**1. Introduction**

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

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 [1]. 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.

determination of the concentration of *E. coli* cells by epifluorescence microscopy method,

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

The bottles were then stirred and placed in a temperature controlled air incubators at three

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

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

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

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

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

(30.0 and 36.5 psu). Bacterial concentration in bottles was targeted to 103

isolated from sewage and wild *E. coli* strain isolated from seagull feces.

measured by optical radiometer PRR800 (Biospherical Inc.).

the dark and every 10 or 20 min in experiments performed in solar light.

 CFU/mL, was kept at 4°C until the beginning of the experiments

Effect of Environmental Conditions on *Escherichia coli* Survival in Seawater

CFU *E. coli*/100 mL. The bottles were then stirred and left

CFU *E. coli*/100 mL.

http://dx.doi.org/10.5772/67912

171

appropriate dilution, cca 103

different temperatures (12, 18 and 24°C).

bottles was also targeted to 103

**2.2. Experiments with natural samples**

(cca 20 h).

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 [8] 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 [14]. 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 [15]. 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.
