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We investigated separate and simultaneous effect of temperature, salinity and solar radiation, as well as bacterial strain and origin on Escherichia coli (E. coli) survival in seawater in experimental conditions. The experiments were carried out by placing the bottles filled with seawater of different salinity (15.0, 30.0 and 36.5 psu) and contaminated by bacterial cultures in three light‐protected air incubators set to different temperatures (6, 12, 18 and 24°C), or by placing the bottles in plastic containers filled with water of controlled temperature and exposing them to direct solar light. In experiments in the dark, two typed and two wild E. coli strains were tested. The mean T90 values were 33.55 h for E. coli ATCC 8739, 42.50 h for E. coli ATCC 35218, 72.8 h for E. coli originating from seagull feces and 278.6 h for E. coli originating from sewage, indicating differences between survival abilities among strains. The effect of temperature on T90 was significant only in seagull E. coli at 36.5 psu and sewage E. coli at 30.0 psu and was positive. The effect of salinity was significant only in seagull strain and also was positive. No interactive effect of temperature and salinity was recorded. Experiments in the presence of solar radiation, carried out with two ATCC E. coli strains, demonstrated its dominate harmful effect on bacterial cells, reducing T90 of both strains to 0.30–0.82 h for E. coli ATCC 35218 and 0.31–5.93 h for E. coli ATCC 8739. Within the ultraviolet A (UVA) and photosynthetically active radiation (PAR) spectrum of solar radiation, the wavelengths of 320–360 nm were found as most bactericidal. By comparing survival of cultivated E. coli cells to those in natural seawater samples, significantly higher survival E. coli cells in natural seawater samples was found.
chapter and author info
Institute of Oceanography and Fisheries, Split, Croatia
Institute of Oceanography and Fisheries, Split, Croatia
Fecal pollution of seawater can present a serious problem due to potentially introducing of intestinal pathogens—bacterial, viral, and parasitic. Because of a wide variety of pathogenic microorganisms that can enter the sea, time‐consuming and complex procedures of their determination, the assessment of microbiological quality of seawater has traditionally been based on the determination of indicator microorganisms, bacteria that suggest the presence of pathogens . In the last decade, European Union member states have accepted Escherichia coli as the indicator for assessment of microbiological quality of bathing and shellfish waters [2, 3]. Apart from its human and warm‐blooded animal feces origin, selection of E. coli as indicator organisms is based on scientific understanding and reliable research of one of the most important criteria which good indicator have to meet: resistance to environmental conditions.
When released into the sea, E. coli cells are exposed to the impact of a very hostile environment. That impact is reflected in the negative effect of the complex array of biotic and abiotic factors of the marine environment. Among abiotic factors, the effect of temperature, salinity, and particularly solar radiation is most pronounced [4–7]. The distribution and abundance of indicator bacteria in seawater depend mostly on their input, but also on the intensity of aforementioned environmental factors  and bacterial cell adaptation capacity. Due to their minor adaptation capacity to marine conditions, E. coli cells suffer a sublethal injury and enter a dormant, viable but nonculturable state, in which they can still maintain some metabolic activity [9–11], infective capacity and potential for pathogenicity [12, 13]. The “viable but nonculturable” state concept was introduced to describe cells that remain metabolically active but are unable to divide in or on nutritional media that normally support their growth. The capacity to form colonies on a solid medium is the first ability that enteric bacteria lose in seawater . That means that they cannot be detected during standard routine monitoring by culturing method unless resuscitation methods on liquid media were used. With the prolonged exposure, particularly in the presence of solar radiation, E. coli cells are irreversibly inactivated and they die . For how long they will maintain culturability on standard media depends on many factors, such as the intensity of environmental factors and the characteristics of the bacterial cell, where besides the origin and previous growth history, the bacterial strain plays an important role [7, 16, 17]. Although there are many studies in which the effects of the aforementioned factors have been addressed, their simultaneous effects have been poorly investigated. In this chapter, the separate and combined effect of temperature, salinity and solar radiation, as well as the growth history, strain and origin of E. coli cells on their survival in seawater were presented. Since microbial pollution of bathing waters is routinely monitored by culturing methods on standard solid media, and viable but nonculturable cells usually cannot be detected, for the purposes of this chapter, the term “survive” means to maintain culturability.
2. Materials and methods
2.1. Experiments with pure bacterial cultures
Bacterial cultures used in experiments were always freshly prepared by incubation of pure culture on mineral‐modified glutamate broth (MMGB). Bacterial suspensions were taken in the exponential phase of growth and serially diluted in phosphate buffer solution. After determination of the concentration of E. coli cells by epifluorescence microscopy method, appropriate dilution, cca 103 CFU/mL, was kept at 4°C until the beginning of the experiments (cca 20 h).
Experiments in the dark were performed in two sets. In the first set, experiments were carried out at six different experimental conditions corresponding to Adriatic sea natural range of temperatures (12°C—mean winter temperature, 18°C—mean spring and autumn temperature and 24°C—mean summer temperature) and salinities (30.0 psu—lower salinity corresponding to areas near the mouth of rivers or sewage outfalls and 36.5 psu—typical salinity in coastal seawater). Two different E. coli strains were used: E. coli ATCC 35218 originating from canine feces and E. coli ATCC 8739, originating from human feces. Bacterial suspension was added to 500 mL borosilicate glass bottles containing autoclaved seawater of appropriate salinity (30.0 and 36.5 psu). Bacterial concentration in bottles was targeted to 103 CFU E. coli/100 mL. The bottles were then stirred and placed in a temperature controlled air incubators at three different temperatures (12, 18 and 24°C).
In the second set, additional experiments were performed at lower temperature (6°C) and lower salinity (15 psu). Two new bacterial strains were also introduced—wild E. coli strain isolated from sewage and wild E. coli strain isolated from seagull feces.
Experiments in the presence of solar radiation were performed with two ATCC E. coli strains, also combining the same temperatures (12, 18 and 24°C) and salinities (30.0 and 36.5 psu). Bacterial suspension was added to 500 mL ultraviolet B (UVB)—blocking borosilicate glass bottles containing autoclaved seawater of appropriate salinity. Bacterial concentration in bottles was also targeted to 103 CFU E. coli/100 mL. The bottles were then stirred and left in dark for 15 min to homogenize. Bacterial cells in bottles were then exposed to different intensities of the natural range and spectrum of solar radiation. The exposure to solar light was carried out in two ways: by placing the bottles in 50 L (60 × 30 × 30 cm) transparent plastic containers, filled with water of appropriate and controlled temperature, and placed on the open space in front of the laboratory;—by hanging the bottles from research vessel, vertically on rope to different depths (0.2, 5, 15 and 30 m) of the water column, to expose them to different intensities of solar light (for E. coli ATCC 8739 only). In both cases, the irradiance, Ed (μWcm−2nm−1), of ultraviolet A (UVA) and photosynthetically active radiation (PAR) was measured by optical radiometer PRR800 (Biospherical Inc.).
All experiments were performed in triplicates. The initial concentrations of culturable E. coli cells in all experiments were determined by taking 10 mL subsamples 15 min after adding bacterial suspension. After exposing the bottles to desired conditions, the concentrations of E. coli were monitored by taking 10 mL subsamples every 24 h in experiments performed in the dark and every 10 or 20 min in experiments performed in solar light.
2.2. Experiments with natural samples
Natural samples of moderately polluted seawater were collected near sewage outfalls and were stored at 4°C. Sampling was performed in the morning (9 o’clock AM) and at noon. The number of culturable E. coli was determined after sampling and every 24 h until their reduction to zero. After isolation and counting on selective agar plates, one colony of E. coli cells that survived T90 (the time required to reduce culturability by 90%) or longest was randomly selected from each plate of samples collected in the morning. Cultures were incubated on MMGB and processed in the same way as pure cultures of ATCC strains. A bacterial suspension was added to 500 mL borosilicate bottles, containing autoclaved, 36.5 psu seawater, and stored in dark at 4°C. The number of culturable E. coli was determined every 24 h until their reduction to zero.
2.3. E. coli determination
The concentration of E. coli in suspensions was determined by the direct method of epifluo-rescence microscopy . The number of culturable E. coli cells in suspension and seawater was determined by the modified ISO/TS 16649‐1 method  using membrane filtration technique and expressed as CFU E. coli/100 mL.
2.4. Statistical analysis
Data were processed using statistical package Statistica 8.0 (StatSoft Inc., 2007). High and significant (R2 > 0.9, p < 0.01) fittings of raw die‐off data of E. coli to exponential function, which can be expressed by Eq. (1), were found in the dark as well as in the presence of solar radiation. Consequently, T90 was derived from Eq. (1) and was calculated by Eq. (2),
where N0 and Nt are the number of culturable E. coli at the beginning of the experiment and at the time of subsampling (t).
The inactivation energy S90 (the insolation required to reduce culturability by 90%) was calculated by Eq. (3),
where En (Wm−2) = the intensity of solar radiation.
3. Results and discussion
3.1. Experiments in the absence of solar radiation
In the natural range of temperature and salinity, the T90 values in this study were 31.9–51.7 h (mean 42.5 h) for E. coli ATCC 35218 and 29.4–37.9 h (mean 33.55 h) for E. coli ATCC 8739 (Figure 1). The results were mostly consistent with the results of previous studies. The T90 values of E. coli reported in earlier studies were from 26 h, which was found in estuarine water at 20°C , to 115 h, which was found for fecal coliforms in seawater at 8–10°C .
The results of two‐way ANOVA revealed that there were no statistically significant (p > 0.05) separate and/or interactive effects of the variations in temperature and salinity in their natural range, on changes in T90 values of ATCC E. coli strains. Unlike the T90 values, these results were not consistent with those of previous studies. In general, most studies showed enhanced stability of indicator bacteria at lower temperatures [4, 5], although Anderson et al.  found a lower stability and negative effect. The increased inactivation of indicator microorganisms at higher temperatures was mostly attributed to increased metabolic activities in terms of reduced nutrient concentration , increasing predation, grazing and the deleterious effect of solar radiation . One of the possible explanations for the absence of clear effect of temperature in our experiments might be the absence of grazing and predation, since seawater was sterilized by autoclaving. Anderson et al.  attributed a positive effect of temperature in their study mostly to sublethal stress that was induced by laboratory manipulation and related to the pre‐exposure history of the used isolates. In our additional experiments with E. coli ATCC 8739 at 6°C, significantly higher T90 values were recorded (Figure 1), indicating more significant negative effect of temperature in its natural range than at lower values. A positive effect of temperature in whole measured range was observed in seagull E. coli at 36.5 psu and sewage E. coli at 30.0 psu (Figure 2).
Most studies showed a negative correlation between survival time of indicator bacteria, including E. coli, and salinity [3, 7, 24, 25]. Troussellier et al.  found negative effect of salinity only in the presence of solar light. An unclear correlation between T90 and salinity in our study was partly attributed to a narrow salinity range. In order to extend the range of salinity and to clarify its effect on T90, we performed additional experiments with E. coli ATCC 8739 at 15 psu. At higher temperatures (18 and 24°C), negative effect of lower salinity was less pronounced than the effect of salinity in its natural range, while at 6°C, lower salinity significantly lowered T90, making the effect of salinity additionally unclear (Figure 3). A negative effect of salinity is attributed to specific characteristics of sea water, such as osmotic pressure  and the toxicity of inorganic salts . Survival largely depends on the osmoregulatory ability of each strain or group of bacteria . In order to equalize osmotic pressure and avoid drastic loss of water from the cytoplasm, bacterial cells can accumulate or synthesize specific osmoprotectant molecules  which increase cell resistance to seawater. This depends, among other things, on preadaptation media. Since E. coli in experiments was incubated on MMGB media, accumulation of glutamate, which is recognized as osmoprotectant , probably caused an unclear effect of salinity on E. coli survival.
Although not statistically significant, E. coli ATCC 35218 survived longer than E. coli ATCC 8739 in almost all tested conditions. A significantly higher T90 was found in pure culture of E. coli isolated from seagull feces, 28.5–173 h (mean 72.8 h) and particularly in E. coli isolated from sewage outfall, 150–390 h (mean 278.6) (Figure 3). This suggested the importance of cell strain and origin to their survival in sea water and partially contributed to better understanding of the variations in results of previous studies. Since all tested E. coli strains were exposed to the same environmental conditions, the obtained results probably illustrated the extensive genetic and phenotypic diversity exhibited within E. coli strains, which could explain the different survival abilities in this study and aquatic environments generally . On the basis of genomic information, E. coli species have been divided into eight phylogenetic groups, A, B1, B2, C, D, E, F, and clade I . Strains belonging to phylogroups A and B1 are highly adapted to humans and vertebrate animals, the A phylogroup strains being predominant in humans and the B1 strains in animals [33, 34]. Significant differences in survival in water environment were found among strains belonging to different phylogroups. Recent studies showed that E. coli B1 strains can persist longer in water than strains of the other phylogroups  and supported the hypothesis that persistent genotypes have an adaptive advantage in the secondary habitat outside the host . Consequently, once released into water, E. coli strains could be selected on the basis of their survival ability, and the resulting population differed from the original one in terms of phenotypic traits. E. coli strain isolated from sewage outfall and used in this study was in the seawater for an unknown period and probably passed selection that explains its superiority in survival over other tested strains.
Anderson et al.  found significant differences in the survival of indicator bacteria, E. coli and enterococci, depending on their origin. Among three investigated sources of bacteria; soil, sewage and dog feces, bacteria that originated from soil showed the highest resistance to environmental factors. These results are very important, particularly because apart human feces, there are many potential sources of enteric bacteria that enter the sea, such as untreated piggery effluents , and the feces of wild birds  and other warm‐blooded animals.
In seagull E. coli, a positive effect of salinity was recorded in whole range of temperature, and at 36.5 psu higher temperature enhanced bacteria survival (Figure 2). Some E. coli cells may adapt to a range of concentrations of sodium chloride, possibly by means of osmoregulatory mechanism induced by salts or some structural modification in the outer membrane of bacteria . Seagulls are marine birds and sea environment is their natural habitat. They feed mainly marine animals that contain high content of sodium chloride and drink both, fresh and seawater. Before being processed and excreted through salt gland in order to maintain osmotic balance, sodium chloride is present in intestinal tract content in a relative high concentration . Consequently, seagull intestinal microflora, including E. coli, is highly adapted to saline environment. Such cells probably survive longer in seawater than other cells. Seagulls present a big problem to shellfish farms because they usually gather on buoys and other farm constructions, and huge quantities of feces enter the sea and contaminate it. Since up to 107 CFU E. coli g−1 was found in seagull feces in this study, and the results suggested better survival of E. coli originating from seagull feces, there is higher probability of their accumulation in filter feeding bivalves than E. coli that survive shorter.
3.2. Experiments in the presence of solar radiation
The intensity of solar radiation in this study was in the range 258–693 Wm−2, with a mean contribution of the UVA spectrum of 9.3–11.1%. In the experiments carried out in the laboratory, a strong (R2 > 0.82) and statistically significant (p < 0.01) negative correlation between the T90 of E. coli and the intensity of the UVA spectrum of solar radiation was found (Figure 4). The T90 of E. coli ATCC 35218 was in the range 0.30–0.82 h, whereas the T90 of E. coli ATCC 8739 ranged from 0.31 to 5.93 h. A significant, 15‐ to 70‐fold reduction in T90, compared with that recorded in the absence of solar radiation indicated the dominant effect of solar radiation on the survival of E. coli in seawater. Multiple linear regressions were used to investigate the combined effect of temperature, salinity, and the intensity of solar radiation on changes in the T90 of E. coli (Table 1). High and statistically significant (p < 0.01) coefficients of multiple determination (R2) were found, which revealed that most of the variance in T90 can be explained by independent variables. However, only variations in solar radiation had a significant effect on T90.
Simultaneous effect of temperature (T), salinity (S), and solar radiation (UVA and PAR) on the T90 of E. coli (rp—coefficient of partial correlation; beta‐regression coefficients; a—intercept; b—coefficient of multiple linear regression; R2—coefficient of multiple determination).
*p < 0.01.
Because of inadequate weather conditions (a relative cloudy sky), in situ experiments were carried out at lower intensities of solar radiation. We also found a strong vertical decline in the intensity of solar radiation (UVA and PAR) and a significant decrease in the contribution of the UVA spectrum in the water column (Figures 5 and 6). Therefore, only a few data from surface layer could be used in our calculations. Unlike the experiments carried out in the laboratory, T90 of E. coli fitted exponential function (Figure 7) which can be expressed by Eq. (4).
According to the function, when EUVA = 0 Wm−2, T90 value of E. coli ATCC 8739 amounted to 31.305, which corresponds to its value in the previous experiments in the absence of sunlight (Figure 1), which confirms the validity of function.
The negative effect of sunlight exposure was also confirmed in the study with wild cultures of E. coli kept at 4°C. A significantly lower survival (T90 = 98 ± 22 h, n = 18) was found in E. coli sampled at noon than in those sampled in the morning (T90 = 153 ± 35 h, n = 18). This might be attributed to prolonged exposure to solar radiation and its higher intensity.
Solar light is considered to be the most important factor to bacterial reduction in the sea, although its effects are restricted to shallow depths , also observed in this study. The negative effects of solar radiation to bacterial cells operate via two different mechanisms: firstly, direct photobiological mechanisms break DNA bonds in bacterial cells [39, 40], while secondly, indirect photochemical mechanisms damage bacterial cells by photosensitized reactions initiated by some of endogenous and/or exogenous sensitizers . Photochemical mechanisms become more important at higher wavelengths, and it is more injurious in the presence of oxygen . Both effects cause a rapid decrease in colony forming ability [25, 26]. Dominant toxic effect of sunlight was also observed in previous studies. Šolić and Krstulović  found that within the investigated range of intensity of solar radiation (510–830 Wm−2), the T90 of fecal coliforms exponentially decreased by about 40% when the intensity of solar radiation increased by 100 Wm−2. Large differences (30‐ to 40‐fold) in survival with or without exposure to sunlight were observed by Fujioka et al. , who noted a T90 of fecal coliforms exposed to solar radiation in the range of 0.5–1.5 h. The reduction in survival time in the presence of solar radiation was also recorded by Chandran and Hatha , where the T90 of E. coli was reduced from 26 h in the dark to 4 h in the presence of solar radiation.
According to Davies‐Colley et al. , T90 is a better indicator for expressing inactivation in the absence of solar radiation and also inactivation caused by solar radiation of an equable and higher irradiance, whereas the S90 better expresses the inactivation caused by solar radiation of variable intensity, as exists in nature. The energy of solar radiation absorbed by bacterial cells and which is responsible for cell injury is a product of the intensity of solar radiation and exposure time.
The mean values of S90 recorded in this study were 250 Whm−2 for E. coli ATCC 35218, and 610 Whm−2 for E. coli ATCC 8739. We found a medium‐strong (R2 = 0.457) and strong (R2 = 0.8282) statistically significant (p < 0.01) negative linear correlation between the intensity of the UVA spectrum of solar radiation and S90 values for both E. coli strains (Figure 8).
A high and significant correlation between the intensity of the UVA spectrum of solar radiation with T90 and S90 in this study suggests that within the studied spectrum of solar radiation, this part of the measured spectrum was most responsible for the inactivation of microorganisms. The linear regression‐slope coefficients suggested the wavelengths 320–360 nm to be the most bactericidal within the UVA spectrum (Table 2).
E. coli ATCC 35218
E. coli ATCC 8739
Contribution of different wavelengths of measured solar radiation to T90 of E. coli expressed through slopes of linear regression (b—coefficient of multiple linear regression; R2—coefficient of multiple determination; p—level of significance).
Acra et al.  found that up to 70% of the solar inactivation of bacteria can be attributed to the effect of the UVA part of the solar radiation spectrum, whereas Sinton et al.  found that 50% of the inactivation of indicator microorganisms could be attributed to solar radiation wavelengths up to 360 nm, with a wavelength of about 330 nm being most bactericidal. Calkins and Barcelo  find the UVB (280–330 nm) portion of the solar spectrum the most bactericidal, causing direct photobiological DNA damage. Although bactericidal effect of solar radiation is mainly associated with shorten wavelengths, the synergistic effect of UVB, UVA, and PAR explained most variations found in culturability of E. coli .
The values of S90 observed in this study were significantly lower than those in similar studies. S90 of fecal coliforms found by Gameson  was 1.290 Whm−2, whereas Sinton et al.  found a significantly higher value, 1.660 Whm−2 (6.0 MJm−2). Significant differences could be attributed to the different intensity of solar radiation and to variations in intensity, but also to different groups of microorganisms tested.
Lower T90 and S90 values in this study might partly be explained by the previous growth history of cell cultures until exposure to the environment, different origin and strain of bacterial cells, as well as by the different intensity of solar radiation and other environmental factors. There are many ways in which bacterial cells can enter the sea. In some cases, they are discharged directly from boats or bathers, and in others, they remain for different periods in wastewater reservoirs and/or are carried out in the sea through natural rivers or artificial conduits . The change from a nutrient‐rich environment to nutrient‐poor one, places E. coli cells under starvation stress, which is less pronounced in bacteria from the stationary‐phase because they have already experienced a starvation adaptation period. As a response to starvation stress, bacterial cells can induce protective mechanisms against UVA stress . Furthermore, starved cultures taken in the stationary‐phase of growth also showed higher osmotic  and temperature  resistance than those taken in a logarithmic‐phase. This is due to specific protein synthesis during stationary‐phase starvation . In this study, E. coli cells were taken during the logarithmic‐phase and were directly transferred to phosphate buffer without washing of nutrients. Since these cells did not experience starvation during the growth phase or during culture transfer, this probably made them less resistant to the negative effects of seawater and oxidative processes of solar radiation than cells that had experienced starvation [47, 49, 50]. Consequently, wild bacterial cells that have not experienced growth in a rich medium and are taken from the stationary phase of growth should show a higher resistance than cultivated cells, as was confirmed in this study. We found a statistically significant, at least threefold lower T90 (50.6 ± 6 h, n = 18) in cultivated wild E. coli cells than in their mother cells that were isolated from seawater (153.3 ± 35 h, n = 18), although the latter were pre‐exposed to solar light and hostile environment for unknown period before seawater samples collected.
As mentioned previously, E. coli ATCC 35218 survived longer than E. coli ATCC 8739 in almost all experimental conditions in the dark, but if exposed to solar radiation, E. coli ATCC 35218 survived significantly shorter than E. coli ATCC 8739. This suggests that these microorganisms probably do not have equally developed mechanisms of protection against various abiotic environmental factors and that the protection rate also depends on which mechanism is more effective under the same conditions.
This study showed that in the absence of solar radiation, there were no statistically significant effects of temperature and salinity in their natural range (12, 18 and 24°C; 30.0 and 36.5 psu) on the survival of two ATCC strains. The survival of E. coli ATCC 8739 was enhanced only at 6°C, suggesting a negative effect of temperature at temperatures lower than natural range. At 15.0 psu, significantly lower T90 values of E. coli ATCC 8739 were observed at lower temperatures (6 and 12°C) than at higher ones (18 and 24°C). In the natural range of temperature and salinity, mean T90 values varied significantly, but only as a function of bacterial strain and origin. The importance of bacterial strain and origin for the survival of E. coli was demonstrated by significantly longer survival of E. coli cells isolated from seagull feces and sewage outfall. In the same conditions, their survival surpassed the survival of ATCC strains up to 10‐fold. A dominate effect of solar light on E. coli survival was confirmed by recording a more rapid E. coli died‐off in cells exposed to a natural range of solar radiation. The T90 was 15‐ to 70‐fold shorter than in the absence of solar radiation. Within the investigated range of solar radiation (320–700 nm), only the effect of UVA spectrum was found to be statistically significant. The wavelengths 320–360 were the most bactericidal. The bacterial strain that had a shorter T90 in the absence of solar radiation showed a significantly higher T90 and S90 when exposed to solar light, suggesting different mechanisms of adaptation to studied abiotic environmental factors and a different efficiency of mechanisms under the same conditions. Cultivated bacteria showed threefold shorter T90 than their mother cells isolated from seawater. This suggested the importance of previous growth history of bacterial cells prior to exposure to the environment, for their adaptation capacity. The observed superiority of wild bacterial cultures over cultivated ones was probably a result of starvation experience during the stationary‐phase, and/or due to a lack of nutrients.
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