance (m0.7 m20) (McManus, 1993).
2.4.
Statistical analyses
Statistical tests such as coefficient of variation (CV), analysis of variance, Student’s t-test, Tukey’s test, linear regression and correlation analyses were carried out according to Zar (1999). Count data were log-transformed to meet parametric assumptions of equality of variances and normal distribution before correlation and linear regression analyses. All data, unless mentioned otherwise, were reported as mean S.D.
3.
Results
3.1.
Abiotic measurements
Table 1 shows the physico-chemical parameters measured at the three stations. Average surface seawater temperature
observed ranged from 29 to 30 C whereas average salinity measured at the estuaries (Klang and Kuantan) was lower than at Port Dickson. Salinity fluctuated over a wider range at the estuaries (CV ¼ 7% and 46%) than Port Dickson (CV ¼ 1%). Dissolved inorganic nitrogen (DIN) measured showed that concentrations at Klang were more than two-fold higher than both Kuantan and Port Dickson. Among the nitrogen species, NH4 was the dominant species at both Klang and Kuantan, accounting for > 70% of DIN. At Port Dickson, NO3 was the dominant species (about 45% of DIN). Average PO4 concentration at Klang was also higher than at both Port Dickson and Kuantan.
3.2.
Biotic measurements
Bacterial abundance ranged from 0.85 to 3.49 106 cell ml1, and was significantly higher at Kuantan than both Klang (q ¼ 4.77, df ¼ 12, p < 0.05) and Port Dickson (q ¼ 5.77, df ¼ 12, p < 0.01) (Table 2). For protist, abundance ranged from 0.58 to 6.64 103 cell ml1, and was about three orders lower than bacteria. Protist abundance at Kuantan was significantly higher than Port Dickson (q ¼ 4.34, df ¼ 12, p < 0.05) but not Klang. Table 2 also shows the bacterial production measured in this study. Bacterial production at both Klang and Port Dickson ranged from 0.72 to 1.69 105 cell ml1 h1 whereas bacterial production at Kuantan ranged from 1.99 to 4.78 105 cell ml1 h1. Similar to patterns exhibited by microbial abundance data, bacterial production at Kuantan was significantly higher than both Klang (q ¼ 4.69, df ¼ 12, p < 0.05) and Port Dickson (q ¼ 5.79, df ¼ 12, p < 0.01). Protistan bacterivory at Kuantan was also higher than both Klang (q ¼ 14.48, df ¼ 9, p < 0.001) and Port Dickson (q ¼ 13.64, df ¼ 9, p < 0.001). Bacterivory rates at Kuantan ranged from 1.60 to 2.15 105 cell ml1 h1 whereas bacterivory rates at both Klang and Port Dickson were about one order lower, and ranged from 1.11 to 4.19 104 cell ml1 h1. E. coli concentration from 1990 until 2005 were also obtained from the Department of Environment Malaysia monitoring stations located near our study area. Although E. coli concentration varied over four-order at all three locations (Fig. 2), analysis of variance showed significant differences among the different locations (F ¼ 19.2, df ¼ 1095, p < 0.001). At Port Dickson and Klang, average E. coli was 1300 3600 MPN (Most Probable Number) per 100 ml, and was significantly higher than Kuantan (640 1900 MPN per 100 ml) (KlangeKuantan: q ¼ 8.52, df ¼ 1095, p < 0.001; Port DicksoneKuantan: q ¼ 6.38, df ¼ 1095, p < 0.001).
Table 1 e Physico-chemical characteristics of the sampling stations in this study. Mean (±S.D.) of surface water temperature, salinity, ammonium (NH4), nitrite (NO2), nitrate (NO3), and phosphorus (PO4). Station Klang (n ¼ 4) Port Dickson (n ¼ 5) Kuantan (n ¼ 5)
Temperature C
Salinity ppt
NH4 mM
NO2 mM
NO3 mM
PO4 mM
30.3 0.5 30.0 1.0
27.7 1.9 28.4 0.3
17.84 24.75 0.42 0.51
2.97 0.64 0.18 0.27
1.24 0.61 0.50 0.28
1.66 1.54 0.12 0.05
29.1 0.8
22.4 10.2
2.89 2.64
0.51 0.29
0.61 0.33
0.60 0.31
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Table 2 e Bacterial abundance, protist abundance, bacterial production and protistan bacterivory rates measured in this study. Date
Bacteria (106 cell ml1)
Protist (103 cell ml1)
Bacterial production (105 cell ml1 h1)
Protistan bacterivory (104 cell ml1 h1)
Klang
24-Apr-06 12-Jun-06 26-Jun-06 31-Jul-06 Average (S.D.)
1.13 0.93 1.29 1.53 1.22 0.26
2.89 4.08 2.57 1.08 2.65 1.23
1.69 0.97 1.66 1.64 1.49 0.35
1.55 1.11 3.70 2.77 2.28 1.18
Port Dickson
04-Jul-06 03-Oct-06 10-Oct-06 17-Oct-06 30-Oct-06 Average (S.D.)
1.10 0.85 0.91 1.11 0.98 0.99 0.12
2.43 1.16 2.01 0.58 1.73 1.58 0.73
e 0.72 1.25 1.36 1.08 1.10 0.28
e e 3.41 4.19 1.95 2.26 2.07
Kuantan
18-Apr-06 17-May-06 19-Jun-06 25-Jul-06 15-Aug-06 Average (S.D.)
2.91 1.31 2.19 2.40 3.49 2.46 0.82
3.02 1.76 5.42 6.64 5.51 4.47 2.01
4.78 1.99 3.60 2.50 2.84 3.14 0.11
21.45 e 16.05 e 16.00 17.83 3.13
Station
3.3.
Bacterial decay rates at different size fractions
Fig. 3 shows the change in E. coli concentration after inoculation in the different fractions of seawater collected from Port Dickson, Kuantan and Klang. There was an apparent decrease in E. coli in most of the larger fraction seawater (i.e. total, <250 mm, <20 mm, <2 mm) whereas in the smaller fraction (i.e. <0.7 mm, <0.2 mm and <0.02 mm), the concentration of E. coli often did not change significantly. Fig. 4 shows the statistically significant ( p < 0.05) decay rates measured in this study, and confirmed that E. coli decay rate was significantly higher in the larger fraction (>0.7 mm) (2.28 0.90 d1) than the smaller fraction (<0.7 mm) (0.58 0.22 d1) (Student’s t-test: t ¼ 12.35, df ¼ 64, p < 0.001). A similar trend was observed for Salmonella where its concentration generally decreased with time, especially in the larger fraction seawater (Fig. 5). Salmonella decay rates in the larger fraction (3.17 1.19 d1) were significantly higher than in the smaller fraction (1.51 0.84 d1) (Student’s t-test: t ¼ 6.65, df ¼ 59, p < 0.001) (Fig. 4). In contrast to E. coli and Salmonella, Vibrio exhibited a different trend (Fig. 6). There was often an increase in Vibrio concentration after one day incubation, and less discernible difference in Vibrio concentration between the larger and smaller fractions of seawater. Few of the experiments gave significant decay rates (Fig. 4), and available Vibrio decay rates ranged 0.90e1.78 d1 at Klang, and 0.54e4.64 d1 at Kuantan. At Port Dickson, only two significant decay rates were observed (1.36 0.21 d1). When we compared E. coli decay rates in the larger fractions among the stations, E. coli decay rate was highest at Klang (1.96e4.90 d1), followed by Kuantan (0.88e2.80 d1) and Port Dickson (0.36e2.93 d1) (KlangeKuantan: q ¼ 6.55, df ¼ 51, p < 0.001; KlangePort Dickson: q ¼ 9.99, df ¼ 51, p < 0.001; KuantanePort Dickson: q ¼ 3.44, df ¼ 51, p < 0.05). However
Fig. 2 e Long term E. coli counts (log MPN/100 ml) from selected Department of Environment monitoring stations near Port Dickson (PD) (n [ 578), Klang (n [ 196) and Kuantan (n [ 322).
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Fig. 3 e E. coli decay (ln cfu mlL1) measured in different fractions at Port Dickson (PD) (n [ 5), Klang (n [ 3) and Kuantan (n [ 5).
there was no significant difference in Salmonella and Vibrio decay rates among the stations.
4.
Discussion
4.1.
Environmental conditions
Surface seawater temperature observed was typical of tropical waters. Klang had the highest nutrient concentrations, and reaffirmed earlier observations (Lee and Bong, 2006, 2008; Lee et al., 2009a). One possible reason is the rapid pace of
development and industrialization taking place upstream of Klang where the capital of Malaysia, Kuala Lumpur is located (Lee and Bong, 2006). In this study, the abundance of both bacteria and protist were within the range for coastal waters of Peninsular Malaysia (Lee et al., 2005; Lee and Bong, 2007, 2008). Protist abundance was about three orders of magnitude lower than bacteria, and this observation was consistent with the analysis by Sanders et al. (1992). Both bacterial production and protistan bacterivory rates measured in this study were also within the range previously published for tropical coastal waters (Lee et al., 2005, 2009a; Lee and Bong, 2006, 2007, 2008).
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Fig. 4 e Summary of decay rates (P < 0.05) measured in this study at Klang, Port Dickson (PD) and Kuantan. Different symbols represent different sampling dates. Error bar denotes the standard error of decay rate and is shown except when smaller than symbol.
However, in contrast to the pattern exhibited by nutrient concentrations where Klang had the highest levels, all biotic variables were highest at Kuantan. It was not clear why biotic variables at Klang were not reflective of its nutrient concentrations as found in earlier studies (Lee and Bong, 2008; Lee et al., 2009a). However both Klang and Kuantan are estuaries, and can experience episodic nutrient inputs that can stimulate microbial biomass and productivity. Long term monitoring data showed the extent of faecal pollution via the indicator E. coli. Although coastal water quality in Malaysia has deteriorated over time (Department of Environment, 2008), there was no apparent increase in E. coli over the 15-year period for all three locations. At present, Malaysia has an interim marine water quality standard that set the criterion for E. coli at 100 MPN per 100 ml (Department of Environment, 2008). From a 15-year period of monitoring data, >60% of the samples at Klang and Port Dickson exceeded the standard whereas at Kuantan, 35% of the samples exceeded the standard. Dow (1995) had earlier reported higher E. coli concentration for stations along the Straits of Malacca. Our study showed that faecal pollution remains a problem for coastal waters here. One reason is many coastal communities in Malaysia lack proper sewage disposal systems and often discharge sewage directly into the sea (Law, 1992). The faecal pollution is accentuated for stations along the Straits of Malacca as the population density is higher along the west coast of Peninsular Malaysia. Although sewage treatment facilities have increased over time, the problem showed no sign of alleviation at both Klang and Port Dickson.
4.2.
Bacterial decay rates at different size fractions
The decay rates obtained in this study were within the range reported by Anderson et al. (2005) but relatively higher than decay rates reported for temperate waters (Lessard and Sieburth, 1983; Rhodes and Kator, 1988). Relative to temperate waters, the higher decay rates obtained in this study could be due to the fact that microbial activity (including protistan bacterivory) in tropical waters is at its optimum (Pomeroy and Wiebe, 2001). For both E. coli and Salmonella, decay rates were generally higher in the larger fraction (>0.7 mm) than in the smaller fraction (<0.7 mm). The main bacterial predators in the larger fractions are nanoflagellates and ciliates (Sanders et al., 1992) whereas the cause of bacterial mortality in the smaller fraction is mainly by viral lysis (Fuhrman, 2000). In the smaller fraction, lytic bacteria may also play a role albeit a minor one (Enzinger and Cooper, 1976). Bacterial decay rates measured in this study suggested that protistan bacterivory was more important, similar to the conclusion by Enzinger and Cooper (1976). A reason why viral lysis played a minor role in this study was because both Salmonella and E. coli are not natural seawater organisms and are not active in the sea (Carlucci and Pramer, 1960b). Bacteriophages require the host physiological activity in order to replicate (Pretorius, 1962). The decay rates in the <0.02 mm fraction is effectively not due to viral lysis or bacterivory as both viruses and protists do not pass through this pore size. The <0.02 mm fraction is therefore suitable to observe the response of halophilic and
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Fig. 5 e Salmonella decay (ln cfu mlL1) measured in different fractions at Port Dickson (PD) (n [ 4), Klang (n [ 3) and Kuantan (n [ 4).
non-halophilic pathogens in seawater. We observed that Vibrio counts in the <0.02 mm fraction increased after day one in all the experiments, and no significant decay of Vibrio could be observed throughout this study. In contrast, the decay rates for E. coli and Salmonella in the <0.02 mm fraction were still statistically significant ( p < 0.05). Our results concurred with earlier reports that non-halophiles are usually not able to grow well in seawater (Gerba and McLeod, 1976). However relative to protistan bacterivory, the decay rates for E. coli and Salmonella in the <0.02 mm fraction were low, and accounted for <20% of total decay rates. These decay rates also did not correlate ( p > 0.15) with salinity, suggesting that bacterivory was more important than salinity for non-halophilic bacterial decay. There were obvious differences in the response of Vibrio in the different seawater fractions relative to both E. coli and Salmonella. In this study, significant Vibrio decay was seldom observed, and rates observed were usually lower than E. coli and Salmonella. One reason why Vibrio decay rates were lower was probably due to the ability of V. parahaemolyticus to grow in a salty environment (Holt et al., 1994). Increase in Vibrio was still apparent after one day incubation before Vibrio counts started decreasing. The latter reduction could be due to other stresses for example limited food availability that resulted in Vibrio growth rates falling below loss rates.
E. coli is widely used as an indicator for faecal pollution and for pathogenic microorganisms (Bonde, 1977). However the validity of E. coli as an indicator is questionable especially in coastal waters (Solo-Gabriele et al., 2000) where presence of E. coli is more likely the balance between supply and loss. This is because E. coli is non-halophilic and is not known to grow well in coastal waters (Gerba and McLeod, 1976). Our results confirmed that decay or loss rates of E. coli did not match that of the halophilic Vibrio and also did not correlate significantly with Salmonella decay rates ( p > 0.50) (Table 3) even though the survival characteristic of E. coli is presumed similar to Salmonella (Bonde, 1977). When we compared the different bacterial responses in the larger fractions, we found that at Klang, Salmonella and E. coli decay rates were significantly higher than Vibrio (SalmonellaeVibrio: q ¼ 6.58, df ¼ 33, p < 0.001; E. colieVibrio: q ¼ 6.16, df ¼ 33, p < 0.001) whereas at Kuantan, Salmonella decay rate was significantly higher than both Vibrio (q ¼ 7.70, df ¼ 46, p < 0.001) and E. coli (q ¼ 4.92, df ¼ 46, p < 0.01). Similarly at Port Dickson, Salmonella decay rate was higher than E. coli (Student’s t-test: t ¼ 4.10, df ¼ 29, p < 0.001). For Port Dickson, only decay rates from Salmonella and E. coli were compared as there were too few decay rates from Vibrio experiments. Generally, we found that Salmonella decay rates were higher
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Fig. 6 e Vibrio decay (ln cfu mlL1) measured in different fractions at Port Dickson (PD) (n [ 3), Klang (n [ 3) and Kuantan (n [ 3).
than E. coli. The persistence of E. coli relative to Salmonella fulfilled one of the criteria for an indicator organism i.e. the indicator organism should survive longer than the pathogen itself (Cabelli, 1978; Allwood et al., 2003). Although our study provided snapshots of the environment, we showed consistently the importance of protistan bacterivory in bacterial decay. E. coli was a poor indicator for halophilic pathogens, and although E. coli decay rates did not correlate with Salmonella, E. coli persisted longer than Salmonella in coastal waters. Our results provided support to the continuous use of E. coli as an indicator organism for nonhalophilic pathogens especially Salmonella.
4.3.
Protistebacteria coupling
Multiple correlation analysis showed that E. coli, Salmonella and Vibrio decay rates did not correlate with each other or other biotic variables (Table 3). Protistan bacterivory also did not correlate with E. coli, Salmonella and Vibrio decay rates. As these pathogens form only a minor fraction of the total bacterial population in the sea (Lee et al., 2009b), their decay rates played only a minor role towards total bacterivory rates. However, we did observe evidences of significant coupling between protist
and bacteria similar to Sanders et al. (1992) and Lee et al. (2005) (Protistan bacterivoryeBacterial Production: r ¼ 0.773, df ¼ 11, p < 0.01; ProtisteBacteria: r ¼ 0.586, df ¼ 12, p < 0.05). Our study showed that protistan bacterivory accounted for 9e56% of bacterial production (Table 2), and was significantly higher at Kuantan (>44% bacterial production) than both Klang (q ¼ 9.68, df ¼ 9, p < 0.001) and Port Dickson (q ¼ 6.68, df ¼ 9, p < 0.01).
Table 3 e Pearson productemoment correlation coefficient (r) between variables measured i.e. bacterial production (BP, cell mlL1 hL1), log bacterial abundance (BA, cell mlL1), grazing (cell mlL1 hL1), log Protist (cell mlL1), decay rates of E. coli (dL1), Salmonella (dL1), and Vibrio (dL1). * is P < 0.05, ** is P < 0.01, *** is P < 0.001. BP
BA
BA 0.896*** Grazing 0.773** 0.794*** Protist 0.546* 0.586* E. coli 0.214 0.120 Salmonella 0.174 0.082 Vibrio 0.129 0.135
Grazing Protist E. coli Salmonella
0.295 0.291 0.120 0.064
0.190 0.121 0.077 0.276 0.040
0.106
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Bacterivory rates at both Klang and Port Dickson accounted for only 9e31% bacterial production. When bacterivory was less than bacterial production, other factors might have played a role in the removal of bacterial production as bacterial abundance is stable and does not change significantly with time (Lee and Bong, 2008). Among the factors that are important include viral lysis (Fuhrman, 2000), removal by benthic filter feeders (Strom, 2000), and sedimentation (Pedro´s-Alio´ and Mas, 1993). Sedimentation is often overlooked as a loss factor for bacteria due to the low sinking rates of microorganisms. However sinking speed can increase for bacteria attached to particles and there might be significant losses by sedimentation (Pedro´s-Alio´ and Mas, 1993). Although these loss factors were not investigated here, studies on viral ecology in tropical waters have suggested that viral lysis may not be an important loss factor (Bettarel et al., 2006; Cissoko et al., 2008). Moreover the higher suspended solids in the coastal waters of Peninsular Malaysia (Bong and Lee, 2008; Lee et al., 2009b) implied that sedimentation is probably more important. However further investigations are needed.
5.
Conclusion
1. Bacterial decay in tropical coastal waters is mainly due to protistan bacterivory. 2. Via the <0.02 mm fraction, our results showed that E. coli and Salmonella do not survive well in seawater. In contrast, Vibrio grows well in seawater. Therefore E. coli is a poor indicator for halophilic pathogens. 3. E. coli decay rates do not correlate with both Salmonella and Vibrio decay rates. However E. coli persists longer than Salmonella in coastal waters. Our results provided support to the continuous use of E. coli as an indicator organism for non-halophilic pathogens especially Salmonella. 4. There is protist‒bacteria coupling where protist counts correlated with bacterial abundance, and protistan bacterivory correlated with bacterial production.
Acknowledgements We are grateful to the Department of Environment, Malaysia for providing the monitoring data of E. coli concentration. Funding for this research was provided by University of Malaya (RG064-09SUS) and Ministry of Science, Technology & Innovation (06-01-03-SF0457). The researchers would also like to thank anonymous reviewers who helped improve the manuscript and University of Malaya for providing the research facilities.
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Alongi, D.M., 1998. Coastal Ecosystem Processes. CRC Press, Boca Raton, FL. Anderson, K.L., Whitlock, J.E., Harwood, V.J., 2005. Persistence and differential survival of fecal indicator bacteria in subtropical waters and sediments. Applied and Environmental Microbiology 71 (6), 3041e3048. Berger, W.H., Smetacek, V.S., Wefer, G., 1989. Ocean productivity and paleoproductivityean overview. In: Berger, W.H., Smetacek, V.S., Wefer, G. (Eds.), Dahlem Workshop on Productivity of the Ocean: Present and Past. John Wiley & Sons, Chichester, pp. 1e34. Bettarel, Y., Bouvy, M., Dumont, C., Sime-Ngando, T., 2006. Virusbacterium interactions in water and sediment of West African inland aquatic systems. Applied and Environmental Microbiology 72 (8), 5274e5282. Bloem, J., Ba¨r-Gilissen, M.J.B., Cappenberg, T.E., 1986. Fixation, counting, and manipulation of heterotrophic nanoflagellates. Applied and Environmental Microbiology 52, 1266e1272. Bonde, G.J., 1977. Bacterial indication of water pollution. Advances in Aquatic Microbiology 1, 273e364. Bong, C.W., Lee, C.W., 2008. Nearshore and offshore comparison of marine water quality variables measured during SESMA 1. Malaysian Journal of Science 27 (3), 25e31. Borrego, J.J., Figueras, M.J., 1997. Microbiological quality of natural waters. Microbiology SEM 1, 169e180. Cabelli, V., 1978. New standards for enteric bacteria. In: Mitchell, R. (Ed.), Water Pollution Microbiology. John Wiley & Sons, New York, pp. 233e271. Carlucci, A.F., Pramer, D., 1959. Factors affecting the survival of bacteria in sea water. Applied and Environmental Microbiology 7, 388e392. Carlucci, A.F., Pramer, D., 1960a. An evaluation of factors effecting the survival of Escherichia coli in sea water. II. Salinity, pH and nutrients. Applied and Environmental Microbiology 8, 247e250. Carlucci, A.F., Pramer, D., 1960b. An evaluation of factors effecting the survival of Escherichia coli in sea water. IV. Bacteriophages. Applied and Environmental Microbiology 8, 254e256. Chick, H., 1908. An investigation of the laws of disinfection. Journal of Hygiene 8, 92e158. Cissoko, M., Desnues, A., Bouvy, M., Sime-Ngando, T., Verling, E., Bettarel, Y., 2008. Effects of freshwater and seawater mixing on virio- and bacterioplankton in a tropical estuary. Freshwater Biology 53, 1154e1162. Colwell, R.R., Seidler, R.J., Kaper, J., Joseph, S.W., Garges, S., Lockman, H., Maneval, D., Bradford, H., Roberts, N., Remmers, E., Huq, I., Huq, A., 1981. Occurrence of Vibrio cholerae serotype O1 in Maryland and Louisiana estuaries. Applied and Environmental Microbiology 41, 555e558. Department of Environment, 2008. Malaysia Environmental Quality Report 2007. Department of Environment, Ministry of Natural Resources and Environment, Malaysia. Dow, K.M., 1995. An overview of pollution issues in the Strait of Malacca. MIMA Issue Paper 5 (95), 1e34. Enzinger, R.M., Cooper, R.C., 1976. Role of bacteria and protozoa in the removal of Escherichia coli from estuarine waters. Applied and Environmental Microbiology 31, 758e763. Fuhrman, J.A., 2000. Impact of viruses on bacterial processes. In: Kirchman, D.L. (Ed.), Microbial Ecology of the Oceans. WileyLiss, New York, pp. 327e350. Fujioka, R.S., Hashimoto, H.H., Siwak, E.B., Young, R.H.F., 1981. Effect of sunlight on survival of indicator bacteria in sea water. Applied and Environmental Microbiology 41, 690e696. Gerba, C.P., McLeod, J.S., 1976. Effects of sediments on the survival of Escherichia coli in marine waters. Applied and Environmental Microbiology 32, 114e120. Hazen, T.C., Toranzos, G.A., 1990. Tropical source water. In: McFeters, G.A. (Ed.), Drinking Water Microbiology: Progress
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and Recent Developments. Springer-Verlag, New York, USA, pp. 32e53. Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T., Williams, S.T., 1994. Bergey’s Manual of Determinative Bacteriology, ninth ed. Williams & Wilkins, Baltimore. Kay, D., Fleischer, J.M., Salomon, R.L., Jones, F., Wyer, M.D., Goodfree, A.F., Zelenauch-Jacquotte, Z., Shore, R., 1994. Predicting likelihood of gastroenteritis from sea bathing results from randomized exposure. Lancet 344, 905e909. Kepner Jr., R.L., Pratt, J.R., 1994. Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiology Reviews 58, 603e615. Law, H.D., 1992. The implementation constraints in waste management in Malaysia. In: Chua, T.E., Garces, L.R. (Eds.), Waste Management in the Coastal Area of the ASEAN Region: Roles of Government, Banking Institutions, Donor Agencies, Private Sector and Communities. ICLARM Conference Proceedings 33. ICLARM, Philippines, pp. 169e172. Lee, C.W., Bong, C.W., 2006. Carbon flux through bacteria in a eutrophic tropical environment: Port Klang waters. In: Wolanski, E. (Ed.), The Environment in Asia Pacific Harbours. Springer, The Netherlands, pp. 329e345. Lee, C.W., Bong, C.W., 2007. Bacterial respiration, growth efficiency and protist grazing rates in mangrove waters in Cape Rachado, Malaysia. Asian Journal of Water Environment and Pollution 4, 11e16. Lee, C.W., Bong, C.W., 2008. Bacterial abundance and production, and their relation to primary production in tropical coastal waters of Peninsular Malaysia. Marine and Freshwater Research 59, 10e21. Lee, C.W., Bong, C.W., Hii, Y.S., 2009a. Temporal variation of bacterial respiration and growth efficiency in tropical coastal waters. Applied and Environmental Microbiology 75, 7594e7601. Lee, C.W., Bong, C.W., Mohamed Yusoff, M.A., Alias, S.A., 2005. Bacterial mediated carbon flux in mangrove waters: a Malaysian perspective. International Journal of Ecology and Environmental Sciences 31, 203e211. Lee, C.W., Ng, A.Y.F., Narayanan, K., Sim, E.U.H., Ng, C.C., 2009b. Isolation and characterization of culturable bacteria from tropical coastal waters. Ciencias Marinas 35, 153e167. Lessard, E.J., Sieburth, JMcN, 1983. Survival of natural sewage populations of enteric bacteria in diffusion and batch chambers in the marine environment. Applied and Environmental Microbiology 45 (3), 950e959. McManus, G.B., 1993. Growth rates of natural populations of heterotrophic nanoplankton. In: Kemp, P.F., Sherr, B.F., Sherr, E.B., Cole, J.J. (Eds.), Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, Boca Raton, pp. 557e562. Moe, C.L., 1997. Waterborne transmission of infectious agents. In: Hurst, C.J., Knudsen, G.R., McInerney, M.J., Stetzenbach, L.D.,
Walter, M.V. (Eds.), Manual of Environmental Microbiology. American Society for Microbiology Press, Washington, D. C., pp. 136e152. Munro, P.M., Gauthier, M.J., Breittmayer, V.A., Bongiovanni, J., 1989. Influence of osmoregulation processes on starvation survival of Escherichia coli in seawater. Applied and Environmental Microbiology 55, 2017e2024. Parsons, T.R., Maita, Y., Lalli, C.M., 1984. A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press, Oxford. Pedro´s-Alio´, C., Mas, J., 1993. Bacterial sinking losses. In: Kemp, P.F., Sherr, B.F., Sherr, E.B., Cole, J.J. (Eds.), Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, Boca Raton, pp. 677e684. Pomeroy, L.R., Wiebe, W.J., 2001. Temperature and substrates as interactive limiting factors for marine heterotrophic bacteria. Aquatic Microbial Ecology 23, 187e204. Presser, K.A., Ross, T., Ratkowsky, D.A., 1998. Modelling the growth limits (growth/no growth interface) of Escherichia coli as a function of temperature, pH, lactic acid concentration, and water activity. Applied and Environmental Microbiology 64, 1773e1779. Pretorius, W.A., 1962. Some observations on the role of coliphage on the number of Escherichia coli in oxidation ponds. Journal of Hygiene 60, 279e281. Rhodes, M.W., Kator, H., 1988. Survival of Escherichia coli and Salmonella spp. in estuarine environments. Applied and Environmental Microbiology 54, 2902e2907. Rozen, Y., Belkin, S., 2001. Survival of enteric bacteria in seawater. FEMS Microbiology Reviews 25, 513e529. Sanders, R.W., Caron, D.A., Berninger, U.G., 1992. Relationship between bacteria and heterotrophic nanoplankton in marine and freshwaters: an inter-ecosystem comparison. Marine Ecology Progress Series 86, 1e14. Sinton, L.W., Hall, C.H., Lynch, P.A., Davies-Colley, R.J., 2002. Sunlight inactivation of fecal indicator bacteria and bacteriophages from waste stabilization pond effluent in fresh and saline waters. Applied and Environmental Microbiology 68, 1122e1131. Solo-Gabriele, H.M., Wolfert, M.A., Desmarais, T.R., Palmer, C.J., 2000. Sources of Escherichia coli in a coastal subtropical environment. Applied and Environmental Microbiology 66, 230e237. Strom, S.L., 2000. Bacterivory: interactions between bacteria and their grazers. In: Kirchman, D.L. (Ed.), Microbial Ecology of the Oceans. Wiley-Liss, New York, pp. 351e386. Wolf, H.W., 1972. The coliform count as a measure of water quality. In: Mitchell, R. (Ed.), Water Pollution Microbiology. Wiley Interscience, New York, pp. 333e345. Zar, J.H., 1999. Biostatistical Analysis, fourth ed. Prentice Hall, Upper Saddle River, NJ.
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Investigating the decay rates of Escherichia coli relative to Vibrio parahemolyticus and Salmonella Typhi in tropical coastal waters Choon Weng Lee a,*, Angie Yee Fang Ng a, Chui Wei Bong a, Kumaran Narayanan b, Edmund Ui Hang Sim c, Ching Ching Ng a a
Laboratory of Microbial Ecology, Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia School of Science, Monash University, Sunway Campus, Selangor, Malaysia c Department of Molecular Biology, Faculty of Resource Science and Technology, University Malaysia Sarawak, Malaysia b
article info
abstract
Article history:
Using the size fractionation method, we measured the decay rates of Escherichia coli, Salmo-
Received 24 June 2010
nella Typhi and Vibrio parahaemolyticus in the coastal waters of Peninsular Malaysia. The size
Received in revised form
fractions were total or unfiltered, <250 mm, <20 mm, <2 mm, <0.7 mm, <0.2 mm and <0.02 mm.
18 November 2010
We also carried out abiotic (inorganic nutrients) and biotic (bacterial abundance, production
Accepted 19 November 2010
and protistan bacterivory) measurements at Port Dickson, Klang and Kuantan. Klang had
Available online 27 November 2010
highest nutrient concentrations whereas both bacterial production and protistan bacterivory rates were highest at Kuantan. We observed signs of protistebacteria coupling via the
Keywords:
following correlations: Protistan bacterivoryBacterial Production: r ¼ 0.773, df ¼ 11, p < 0.01;
Bacterial decay rate
ProtistBacteria: r ¼ 0.586, df ¼ 12, p < 0.05. However none of the bacterial decay rates were
Size fractionation
correlated with the biotic variables measured. E. coli and Salmonella decay rates were
Top-down control
generally higher in the larger fraction (>0.7 mm) than in the smaller fraction (<0.7 mm) sug-
Straits of Malacca
gesting the more important role played by protists. E. coli and Salmonella also decreased in the
South China sea
<0.02 mm fraction and suggested that these non-halophilic bacteria did not survive well in seawater. In contrast, Vibrio grew well in seawater. There was usually an increase in Vibrio after one day incubation. Our results confirmed that decay or loss rates of E. coli did not match that of Vibrio, and also did not correlate with Salmonella decay rates. However E. coli showed persistence where its decay rates were generally lower than Salmonella. ª 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Coastal waters account for less than 10% of the ocean area. However they are highly productive and account for 25% of primary production in the ocean (Berger et al., 1989). Coastal waters are increasingly exploited by humans for food, recreation, transport and other needs, and at present most are in various stages of degradation (Alongi, 1998). There is also an increasing public health threat from pathogens (Hazen and
Toranzos, 1990; Moe, 1997). Disposal of inadequately treated waste is considered faecal pollution and a main source of bacterial pathogens in the sea (Solo-Gabriele et al., 2000). For faecal pollution studies, the concept of bacterial indicator is standard (Wolf, 1972). A fundamental assumption to this concept is the parity in the survival of indicator and enteric pathogens over a wide range of aquatic environments (Bonde, 1977). It is however acknowledged that these indicators are inadequate to predict the presence of pathogenic
* Corresponding author. E-mail address: [email protected] (C.W. Lee). 0043-1354/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2010.11.025
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microorganisms (Rhodes and Kator, 1988; Kay et al., 1994; Borrego and Figueras, 1997). At present, the coliform group of microorganisms or microorganisms found in the intestines of all warm blooded animals especially Escherichia coli is used as a standard indicator of faecal contamination in many countries (Hazen and Toranzos, 1990) including Malaysia (Department of Environment, 2008). The value of E. coli as an indicator microorganism is significantly enhanced by the ease with which it can be detected and cultured (Wolf, 1972) when compared with other bacterial pathogens. In order to use E. coli as an indicator bacterium in seawater, knowledge of its survival must be acquired as it is not a halophilic microorganism. Loss rates of indicator microorganisms pose an interesting problem especially in coastal waters as many factors affect the survival times of E. coli for example temperature, light, salinity, predation, nutrients and pollutants (Fujioka et al., 1981; Munro et al., 1989; Presser et al., 1998; Solo-Gabriele et al., 2000; Rozen and Belkin, 2001; Sinton et al., 2002). Since the 1950s, Carlucci and Pramer (1959; 1960a; 1960b) have studied the survival of bacteria including E. coli in seawater. Numerous studies have shown that decay rates of E. coli do not reflect Vibrio cholerae in estuarine waters and also Salmonella spp. (Colwell et al., 1981; Rhodes and Kator, 1988). However to the best of our knowledge, there is no study that compares the relative loss rates of E. coli with both halophilic and non-halophilic bacterial pathogens. In this study, we investigated whether the decay rates for E. coli were similar to a non-halophilic bacterial pathogen (i.e. Salmonella Typhi) and a halophilic bacterial pathogen (i.e. Vibrio parahaemolyticus)? Our results could help properly evaluate the risk posed by such bacteria either to the health of bathers in recreational waters or to the safety of fisheries or marine aquaculture.
2.
Materials and methods
2.1.
Sampling
We sampled at three stations located on the east (Kuantan: 03 48.40 N 103 20.60 E) and west (Klang: 03 00.10 N 101 23.40 E and Port Dickson: 02 29.50 N 101 50.30 E) of Peninsular Malaysia from April until October 2006 (Fig. 1). The stations at Kuantan and Klang were located in estuaries whereas the station at Port Dickson was sandy coast. Surface seawater samples were collected during high tide, using an acid-cleaned bucket, and in-situ measurements of temperature (0.1 C) and salinity (0.1 ppt) were carried out using a salinometer (YSI-30, US). Seawater samples were then kept in a cooler box for no more than 4 h until processing in the laboratory. In the laboratory, seawater samples for dissolved nutrient analyses were filtered through pre-combusted (450 C for 5 h) Whatman GF/F filters, and stored at 20 C until analysis.
2.2.
Environmental conditions
Dissolved inorganic nitrogen (nitrate (NO3), nitrite (NO2), ammonium (NH4)), and phosphate (PO4) concentrations were measured using a spectrophotometer (Parsons et al., 1984). All nutrient measurements above were carried out in triplicates.
Fig. 1 e Map showing the location of the sampling sites, east (Kuantan) and west (Klang and Port Dickson) of Peninsular Malaysia.
Coefficient of variation (CV) for NH4, NO2 and PO4 analyses were <5%, and <10% for NO3 analysis. Bacteria and protist were determined using the direct count method by an epifluorescence microscope (Olympus BX60, Japan) with a U-MWU filter cassette (excitor 330385 nm, dichroic mirror 400 nm, barrier 420 nm). For protist, 10 ml sample was filtered onto a black 0.8 mm pore size Isopore filter (Millipore, Ireland), and then stained with the fluorochrome primulin (40 mg ml1 final concentration) for 5 min (Bloem et al., 1986) whereas for bacteria, 2 ml sample was filtered onto a black 0.2 m pore size Isopore filter, and then stained with 40 6diamidino-2-phenylindole (DAPI, 1 mg ml1 final concentration) for 10 min (Kepner and Pratt, 1994). Slides were kept frozen for < 3 days before enumeration. A minimum of 10 microscope fields or 500 cells were counted for bacteria and for protist, at least 30 microscope fields were observed.
2.3.
Bacterial decay rates
For bacterial decay experiments, seawater samples were sizefractionated as total or unfiltered, <250 mm (through a 250 mm stainless steel mesh), <20 mm (20 mm pore size nylon mesh), <2 mm (2.0 mm polycarbonate membrane filter), <0.7 mm (GF/F filter), <0.2 mm (0.2 mm polycarbonate membrane filter) and <0.02 mm (0.02 mm Whatman Anodisc). The size fractions were inoculated separately with 1% (v/v) fresh cultures of E. coli, Salmonella Typhi (hereafter referred to as Salmonella) and V. parahaemolyticus (hereafter referred to as Vibrio), and then incubated at 30 C for about three days. Both E. coli and Salmonella inocula were prepared on nutrient broth whereas
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for Vibrio, 3% NaCl (final concentration) was added to the nutrient broth. From the inoculated size fractions, the initial E. coli, Salmonella and Vibrio concentration was determined as colony forming unit (cfu) ml1 via spread plating. The number of cfus was observed one day after spread plating on MacConkey agar (for E. coli and Salmonella) and Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar with 3% NaCl (for Vibrio). A portion of the sample was also removed every day to determine the change in E. coli, Salmonella and Vibrio concentration. Bacterial decay rate was modeled by the standard differential equation: dN/dt ¼ kN (Chick, 1908) where k ¼ decay constant, N is the cfu ml1, and t ¼ time of incubation. Integrating the differential equation gave the following ln N ¼ kt þ C. Hence for decay rate assessment, we transformed the cfu data by natural logarithm and plotted the ln cfu ml1 against incubation time. Linear regression analysis was then used to find the best-fit slope i.e. decay rate. We also measured separately, the concurrent bacterial production and protistan bacterivory rates by measuring bacterial growth rate in both <0.7 mm and <20 mm fractions after 12 h incubation. Bacterial growth rate in each fraction was calculated as the increase in natural logarithmic bacterial abundance over time. The bacterial growth rate in the <0.7 mm fraction was assumed without grazing (m0.7) whereas growth rate in the <20 mm fraction (m20) was the product of both growth and grazing. Bacterial production (BP) was then estimated by the following equation: BP ¼ Bacterial abundance m0.7 (Lee et al., 2009a) whereas bacterivory rate was estimated by Bacterial abundance (m0.7 m20) (McManus, 1993).
2.4.
Statistical analyses
Statistical tests such as coefficient of variation (CV), analysis of variance, Student’s t-test, Tukey’s test, linear regression and correlation analyses were carried out according to Zar (1999). Count data were log-transformed to meet parametric assumptions of equality of variances and normal distribution before correlation and linear regression analyses. All data, unless mentioned otherwise, were reported as mean S.D.
3.
Results
3.1.
Abiotic measurements
Table 1 shows the physico-chemical parameters measured at the three stations. Average surface seawater temperature
observed ranged from 29 to 30 C whereas average salinity measured at the estuaries (Klang and Kuantan) was lower than at Port Dickson. Salinity fluctuated over a wider range at the estuaries (CV ¼ 7% and 46%) than Port Dickson (CV ¼ 1%). Dissolved inorganic nitrogen (DIN) measured showed that concentrations at Klang were more than two-fold higher than both Kuantan and Port Dickson. Among the nitrogen species, NH4 was the dominant species at both Klang and Kuantan, accounting for > 70% of DIN. At Port Dickson, NO3 was the dominant species (about 45% of DIN). Average PO4 concentration at Klang was also higher than at both Port Dickson and Kuantan.
3.2.
Biotic measurements
Bacterial abundance ranged from 0.85 to 3.49 106 cell ml1, and was significantly higher at Kuantan than both Klang (q ¼ 4.77, df ¼ 12, p < 0.05) and Port Dickson (q ¼ 5.77, df ¼ 12, p < 0.01) (Table 2). For protist, abundance ranged from 0.58 to 6.64 103 cell ml1, and was about three orders lower than bacteria. Protist abundance at Kuantan was significantly higher than Port Dickson (q ¼ 4.34, df ¼ 12, p < 0.05) but not Klang. Table 2 also shows the bacterial production measured in this study. Bacterial production at both Klang and Port Dickson ranged from 0.72 to 1.69 105 cell ml1 h1 whereas bacterial production at Kuantan ranged from 1.99 to 4.78 105 cell ml1 h1. Similar to patterns exhibited by microbial abundance data, bacterial production at Kuantan was significantly higher than both Klang (q ¼ 4.69, df ¼ 12, p < 0.05) and Port Dickson (q ¼ 5.79, df ¼ 12, p < 0.01). Protistan bacterivory at Kuantan was also higher than both Klang (q ¼ 14.48, df ¼ 9, p < 0.001) and Port Dickson (q ¼ 13.64, df ¼ 9, p < 0.001). Bacterivory rates at Kuantan ranged from 1.60 to 2.15 105 cell ml1 h1 whereas bacterivory rates at both Klang and Port Dickson were about one order lower, and ranged from 1.11 to 4.19 104 cell ml1 h1. E. coli concentration from 1990 until 2005 were also obtained from the Department of Environment Malaysia monitoring stations located near our study area. Although E. coli concentration varied over four-order at all three locations (Fig. 2), analysis of variance showed significant differences among the different locations (F ¼ 19.2, df ¼ 1095, p < 0.001). At Port Dickson and Klang, average E. coli was 1300 3600 MPN (Most Probable Number) per 100 ml, and was significantly higher than Kuantan (640 1900 MPN per 100 ml) (KlangeKuantan: q ¼ 8.52, df ¼ 1095, p < 0.001; Port DicksoneKuantan: q ¼ 6.38, df ¼ 1095, p < 0.001).
Table 1 e Physico-chemical characteristics of the sampling stations in this study. Mean (±S.D.) of surface water temperature, salinity, ammonium (NH4), nitrite (NO2), nitrate (NO3), and phosphorus (PO4). Station Klang (n ¼ 4) Port Dickson (n ¼ 5) Kuantan (n ¼ 5)
Temperature C
Salinity ppt
NH4 mM
NO2 mM
NO3 mM
PO4 mM
30.3 0.5 30.0 1.0
27.7 1.9 28.4 0.3
17.84 24.75 0.42 0.51
2.97 0.64 0.18 0.27
1.24 0.61 0.50 0.28
1.66 1.54 0.12 0.05
29.1 0.8
22.4 10.2
2.89 2.64
0.51 0.29
0.61 0.33
0.60 0.31
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Table 2 e Bacterial abundance, protist abundance, bacterial production and protistan bacterivory rates measured in this study. Date
Bacteria (106 cell ml1)
Protist (103 cell ml1)
Bacterial production (105 cell ml1 h1)
Protistan bacterivory (104 cell ml1 h1)
Klang
24-Apr-06 12-Jun-06 26-Jun-06 31-Jul-06 Average (S.D.)
1.13 0.93 1.29 1.53 1.22 0.26
2.89 4.08 2.57 1.08 2.65 1.23
1.69 0.97 1.66 1.64 1.49 0.35
1.55 1.11 3.70 2.77 2.28 1.18
Port Dickson
04-Jul-06 03-Oct-06 10-Oct-06 17-Oct-06 30-Oct-06 Average (S.D.)
1.10 0.85 0.91 1.11 0.98 0.99 0.12
2.43 1.16 2.01 0.58 1.73 1.58 0.73
e 0.72 1.25 1.36 1.08 1.10 0.28
e e 3.41 4.19 1.95 2.26 2.07
Kuantan
18-Apr-06 17-May-06 19-Jun-06 25-Jul-06 15-Aug-06 Average (S.D.)
2.91 1.31 2.19 2.40 3.49 2.46 0.82
3.02 1.76 5.42 6.64 5.51 4.47 2.01
4.78 1.99 3.60 2.50 2.84 3.14 0.11
21.45 e 16.05 e 16.00 17.83 3.13
Station
3.3.
Bacterial decay rates at different size fractions
Fig. 3 shows the change in E. coli concentration after inoculation in the different fractions of seawater collected from Port Dickson, Kuantan and Klang. There was an apparent decrease in E. coli in most of the larger fraction seawater (i.e. total, <250 mm, <20 mm, <2 mm) whereas in the smaller fraction (i.e. <0.7 mm, <0.2 mm and <0.02 mm), the concentration of E. coli often did not change significantly. Fig. 4 shows the statistically significant ( p < 0.05) decay rates measured in this study, and confirmed that E. coli decay rate was significantly higher in the larger fraction (>0.7 mm) (2.28 0.90 d1) than the smaller fraction (<0.7 mm) (0.58 0.22 d1) (Student’s t-test: t ¼ 12.35, df ¼ 64, p < 0.001). A similar trend was observed for Salmonella where its concentration generally decreased with time, especially in the larger fraction seawater (Fig. 5). Salmonella decay rates in the larger fraction (3.17 1.19 d1) were significantly higher than in the smaller fraction (1.51 0.84 d1) (Student’s t-test: t ¼ 6.65, df ¼ 59, p < 0.001) (Fig. 4). In contrast to E. coli and Salmonella, Vibrio exhibited a different trend (Fig. 6). There was often an increase in Vibrio concentration after one day incubation, and less discernible difference in Vibrio concentration between the larger and smaller fractions of seawater. Few of the experiments gave significant decay rates (Fig. 4), and available Vibrio decay rates ranged 0.90e1.78 d1 at Klang, and 0.54e4.64 d1 at Kuantan. At Port Dickson, only two significant decay rates were observed (1.36 0.21 d1). When we compared E. coli decay rates in the larger fractions among the stations, E. coli decay rate was highest at Klang (1.96e4.90 d1), followed by Kuantan (0.88e2.80 d1) and Port Dickson (0.36e2.93 d1) (KlangeKuantan: q ¼ 6.55, df ¼ 51, p < 0.001; KlangePort Dickson: q ¼ 9.99, df ¼ 51, p < 0.001; KuantanePort Dickson: q ¼ 3.44, df ¼ 51, p < 0.05). However
Fig. 2 e Long term E. coli counts (log MPN/100 ml) from selected Department of Environment monitoring stations near Port Dickson (PD) (n [ 578), Klang (n [ 196) and Kuantan (n [ 322).
w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 6 1 e1 5 7 0
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Fig. 3 e E. coli decay (ln cfu mlL1) measured in different fractions at Port Dickson (PD) (n [ 5), Klang (n [ 3) and Kuantan (n [ 5).
there was no significant difference in Salmonella and Vibrio decay rates among the stations.
4.
Discussion
4.1.
Environmental conditions
Surface seawater temperature observed was typical of tropical waters. Klang had the highest nutrient concentrations, and reaffirmed earlier observations (Lee and Bong, 2006, 2008; Lee et al., 2009a). One possible reason is the rapid pace of
development and industrialization taking place upstream of Klang where the capital of Malaysia, Kuala Lumpur is located (Lee and Bong, 2006). In this study, the abundance of both bacteria and protist were within the range for coastal waters of Peninsular Malaysia (Lee et al., 2005; Lee and Bong, 2007, 2008). Protist abundance was about three orders of magnitude lower than bacteria, and this observation was consistent with the analysis by Sanders et al. (1992). Both bacterial production and protistan bacterivory rates measured in this study were also within the range previously published for tropical coastal waters (Lee et al., 2005, 2009a; Lee and Bong, 2006, 2007, 2008).
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Fig. 4 e Summary of decay rates (P < 0.05) measured in this study at Klang, Port Dickson (PD) and Kuantan. Different symbols represent different sampling dates. Error bar denotes the standard error of decay rate and is shown except when smaller than symbol.
However, in contrast to the pattern exhibited by nutrient concentrations where Klang had the highest levels, all biotic variables were highest at Kuantan. It was not clear why biotic variables at Klang were not reflective of its nutrient concentrations as found in earlier studies (Lee and Bong, 2008; Lee et al., 2009a). However both Klang and Kuantan are estuaries, and can experience episodic nutrient inputs that can stimulate microbial biomass and productivity. Long term monitoring data showed the extent of faecal pollution via the indicator E. coli. Although coastal water quality in Malaysia has deteriorated over time (Department of Environment, 2008), there was no apparent increase in E. coli over the 15-year period for all three locations. At present, Malaysia has an interim marine water quality standard that set the criterion for E. coli at 100 MPN per 100 ml (Department of Environment, 2008). From a 15-year period of monitoring data, >60% of the samples at Klang and Port Dickson exceeded the standard whereas at Kuantan, 35% of the samples exceeded the standard. Dow (1995) had earlier reported higher E. coli concentration for stations along the Straits of Malacca. Our study showed that faecal pollution remains a problem for coastal waters here. One reason is many coastal communities in Malaysia lack proper sewage disposal systems and often discharge sewage directly into the sea (Law, 1992). The faecal pollution is accentuated for stations along the Straits of Malacca as the population density is higher along the west coast of Peninsular Malaysia. Although sewage treatment facilities have increased over time, the problem showed no sign of alleviation at both Klang and Port Dickson.
4.2.
Bacterial decay rates at different size fractions
The decay rates obtained in this study were within the range reported by Anderson et al. (2005) but relatively higher than decay rates reported for temperate waters (Lessard and Sieburth, 1983; Rhodes and Kator, 1988). Relative to temperate waters, the higher decay rates obtained in this study could be due to the fact that microbial activity (including protistan bacterivory) in tropical waters is at its optimum (Pomeroy and Wiebe, 2001). For both E. coli and Salmonella, decay rates were generally higher in the larger fraction (>0.7 mm) than in the smaller fraction (<0.7 mm). The main bacterial predators in the larger fractions are nanoflagellates and ciliates (Sanders et al., 1992) whereas the cause of bacterial mortality in the smaller fraction is mainly by viral lysis (Fuhrman, 2000). In the smaller fraction, lytic bacteria may also play a role albeit a minor one (Enzinger and Cooper, 1976). Bacterial decay rates measured in this study suggested that protistan bacterivory was more important, similar to the conclusion by Enzinger and Cooper (1976). A reason why viral lysis played a minor role in this study was because both Salmonella and E. coli are not natural seawater organisms and are not active in the sea (Carlucci and Pramer, 1960b). Bacteriophages require the host physiological activity in order to replicate (Pretorius, 1962). The decay rates in the <0.02 mm fraction is effectively not due to viral lysis or bacterivory as both viruses and protists do not pass through this pore size. The <0.02 mm fraction is therefore suitable to observe the response of halophilic and
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Fig. 5 e Salmonella decay (ln cfu mlL1) measured in different fractions at Port Dickson (PD) (n [ 4), Klang (n [ 3) and Kuantan (n [ 4).
non-halophilic pathogens in seawater. We observed that Vibrio counts in the <0.02 mm fraction increased after day one in all the experiments, and no significant decay of Vibrio could be observed throughout this study. In contrast, the decay rates for E. coli and Salmonella in the <0.02 mm fraction were still statistically significant ( p < 0.05). Our results concurred with earlier reports that non-halophiles are usually not able to grow well in seawater (Gerba and McLeod, 1976). However relative to protistan bacterivory, the decay rates for E. coli and Salmonella in the <0.02 mm fraction were low, and accounted for <20% of total decay rates. These decay rates also did not correlate ( p > 0.15) with salinity, suggesting that bacterivory was more important than salinity for non-halophilic bacterial decay. There were obvious differences in the response of Vibrio in the different seawater fractions relative to both E. coli and Salmonella. In this study, significant Vibrio decay was seldom observed, and rates observed were usually lower than E. coli and Salmonella. One reason why Vibrio decay rates were lower was probably due to the ability of V. parahaemolyticus to grow in a salty environment (Holt et al., 1994). Increase in Vibrio was still apparent after one day incubation before Vibrio counts started decreasing. The latter reduction could be due to other stresses for example limited food availability that resulted in Vibrio growth rates falling below loss rates.
E. coli is widely used as an indicator for faecal pollution and for pathogenic microorganisms (Bonde, 1977). However the validity of E. coli as an indicator is questionable especially in coastal waters (Solo-Gabriele et al., 2000) where presence of E. coli is more likely the balance between supply and loss. This is because E. coli is non-halophilic and is not known to grow well in coastal waters (Gerba and McLeod, 1976). Our results confirmed that decay or loss rates of E. coli did not match that of the halophilic Vibrio and also did not correlate significantly with Salmonella decay rates ( p > 0.50) (Table 3) even though the survival characteristic of E. coli is presumed similar to Salmonella (Bonde, 1977). When we compared the different bacterial responses in the larger fractions, we found that at Klang, Salmonella and E. coli decay rates were significantly higher than Vibrio (SalmonellaeVibrio: q ¼ 6.58, df ¼ 33, p < 0.001; E. colieVibrio: q ¼ 6.16, df ¼ 33, p < 0.001) whereas at Kuantan, Salmonella decay rate was significantly higher than both Vibrio (q ¼ 7.70, df ¼ 46, p < 0.001) and E. coli (q ¼ 4.92, df ¼ 46, p < 0.01). Similarly at Port Dickson, Salmonella decay rate was higher than E. coli (Student’s t-test: t ¼ 4.10, df ¼ 29, p < 0.001). For Port Dickson, only decay rates from Salmonella and E. coli were compared as there were too few decay rates from Vibrio experiments. Generally, we found that Salmonella decay rates were higher
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Fig. 6 e Vibrio decay (ln cfu mlL1) measured in different fractions at Port Dickson (PD) (n [ 3), Klang (n [ 3) and Kuantan (n [ 3).
than E. coli. The persistence of E. coli relative to Salmonella fulfilled one of the criteria for an indicator organism i.e. the indicator organism should survive longer than the pathogen itself (Cabelli, 1978; Allwood et al., 2003). Although our study provided snapshots of the environment, we showed consistently the importance of protistan bacterivory in bacterial decay. E. coli was a poor indicator for halophilic pathogens, and although E. coli decay rates did not correlate with Salmonella, E. coli persisted longer than Salmonella in coastal waters. Our results provided support to the continuous use of E. coli as an indicator organism for nonhalophilic pathogens especially Salmonella.
4.3.
Protistebacteria coupling
Multiple correlation analysis showed that E. coli, Salmonella and Vibrio decay rates did not correlate with each other or other biotic variables (Table 3). Protistan bacterivory also did not correlate with E. coli, Salmonella and Vibrio decay rates. As these pathogens form only a minor fraction of the total bacterial population in the sea (Lee et al., 2009b), their decay rates played only a minor role towards total bacterivory rates. However, we did observe evidences of significant coupling between protist
and bacteria similar to Sanders et al. (1992) and Lee et al. (2005) (Protistan bacterivoryeBacterial Production: r ¼ 0.773, df ¼ 11, p < 0.01; ProtisteBacteria: r ¼ 0.586, df ¼ 12, p < 0.05). Our study showed that protistan bacterivory accounted for 9e56% of bacterial production (Table 2), and was significantly higher at Kuantan (>44% bacterial production) than both Klang (q ¼ 9.68, df ¼ 9, p < 0.001) and Port Dickson (q ¼ 6.68, df ¼ 9, p < 0.01).
Table 3 e Pearson productemoment correlation coefficient (r) between variables measured i.e. bacterial production (BP, cell mlL1 hL1), log bacterial abundance (BA, cell mlL1), grazing (cell mlL1 hL1), log Protist (cell mlL1), decay rates of E. coli (dL1), Salmonella (dL1), and Vibrio (dL1). * is P < 0.05, ** is P < 0.01, *** is P < 0.001. BP
BA
BA 0.896*** Grazing 0.773** 0.794*** Protist 0.546* 0.586* E. coli 0.214 0.120 Salmonella 0.174 0.082 Vibrio 0.129 0.135
Grazing Protist E. coli Salmonella
0.295 0.291 0.120 0.064
0.190 0.121 0.077 0.276 0.040
0.106
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Bacterivory rates at both Klang and Port Dickson accounted for only 9e31% bacterial production. When bacterivory was less than bacterial production, other factors might have played a role in the removal of bacterial production as bacterial abundance is stable and does not change significantly with time (Lee and Bong, 2008). Among the factors that are important include viral lysis (Fuhrman, 2000), removal by benthic filter feeders (Strom, 2000), and sedimentation (Pedro´s-Alio´ and Mas, 1993). Sedimentation is often overlooked as a loss factor for bacteria due to the low sinking rates of microorganisms. However sinking speed can increase for bacteria attached to particles and there might be significant losses by sedimentation (Pedro´s-Alio´ and Mas, 1993). Although these loss factors were not investigated here, studies on viral ecology in tropical waters have suggested that viral lysis may not be an important loss factor (Bettarel et al., 2006; Cissoko et al., 2008). Moreover the higher suspended solids in the coastal waters of Peninsular Malaysia (Bong and Lee, 2008; Lee et al., 2009b) implied that sedimentation is probably more important. However further investigations are needed.
5.
Conclusion
1. Bacterial decay in tropical coastal waters is mainly due to protistan bacterivory. 2. Via the <0.02 mm fraction, our results showed that E. coli and Salmonella do not survive well in seawater. In contrast, Vibrio grows well in seawater. Therefore E. coli is a poor indicator for halophilic pathogens. 3. E. coli decay rates do not correlate with both Salmonella and Vibrio decay rates. However E. coli persists longer than Salmonella in coastal waters. Our results provided support to the continuous use of E. coli as an indicator organism for non-halophilic pathogens especially Salmonella. 4. There is protist‒bacteria coupling where protist counts correlated with bacterial abundance, and protistan bacterivory correlated with bacterial production.
Acknowledgements We are grateful to the Department of Environment, Malaysia for providing the monitoring data of E. coli concentration. Funding for this research was provided by University of Malaya (RG064-09SUS) and Ministry of Science, Technology & Innovation (06-01-03-SF0457). The researchers would also like to thank anonymous reviewers who helped improve the manuscript and University of Malaya for providing the research facilities.
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