**2.** *Escherichia coli*

*Escherichia coli* is one of the best studied organisms. It belongs to the family of *Enterobacteriaceae*. It is a Gram-negative rod-shaped bacterium, non-sporulating, nonmotile or motile by peritrichous flagella, chemoorganotrophic, facultative anaerobic, producing acid from glucose, catalase positive, oxidase negative and mesophilic [6].

It is a well-known commensal bacterium that is part of the gut microbiota of humans and other warm-blooded organisms. However, also pathogenic strains of *E. coli* do exist and can cause a variety of intestinal and extraintestinal infections in humans and many animal hosts. *E. coli* is considered to be one of the most important pathogens; it is the most frequently isolated species in clinical microbiology laboratories [7]. Intestinal pathogenic *E. coli* (IPEC) strains, also called diarrhoeagenic *E. coli* (DEC) strains, are divided into six different well-described categories, i.e*.* pathotypes: enteropathogenic *E. coli* (EPEC), enterohaemorrhagic *E. coli* (EHEC), enterotoxigenic *E. coli* (ETEC), enteroaggregative *E. coli* (EAEC), enteroinvasive *E. coli* (EIEC) and diffusely adherent *E. coli* (DAEC) [8]. DEC causes diarrhoea syndromes that vary in clinical presentation and pathogenesis depending on the strain's pathotype [7]. *E. coli* strains involved in diarrhoeal diseases are one of the most important among the various etiological agents of diarrhoea [9]. The extraintestinal pathogenic *E. coli* (ExPEC) strain group is comprised of different *E. coli* associated with infections of extraintestinal anatomic sites [10]. Traditionally, the ExPEC isolates are separated into groups determined by disease association, i.e*.* uropathogenic *E. coli* (UPEC), neonatal meningitis-associated *E. coli* (NMEC) and sepsis-causing *E. coli* (SEPEC), naming the most important ExPEC groups. But ExPEC strains are also implicated in infections originating from abdominal and pelvic sources (e.g. biliary infections, infective peritonitis and pelvic inflammatory disease) and also associated with skin and soft tissue infections and hospital-acquired pneumonia [11]. Due to its genotypic and phenotypic diversity, *E. coli* is known as the paradigm for a versatile bacterial species [12].

The pathogenic strains possess specialised virulence factors such as adhesins, toxins, iron acquisition systems, polysaccharide coats and invasins that are not present in commensal strains [7].

### **3. Adhesins**

Adhesins play a very important role in the host-microbe interactions, as they convey the adherence to the epithelial host's cells, surface structures or molecules.

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Adhesion is the essential first step for most commensal and pathogenic bacteria in order to colonise and persist within the host [13]. While adhesion to abiotic surfaces is usually mediated by non-specific interactions, adhesion to biotic surfaces typically involves specific receptor-ligand interaction [14]. Adhesins are structures on the bacte-

*Scanning electron microscopy of Escherichia coli strain 963 adhering to 19-day-old Caco-2 cells [5]. The fimbrial structures on the bacterial surfaces are promoting the bacterial adherence to receptors on host cells.*

rial surface that help the bacteria to bind to receptors on host's cells (**Figure 1**). Adhesins are not just involved in adherence but also in bacterial invasion, survival, biofilm formation, serum resistance and cytotoxicity [15]. Moreover, they are also involved in bacterial motility and DNA transfer [13]. They differ in their architecture and receptor specificities. Types of adhesin vary depending on the

Adhesins are among the most important virulence-associated properties of *E. coli*, as they are the main virulence factors of bacteria needed in bacterial colonisation. There are two types of bacterial adhesins: fimbrial and afimbrial [16]. Fimbrial adhesins, i.e. fimbriae, are rodlike structures with a diameter of 5–7 nm. Each fimbria consists out of several hundred copies of a protein, whose generic name is 'major subunit', and other proteins, present in one or a very few copy number and called 'minor subunits' that are positioned either at the basis or at the top of the fimbriae or intercalated between the 'major subunits' [16]. Fimbriae can be even longer than 1 μm [13]. On the bacterial surface of wild-type *E. coli* strains, there are around 500 fimbriae [17]. P-fimbriae and F-17 fimbriae belong to

Non-fimbrial adhesins are monomeric or trimeric structures that decorate the surface of bacteria. These adhesins are anchored to the surface of the outer membrane and due to their small size, the size of non-fimbrial adhesins is approximately 15 nm, allow an intimate contact between the bacterial cell surface and specific substrates. One of the major classes of non-fimbrial adhesins is autotransporter

P-fimbriae are the most extensively studied adhesins. They are also the first virulence-associated factor found among uropathogenic *E. coli*. These fimbriae bind to Gal(α1–4)Galβ moieties of the membrane glycolipids on human erythrocytes

*DOI: http://dx.doi.org/10.5772/intechopen.85164*

Gram nature of bacteria [15].

**Figure 1.**

the fimbrial adhesins.

adhesins [13].

**3.1 P-fimbriae**

*Annealing Temperature of 55°C and Specificity of Primer Binding in PCR Reactions DOI: http://dx.doi.org/10.5772/intechopen.85164*

#### **Figure 1.**

*Synthetic Biology - New Interdisciplinary Science*

to obtain a certain protein.

**2.** *Escherichia coli*

such subsequent experiments are nucleotide sequencing, in order to determine the nucleotide sequence of the insert or in vitro transcription, and translation, in order

*Escherichia coli* is one of the best studied organisms. It belongs to the family of *Enterobacteriaceae*. It is a Gram-negative rod-shaped bacterium, non-sporulating, nonmotile or motile by peritrichous flagella, chemoorganotrophic, facultative anaerobic, producing acid from glucose, catalase positive, oxidase negative and mesophilic [6]. It is a well-known commensal bacterium that is part of the gut microbiota of humans and other warm-blooded organisms. However, also pathogenic strains of *E. coli* do exist and can cause a variety of intestinal and extraintestinal infections in humans and many animal hosts. *E. coli* is considered to be one of the most important pathogens; it is the most frequently isolated species in clinical microbiology laboratories [7]. Intestinal pathogenic *E. coli* (IPEC) strains, also called diarrhoeagenic *E. coli* (DEC) strains, are divided into six different well-described categories, i.e*.* pathotypes: enteropathogenic *E. coli* (EPEC), enterohaemorrhagic *E. coli* (EHEC), enterotoxigenic *E. coli* (ETEC), enteroaggregative *E. coli* (EAEC), enteroinvasive *E. coli* (EIEC) and diffusely adherent *E. coli* (DAEC) [8]. DEC causes diarrhoea syndromes that vary in clinical presentation and pathogenesis depending on the strain's pathotype [7]. *E. coli* strains involved in diarrhoeal diseases are one of the most important among the various etiological agents of diarrhoea [9]. The extraintestinal pathogenic *E. coli* (ExPEC) strain group is comprised of different *E. coli* associated with infections of extraintestinal anatomic sites [10]. Traditionally, the ExPEC isolates are separated into groups determined by disease association, i.e*.* uropathogenic *E. coli* (UPEC), neonatal meningitis-associated *E. coli* (NMEC) and sepsis-causing *E. coli* (SEPEC), naming the most important ExPEC groups. But ExPEC strains are also implicated in infections originating from abdominal and pelvic sources (e.g. biliary infections, infective peritonitis and pelvic inflammatory disease) and also associated with skin and soft tissue infections and hospital-acquired pneumonia [11]. Due to its genotypic and phenotypic diversity, *E. coli* is known

as the paradigm for a versatile bacterial species [12].

ent in commensal strains [7].

The pathogenic strains possess specialised virulence factors such as adhesins, toxins, iron acquisition systems, polysaccharide coats and invasins that are not pres-

Adhesins play a very important role in the host-microbe interactions, as they convey the adherence to the epithelial host's cells, surface structures or molecules.

In our experiments, the aim was to determine the nucleotide sequence of several fimbrial genes from different *Escherichia coli* (*E. coli*) strains isolated from faecal samples of dogs with diarrhoea. The genes of interest were *papA*, *papG*, *papEF* of the P-fimbriae and *F17G* of the F17-fimbriae. Therefore, from a collection of 24 clinical haemolytic *E. coli* strains from faecal samples of dogs with diarrhoea [5], genomic DNA was isolated and used as the matrix DNA to amplify these genes of interest with gene-specific primers with PCR. Further, the obtained PCR products were cloned into a TA cloning vector, and the nucleotide sequence was determined.

**166**

**3. Adhesins**

*Scanning electron microscopy of Escherichia coli strain 963 adhering to 19-day-old Caco-2 cells [5]. The fimbrial structures on the bacterial surfaces are promoting the bacterial adherence to receptors on host cells.*

Adhesion is the essential first step for most commensal and pathogenic bacteria in order to colonise and persist within the host [13]. While adhesion to abiotic surfaces is usually mediated by non-specific interactions, adhesion to biotic surfaces typically involves specific receptor-ligand interaction [14]. Adhesins are structures on the bacterial surface that help the bacteria to bind to receptors on host's cells (**Figure 1**).

Adhesins are not just involved in adherence but also in bacterial invasion, survival, biofilm formation, serum resistance and cytotoxicity [15]. Moreover, they are also involved in bacterial motility and DNA transfer [13]. They differ in their architecture and receptor specificities. Types of adhesin vary depending on the Gram nature of bacteria [15].

Adhesins are among the most important virulence-associated properties of *E. coli*, as they are the main virulence factors of bacteria needed in bacterial colonisation. There are two types of bacterial adhesins: fimbrial and afimbrial [16].

Fimbrial adhesins, i.e. fimbriae, are rodlike structures with a diameter of 5–7 nm. Each fimbria consists out of several hundred copies of a protein, whose generic name is 'major subunit', and other proteins, present in one or a very few copy number and called 'minor subunits' that are positioned either at the basis or at the top of the fimbriae or intercalated between the 'major subunits' [16]. Fimbriae can be even longer than 1 μm [13]. On the bacterial surface of wild-type *E. coli* strains, there are around 500 fimbriae [17]. P-fimbriae and F-17 fimbriae belong to the fimbrial adhesins.

Non-fimbrial adhesins are monomeric or trimeric structures that decorate the surface of bacteria. These adhesins are anchored to the surface of the outer membrane and due to their small size, the size of non-fimbrial adhesins is approximately 15 nm, allow an intimate contact between the bacterial cell surface and specific substrates. One of the major classes of non-fimbrial adhesins is autotransporter adhesins [13].

#### **3.1 P-fimbriae**

P-fimbriae are the most extensively studied adhesins. They are also the first virulence-associated factor found among uropathogenic *E. coli*. These fimbriae bind to Gal(α1–4)Galβ moieties of the membrane glycolipids on human erythrocytes

#### **Figure 2.**

*Scheme of pap and F17 operon and annealing sites of the used primers. Genes in the operon are presented as boxes. The positions of used primers to amplify the studied genes are marked with arrows. (A) Scheme of pap operon. The scheme of pap operon was drawn based on the GenBank deposited nucleotide sequence X61239.1 [20] and (B) scheme of F17 operon. The scheme of F17 operon was drawn based on the GenBank deposited nucleotide sequence L77091.1 [26].*

of the P blood group and on uroepithelial cell fimbriae [18]. Further receptors for P-fimbriae are present on erythrocytes from pigs, pigeon, fowl, goats and dogs but not on those from cows, guinea pigs or horses [19]. These fimbriae are encoded in the *pap* operon, consisting of 11 different genes (see **Figure 2A**): *papA* (558 bp), *papB* (315 bp), *papC* (2511 bp), *papD* (720 bp), *papE* (522 bp), *papF* (504 bp), *papG* (1008 bp), *papH* (588 bp), *papI* (234 bp), *papJ* (582 bp) and *papK* (537 bp) [20].

The product of the *papA* gene is the major subunit protein A (19.5 kDa) [19]. In *papB* a regulatory protein (13 kDa) is encoded. PapB is necessary for the activation of the *papA* expression [21]. PapC (80 kDa) is located in the outer membrane and forms the assembly platform for fimbrial growth. PapD (27.5 kDa) is present in the periplasmic space and is involved in the translocation of fimbrial subunits across the periplasmic space to the outer membrane prior to assembly. PapE (16.5 kDa), PapF (15 kDa) and PapG (35 kDa) are minor fimbrial components. PapG is the adhesin molecule conferring the binding specificity [19]. PapH (20 kDa) terminates fimbrial assembly and helps anchor the fully grown fimbriae to the cell surface [22]. PapI (12 kDa) is another regulatory protein involved in *papA* expression due to activation of *papB* promoter [21]. PapJ (18 kDa) is a periplasmic protein required to maintain the integrity of P-fimbriae [23]. PapK (20 kDa) regulates the length of the tip fibrillum and joins it to the rod [24].

Many variants of P-fimbriae exist. PapA molecules from different P-fimbrial serovariants have a high degree of similarity at the N and C termini, while the central portions of PapA exhibit a great variation in the primary structure. This central part of PapA is hydrophilic and exposed and hence under selective pressure from the host immune system. Substantial heterogeneity is also between different minor fimbrial subunits (PapE, PapF and PapG) [19]. In addition also P-fimbriarelated fimbriae, the so-called Prs-fimbriae, exist. Prs-fimbriae are encoded in the *prs* (*pap*-related sequence) operon [18].

#### **3.2 F17-fimbriae**

F17-fimbriae are found on pathogenic *E. coli* strains, isolated from infections in domestic animals. They are mainly detected on bovine and ovine *E. coli* associated with diarrhoea or septicaemia but also on *E. coli* from other hosts, including humans. The F17 adhesin binds to N-acetyl-d-glucosamin receptors of bovine intestinal cells;

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however, F17 subtypes were also found to bind to N-acetyl-d-glucosamin receptors of human uroepithelial and intestinal cells [25]. The F17-fimbriae are encoded in the *F17* operon, consisting of four genes: *F17A* (546 bp), *F17D* (723 bp), *F17C* (2469 bp)

F17A protein (20 kDa [25]) is the structural component of the F17-fimbriae (major subunit protein). The F17A protein is homologous to PapA protein of the P-fimbriae [27]. F17C protein (90 kDa) probably functions as a base protein on which the fimbrial subunits are polymerised. F17D protein (28 kDa) has a close homology to the PapD protein of the P-fimbriae [28]. It functions as the periplasmic transport protein [29]. F17G protein (36 kDa [25]) is the minor fimbrial component required for the binding of the F17-fimbriae to its receptor on the host cell [30]. Several variants of F17-fimbriae exist. The diversity is based on differences in F17A and F17G genes. The variant of F17-fimbriae found in humans is designated as

The analysed 24 clinical haemolytic *E. coli* strains [5] originated from dogs with diarrhoea and were isolated at the Veterinary Microbiological Diagnostics Centre of Utrecht University, the Netherlands. Some more details about the strains are given in **Table 1**. As positive control strains, a dog uropathogenic *E. coli* strain (strain 1473) and a cattle mastitis *E. coli* strain (strain E5) from Wim Gaastra's *E. coli* collec-

All used bacterial strains were stored at −80°C as a suspension in a 1:1 mixture of L-broth and glycerol as published by Garcia et al. [32]. The strains were grown overnight on LB plates and in liquid LB medium at 37°C. When grown in liquid LB medium, the flasks with the bacterial culture were incubated with aeration.

Chromosomal DNA was isolated from all 24 clinical haemolytic *E. coli* strains [5] and strains used for positive controls [31] using a slightly modified protocol based on the protocol of miniprep of bacterial genomic DNA published by Ausubel et al. [33]. To summarise, 2 ml of an overnight bacterial culture was centrifuged for 2 min at 14,000 rpm at room temperature. The obtained bacterial pellet was resuspended in 567 μl of buffer TE and 6 μl of 0.5 M EDTA. The suspension was incubated for 15 min at −80°C. Following the incubation at −80°C, the suspension was thawed, and 10 μl of 25 mg/ml proteinase K solution was added. The suspension was mixed thoroughly, and 30 μl of 10% SDS was added to the suspension and mixed thoroughly again. A 2-hour incubation at 37°C followed, and then 100 μl of 5 M NaCl was added to the suspension and mixed thoroughly. Next 80 μl of CTAB/NaCl was added, mixed thoroughly again and incubated at 65°C for 10 min. After the incubation the suspension was treated with 200 μl of chloroform/isoamyl alcohol and centrifuged for 5 min at 14,000 rpm at room temperature. The aqueous supernatant was transferred to a fresh microcentrifuge tube and treated with 100 μl of phenol/ chloroform/isoamyl alcohol and centrifuged for 5 min at 14,000 rpm at room temperature. The aqueous supernatant was transferred to a fresh microcentrifuge tube, and the DNA in the aqueous supernatant was precipitated with addition of 0.6 volume of isopropanol. The precipitated chromosomal DNA was transferred to a fresh microcentrifuge tube containing 100 μl of 70% ethanol. The precipitated

*DOI: http://dx.doi.org/10.5772/intechopen.85164*

and *F17G* (1035 bp) (see **Figure 2B**) [26].

G-fimbriae, encoded in the *gaf* operon [25].

**4.1 Bacterial strains, growth media and conditions**

**4. Materials and methods**

tion were used [31].

**4.2 Isolation of chromosomal DNA**

*Annealing Temperature of 55°C and Specificity of Primer Binding in PCR Reactions DOI: http://dx.doi.org/10.5772/intechopen.85164*

however, F17 subtypes were also found to bind to N-acetyl-d-glucosamin receptors of human uroepithelial and intestinal cells [25]. The F17-fimbriae are encoded in the *F17* operon, consisting of four genes: *F17A* (546 bp), *F17D* (723 bp), *F17C* (2469 bp) and *F17G* (1035 bp) (see **Figure 2B**) [26].

F17A protein (20 kDa [25]) is the structural component of the F17-fimbriae (major subunit protein). The F17A protein is homologous to PapA protein of the P-fimbriae [27]. F17C protein (90 kDa) probably functions as a base protein on which the fimbrial subunits are polymerised. F17D protein (28 kDa) has a close homology to the PapD protein of the P-fimbriae [28]. It functions as the periplasmic transport protein [29]. F17G protein (36 kDa [25]) is the minor fimbrial component required for the binding of the F17-fimbriae to its receptor on the host cell [30].

Several variants of F17-fimbriae exist. The diversity is based on differences in F17A and F17G genes. The variant of F17-fimbriae found in humans is designated as G-fimbriae, encoded in the *gaf* operon [25].
