**2. Collection and storage of sample for** *E. coli* **isolation**

### **2.1. Sample collection**

*E*. *coli* predominantly inhabit the gastrointestinal tract of mammals and are shed to the environment through feces. The feces from mammals can be collected for the purpose of *E*. *coli* isolation. In this case, fresh fecal material from individual humans or animals can be used. Dry or sunburnt fecal samples may lead to false negative results. Shading of *E*. *coli* in feces makes this microorganism abundantly available in the environment. As a result, *E*. *coli* can be recovered from water, soil, contaminated food material, and surfaces.

Sampling of the soil for isolation of *E*. *coli* requires taking the sample 2–5 cm beneath the surface. Top soils may contain dead bacteria. Water samples can be collected for *E*. *coli* detection. *E*. *coli* can also be isolated from contaminated surfaces of both animate and inanimate materials. Animate surfaces include human or animal body surface. Food surfaces or working structures such as table, knives, and clothes can be a good source of *E*. *coli*. Food surfaces such as meat, eggs, or fish can be used to isolate *E*. *coli*, depending on the objective of the study. Animal fecal sample can be taken from the rectum (large animals) or fresh droppings can be collected by fingers of a gloved hand. Human stool can be put in a container with a stopper. Water samples can be collected by different methods according to nature of the water body. Still surface can be collected by hand deep method, whereas flowing water sample collection requires depthand-width-integrating methods. In this type of water body, for example, a stream, 5–10, or more samples are collected across the vertical depth and width [3]. Samples from surfaces such as hide, table, knife, and the likes can be obtained by sweeping a buffered peptone water with premoistened swabs or sponge on the sampling surface in a Z-pattern [4]. The sponge or swabs that covers approximately 400–1000 cm2 are then put in 100 ml of tryptic soya broth for further processing.

### **2.2. Sample storage**

tract are intestinal pathogenic *E*. *coli* (IPEC). Those that cause disease syndromes in systems other than gastrointestinal tract are called extra-intestinal *E*. *coli* (EXPEC). The commensal group form part of gut microbiota and is used as indicator bacteria for fecal contamination.

188 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

Pathogenic *E*. *coli* group consist of many strains, which for simplicity, can be grouped according to the virulence factors they possess or pathological effects they cause. The intestinal pathogenic *E*. *coli* include enterotoxigenic *E*. *coli* (ETEC), enteroaggregative *E*. *coli* (EAEC), enteropathogenic *E*. *coli* (EPEC), enteroinvasive *E*. *coli* (EIEC), diffusely adherent *E*. *coli* (DAEC), and verocytotoxigenic *E*. *coli* (VTEC) according to O'Sullivan et al. [24]. Extraintestinal pathogenic *E*. *coli* includes uropathogenic *E*. *coli* (UPEC), neonatal meningitis-asso-

Most pathogenic *E*. *coli* are transmitted by fecal-oral route from food materials, water, animals, and environment. Depending on the pathotype and the system, *E*. *coli* infection may cause a range of syndromes including watery, mucoid, or bloody diarrhea; abdominal cramps; urinary tract infection syndromes; and meningitis. Complications to pathogenic *E*. *coli* infection may lead to hemorrhagic uremic syndrome (HUS). These syndromes have been reported as food poisoning outbreak, travel-related illness, or animal or contaminated environment contact-related diseases. Global *E*. *coli*-related morbidities and mortalities are high. The estimates for the year 2010 show that there were 321,969,086 cases of *E*. *coli* foodborne illness which is 16.1% of global food-borne diseases. Also there were 196,617 deaths attributable to *E*. *coli*-related food-borne poisoning which is 0.02% of global mortalities due to food poisoning [2]. This situation calls for regular and continuous investigations to diag-

Inappropriate planning of research due to lack of knowledge may lead to undesired outcomes. For instance, if one aims at assessing the magnitude of shading of diarrheagenic *E*. *coli* in cattle feces, he or she may end up with underestimated results if he or she chooses to use sorbitol MacConkey agar as a screening media because not all diarrheagenic *E*. *coli* are sorbitol fermenters. Likewise, if one is looking for *E*. *coli* O157:H7 in a sample, the use of media that discriminate bacteria according to the presence of β-glucuronidase activities may lead to missing the desired outcome since *E*. *coli* O157:H7 do not possess such an enzyme. This chapter, therefore, outlines approaches to isolate and characterize *E*. *coli* from animals, humans, and the environment so that planning and implementation of *E*. *coli*-related research can

*E*. *coli* predominantly inhabit the gastrointestinal tract of mammals and are shed to the environment through feces. The feces from mammals can be collected for the purpose of *E*. *coli* isolation. In this case, fresh fecal material from individual humans or animals can be used.

ciated *E*. *coli* (NMEC), and sepsis-causing *E*. *coli* (SEPEC) [1].

nose, treat, and prevent *E*. *coli*-related diseases.

match the set objectives and desired outcome.

**2.1. Sample collection**

**2. Collection and storage of sample for** *E. coli* **isolation**

Samples for *E*. *coli* isolation are best processed right after collection, normally within 24 h. This includes inoculation into enrichment or inoculation onto solid culture media. When situation does not allow, a sample can be stored at low temperatures that restrict further cell division, but at the same time, allows survival of the bacteria. Surface water samples for *E*. *coli* isolation stored at below 10°C, but not freezing, can give comparably good results for up to 48 h after collection [5].

Sometimes analysis of fecal samples immediately after collection is impractical due to temporal and spatial challenges or assessment of old samples can be a requirement. In this case, fecal/stool samples should be stored for later laboratory isolation or old samples that were appropriately stored are recalled. Fecal samples will maintain *E*. *coli* population density, clonal characteristics, and diversity as fresh samples when stored in glycerol broth at lower temperatures than −70°C for 30 days up to 1 year. The fecal sample may form 10% of final concentration in 10% glycerol broth. However, storage of this sample at −20°C for the same time period will lead to a decrease in bacteria population density but increased diversity [6, 7]. Moreover, samples stored in glycerol broth will have more similar *E*. *coli* isolates to isolates from the fresh original sample than those from samples stored without mixing with glycerol, and if samples are repeatedly thawed, then addition of glycerol broth is recommended. Pure samples stored for a long time without glycerol lead to decrease in *E*. *coli* number [6]. Therefore, longer storage of fecal samples without appropriate processing may lead to inaccurate results.
