Diffusely Adhering Escherichia Coli

Escherichia coli is a facultative anaerobe that is part of the human colon’s microbiota. This organism colonizes the intestines of newborns as early as in the first hours of their life. Most E. coli rods are harmless and its presence is limited to the digestive tract. Nevertheless, in weakened people, patients suffering from immunosuppression and as a result of damage to the intestinal epithelial layer, even these non-pathogenic strains of E. coli may cause extraintestinal infections in humans, e.g. infections of the urinary ract, blood, wounds and other. In addition to the non-pathogenic strains of E. coli that colonize the human intestine, a large group of strains is pathogenic to humans and animals [18, 61]. Pathogenic E. coli rods include strains associated with infections of the digestive tract, as well as strains which cause extrainetstinal infections. E. coli strains responsible for intestinal infections are divided into the following pathovars depending on the virulence factors they generate: enteropathogenic E. coli (EPEC), Shiga toxinproducing E. coli (STEC) along with the subgroup of enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), adherent-invasive E. coli (AIEC) and diffusely-adherent E. coli (DAEC). Those E. coli strains that cause extraintestinal infections (ExPEC; Extraintestinal Pathogenic Escherichia coli), comprise uropathogenic E. coli strains (UPEC; Uropathogenic Escherichia coli) responsible for urinary tract infections, as well as such E. coli strains (NMEC; Neonatal Meningitis Escherichia coli) that have a K1 capsule and cause meningitis in newborns [20, 38].

DEAC strains are an extremely heterogeneous group of potentially pathogenic E. coli, which are characterized by their diffuse adherence to epithelial cells, regardless of the type of adhesins produced from the Afa/Dr family of adhesins (Fig. 1).

Fig. 1.

The Afa/Dr family of adhesins includes fimbrial adhesins, specific for the DAEC pathotype, i.e. Dr and F1845 fimbriae, as well as adhesins related to adhesins specific for the EAEC pathotype, i.e. AAF-I, AAF-II, AAF-III and HdaA fimbriae, in addition to non-fimbrial adhesins (AfaE-I, AfaE-22, AfaE-III, AfaE-V, AfaE-VII, AfaE-VIII, Dr-II and NFA-1) (Tab. I) [63, 76, 77]. The AfaE-VII adhesin is present in DAEC strains isolated from cattle, while AfaE-VIII, in strains isolated from humans and animals. In turn, the AAF-I, AAF-II and AAF-III aggregative fimbriae typical of the EAEC pathovar constitute distantly related adhesins from the Afa/Dr family. The receptor for many Afa/Dr adhesins is hDAF (CD55) (human Decay-Accelerating Factor). Furthermore, the AfaE-III, Dr and F1845 adhesins bind to human carcinoembryogenic antigens hCEACAM-1 and hCEACAM-6 (human Carcinoembryogenic Antigen related Cell Adherence Molecules), as well as CEA (Carcinoembryogenic Antigen) which is also a receptor for the NFA-1 adhesin. In addition, the Dr adhesin binds to type IV collagen. Meanwhile, the receptor of the AfaE-VIII adhesin remains unknown [51] (Fig. 2).

Fig. 2.

The operons encoding the Afa, Dr and F1845 adhesisns, i.e. the afa, dra and daa operons, have a similar genetic organization and consist of at least five genes (a-d), conservative in the Afa/Dr family of adhesins which encode chaperone proteins and the variable afaE gene which encodes the AfaE structural adhesive protein [76]. Thus far, only the AfaE-I, AfaE-III, Dr, Dr-II and F1845 adhesins have been studied thoroughly in terms of their genetic organization, identified receptors and the pathogenesis in humans.

AfaE-I is an afimbrial adhesin of typical DAEC strains which was first described in the K552 E. coli strain, isolated from a urinary tract infection case. AfaE-I is also present in DAEC strains responsible for diarrhoea. This adhesin exhibits mannose-resistant haemagglutination and has 32% identity with the Dr adhesin in terms of its sequence of amino acids. Furthermore, an adhesin which has 98% identity to the AfaE-I adhesin in terms of its sequence of amino acids has been described in a prototype strain representing EPEC, defined on the basis of its electrophoretic type as type ET5 which is included among atypical DAEC. The ET5 type includes the O55: H- and O55: H7 E. coli serotypes. In addition to the diffuse adherence ability, strains included in the ET5 type have virulence genes typical of EPEC. These are genes associated with the ability to induce histopathological changes in the epithelial cells, the so-called AE (Attaching Effacing), which consists of blurring the structure of microvilli similarly to those induced by EPEC adherence. In EPEC, lesions of the AE type confer LA (Localized Adherence), hence type ET5 presents a mixed type of LA-DA adherence [40, 76].

The AfaE-III DAEC afimbrial adhesin was first described in the A30 E. coli strain responsible for urinary tract infections [51]. This adhesin shows 98% homology to the Dr adhesin in terms of its sequence of amino acids but, unlike it, it determines adherence to epithelial cells, inhibited by chloramphenicol. Furthermore, the set of genes encoding AfaE-III is closely related to the daa operon which encodes the F1845 fimbriae. Products of the afaA and afaF genes of the afa-3 operon are homologous to the PapI-PapB UPEC proteins [23].

The Dr adhesin is encoded by a set of genes of the dra operon that determines the mannose-resistant adherence of these strains to epithelial cells. A prototype E. coli strain in which Dr adhesin has been described is the strain IH11128 (O75: K5: H-). In turn, Dr-II adhesin was first described in an EC7372 E. coli strain isolated from a case of glomerulonephritis. The Dr-II adhesin demonstrates only 20% similarity with adhesins from the Afa/Dr family, but as much as 96% homology in terms of the sequence of amino acids with the NFA-1 adhesin, present among E. coli strains responsible for urinary tract infections. Nevertheless, despite the low degree of similarity between the Dr-II adhesin and Afa/Dr adhesins, both types of adhesins are characterized by a similar genetic organization of operons encoding these antigens. In addition, strain EC7372 has a PAI (Pathogenicity Island) similar to PAI_CFT073 specific for UPEC, along with genes encoding the HlyA haemolysin and operons encoding the Pap fimbriae. In contrast to other Afa/Dr-positive E. coli, the EC7372 strain induces apoptosis and necrosis of epithelial cells [30, 70, 76].

The F1845 fimbrial adhesin of DAEC strains is responsible for the diffuse adherence of the C1845 E. coli strain, which has been isolated from a case of chronic diarrhoea in children. Research on the C1845 strain on an animal model has shown that it induces loss of colonic epithelial cells in piglets [7]. The operon encoding F1845 fimbriae comprises 5 daaA, daaB, daaC, daaD, and daaE genes located on the plasmid or on the chromosome in DAEC strains similar to the C1845 E. coli and responsible for diarrhoea. The F1845 adhesin is 57% identical to the Dr fimbriae in terms of the sequence of amino acids [6, 48]. In 1989, Bilge et al. [7] developed a diagnostic DNA probe for the DAEC pathotype that contains a fragment of the daaC gene belonging to the operon encoding fimbriae F1845. This probe, however, only identifies part of DAEC strains, among others, strains producing adhesins that belong to the Afa/Dr family, including the F1845 fimbriae. Furthermore, the daaC probe cross-hybridizes with the aafC gene which encodes the AAF-II aggregative fimbriae. In addition, a positive cross-hybridization of the daaC probe was indicated with a small percentage of strains that did not demonstrate adherence to epithelial cells [7]. Nevertheless, epidemiological studies involving the daaC DNA probe shown a relationship between the Afa/Dr DAEC strains and diarrhoea in children of over 2 years of age [76]. Although this probe’s specificity is estimated at over 95%, its sensitivity only amounts to approx. 60%. According to studies by Scaletsky et al. [73], the sensitivity of the daaC probe in a hybridization test involving 185 E. coli strains with diffuse adherence amounted to 64.3%. Lozer et al. [55], in turn, compared the frequency of detecting strains of the DAEC pathotype using the method of hybridization with the daaC probe and in a PCR reaction with genes encoding afa adhesins. The DAEC pathotype was confirmed by these researchers in 19.1% of the 560 E. coli strains tested using the daaC probe and in 25.5% of the strains tested using the PCR reaction.

The AIDA-I adhesin is an autotransporter protein of the external membrane with a molecular mass of 100 kDa, which was first described in the O126:H27 E. coli strain, isolated from a case of infant diarrhoea by Benz et al. [5]. The AIDA-I protein is a three-component antigen consisting of a functional α-domain that mediates in the specific binding of E. coli with the host cell and the β translocator (AIDA^C) [42, 43, 44, 62]. The expression of AIDA-I depends on the activity of two plasmid genes, i.e. aidA, which encodes the precursor protein with a molecular mass of 132 kDa, post-translationally modified by a product of the aah gene (autotransporter adhesin heptosyltransferase) into a mature AIDA-I glycoprotein [44]. After secretion, AIDA-I remains non-covalently bound to the surface of the bacterium. The ability of AIDA-I to self-associate into aggregates caused this protein to be included in a new group of autotransporter proteins labeled as SAATs (self-associating autotransporters) which, apart from AIDA-I, includes the Ag43 antigen responsible for the autoaggregation of numerous, pathogenic and nonpathogenic E. coli strains and TibA, an ETEC adhesin/Invasin [16]. In addition to adherence to epithelial cells, AIDA-I is involved in the formation of a biofilm by E. coli rods [78].

Besides the DAEC pathovar, the AIDA-I adhesin was detected in other pathogenic E. coli strains, among others, in ETEC that produce the STb heat-stable enterotoxin and, in strains of the STEC pathotype, i.e. Stx2 Shiga toxin and he F18 fimbriae-producing strains. The presence of AIDA-I was also found among atypical EPEC strains [22, 76, 80]. The receptor for the AIDA-I adhesin is the N-glycosylated glycoprotein pg119, present on many types of human and animal cells [44]. AIDA-I-positive DAEC strains are responsible for diarrhoea in infants. Adherence to intestinal epithelial cells by via of AIDA-I stimulates synthesis and secretion of the EspA, EspB and EspD effector proteins into the cytosol of the host cell that induce the reorganization of a cell’s cytoskeleton combined with the accumulation of actin underneath the adherent E. coli. The resulting histopathological changes in epithelial cells resemble A/E changes induced by EPEC adherence, hence strains which have this adhesin are included in the EPEC pathovar [76, 77].

On the basis of the presented adhesins and their receptors, DAEC strains have been divided into two basic groups, i.e. typical DAEC, which bind to the hDAF receptor, and atypical DAEC, which do not bind the hDAF factor. Each of these groups is divided into two subclasses. Typical DAEC of the 1st subclass include strains that have Afa/Dr adhesins which bond to the CEA antigen (Afa/Dr_CEA) i.e. the Afa-III, Dr and F1845 strains. The second subclass of typical DAECs includes strains that do not bond to the CEA antigen, i.e. the AfaE-I and Dr-II strains. Atypical DAECs of the 1st subclass include strains that demonstrate the presence of the AfaE-VII, AfaE-VIII, AAF-I, AAF-II and AAF-III adhesins. The second subclass of atypical DAEC has Afa/Dr adhesins and other ones that demonstrate diffuse adherence. This group comprises DA-EPEC strains that demonstrate the presence of the AIDA-I adhesin and have a pathogenicity island homologous to the LEE pathogenicity island (locus of enterocyte effacement) EPEC, as well as the ET5 type, which also possesses the LEE pathogenicity island and the AfaE-I adhesin [76] (Fig. 3).

Fig. 3.

Many strains of the DEAC pathovar contain numerous genes encoding uptake systems of iron ions. Almost half of these strains demonstrate the presence of the irp2 gene which belongs to the operon encoding yersiniabactin, i.e. a siderophore specific of rods from the Yersinia genus. Furthermore, among DAEC isolates, the icuB gene has been found, which is part of the operon that encodes aerobactin, i.e. a siderophore synthesized by some E. coli and Shigella strains, followed by the shuA gene of the operon enabling the use of haem, which is specifically present in Shigella and the iroN gene encoding the catecholate siderophore salmochelin, common among E. coli responsible for blood and urinary tract infections. In addition, some DAEC strains have the modD gene which belongs to the operon encoding the molybdenum transport system [9, 22, 76]. Many DAEC strains also demonstrate the ability to synthesize the Sat toxin which belongs to the sub-family of serine protease autotransporters of Enterobactericeae SPATE (Secreted Autotrasporter Toxin of Enterobacteriaeae) [31].

Infection with DAEC strains induces a strong immune response. In their in vitro studies, Betis et al. [64] demonstrated that the adherence of Afa/Dr DEAC strains to polarized intestinal epithelial cells by means of the hDAF receptor stimulates the release of IL-8 into basolateral spaces, which induces the migration of PMNL (Polymorphonuclear Leukocytes) through the intestinal epithelium [75]. The migration of PMNL through the epithelial layer is associated with the secretion of TNFα and IL-1β cytokines by enterocytes. In turn, the secreted pro-inflammatory cytokines increase the expression of the hDAF factor on the surface of enterocytes, thus enhancing DAEC adherence. Hardin et al. [34] demonstrated that IL-1β contributes to physiological changes in the intestinal epithelium under the influence of inflammation induced by an infection. That is because this cytokine stimulates the secretion of anions, inhibits the absorption of Na⁺ and Cl^– ions and the transport of glucose, dependent on the SGLT-1 transporter of sodium and glucose ions (Sodium-Glucose Transport protein-1), which ultimately contributes to the development of diarrhoea. However, Brest et al. [12] demonstrated that although DAEC strains induce the inflow of neutrophil in the intestinal mucosa, they are phagocytosed by leukocytes to a much smaller extent compared to the non-pathogenic DH5α control E. coli strain. Infection of MNL with Afa/Dr strains induces a spontaneous apoptosis of most of the leukocytes, one that is characterized by morphological changes in a cell nucleus, DNA fragmentation, activation of caspases and high expression of annexin V. Recombinant E. coli strains into which Afa/Dr adhesin genes (Afa-1, Afa-III, Dr and F1845) have been introduced, although they adhere to neutrophils, they are not engulfed. These studies suggest that the interaction of Afa/Dr E. coli with PMNL increases the virulence of these strains by inducing the apoptosis of leukocytes and decreasing their phagocytic capacities [12]. Tieng et al. [82] presented evidence of the influence of DAEC adherence and their interaction with the hDAF receptor on triggering a non-specific immune response using MICA particles (MHC class I polypeptide-related sequence). MICA are homologues of class I histocompatibility antigens (MHC-I) that are present on cells of the intestinal epithelium and constitute ligands for cell receptors involved in a non-specific, innate immune response, i.e. Tγδ lymphocytes, CD8+ cells, Tαβ lymphocytes and NK cells. The interaction of DAEC with the hDAF receptor increases the expression of MICA particles, which, in turn, stimulates the secretion of the interferon γ (IFNγ) by NK (natural killer) cells, increasing the inflammation of the intestinal epithelium. By binding to hDAF, Afa/Dr DAEC strains activate signalling pathways that engage Erk/MAP protein kinases, which stimulates epithelial cells to synthesize and secrete IL-8 [15]. In addition, the interaction of DEAC strains with the hDAF receptor induces an increase in the expression and synthesis of the VEGF bioactive endothelial growth factor by epithelial cells. Epidemiological studies indicate that Afa/Dr strains often colonize the intestinal mucosa of patients suffering from inflammatory bowel disease and patients diagnosed with adenocarcinoma of the colon, although their role in the development of these chronic intestinal diseases remains unexplained [57].

Yamamoto et al. [85] were the first to observe changes in the cytoskeleton of human epithelial cells (HeLa) under the influence of DAEC adherence. Subsequently, Peiffer et al. [69] demonstrated that the interaction of the C1845 strain with the hDAF receptor on the surface of epithelial cells promotes the extension of microvilli and this process is accompanied by an increased epithelial permeability and changes in the distribution of tight junction proteins. A DAF particle contains four SCR domains, a serine/threonine region and a glycosylphosphatidylinositol portion anchoring hDAF in the cell membrane. The SCR-3 domain, which is responsible for the protective effect of hDAF against the toxic effects of the complement cascade on cells, is also the receptor that binds Dr adhesins. The interaction between DAEC and hDAF induces the accumulation of hDAF particles underneath the adjacent bacteria, which activates a cascade of signals that engage tyrosine kinases, Cγ phospholipase, phosphatidylinositol 3-kinase (PI3K), kinase C, as well as leads to an intracellular increase in the concentration of calcium ions. The increase in the concentration of calcium ions modulates the conformation of proteins in the cytoskeleton of the brush border through calcium-dependent proteins binding actin fibres, namely villin and fimbrin. Villin is involved in organizing the actin and binds actin filaments at a low concentration of calcium ions. However, an increase in the concentration of calcium ions induces vilin fragmentation which, in turn, leads to the depolymerization of F-actin in the uppermost part of microvilli. The redistribution of proteins in the cellular cytoskeleton is accompanied by a decrease in the expression of the digestive enzymes of the brush border, i.e. sucrase-isomaltase (SI), dipeptidyl peptidase IV (DPPIV), as well as glucose (SGLT-1) and fructose (GLUT-5) transporters, which may lead to the development of diarrhoea [4, 28, 51, 68, 69]. According to researchers, the change in the electrochemical gradient of the intestinal epithelium resulting in the development of diarrhoea or the induction of an immune response may be a pathophysiological consequence of the observed phenomena. Studies by Taddei et al. [81] demonstrated that the responsibility for the rearrangement of tight junction proteins, i.e. ZO-1, ZO-3 and occludin, which plays a key role in sealing the junction between epithelial cells, lies with the serine group of the Sat toxin. Guignot et al. [31] demonstrated that the majority, namely 88%, of the DAEC strains associated with urinary tract infections, as well as about 46% of the DAEC strains responsible for diarrhoea in children carry the sat gene encoding this toxin [31]. However, these researchers failed to demonstrate the gene encoding the Sat toxin in the DAEC strains isolated from healthy children.

In turn, studies by Selvarangan et al. [74] on a mouse model demonstrated that DAEC binding to collagen is of vital importance in the pathomechanism of chronic glomerulonephritis. Isogenic mutants lacking the DraE antigen and, therefore, the ability to bind to collagen, although still capable of binding the hDAF factor, are rapidly removed from the animals’ kidneys, in contrast to the wild parental DraE-positive strain. Dr-positive strains demonstrate tropism to the basal membranes of glomeruli and the ability to cause chronic nephritis. Nowicki et al. [63] demonstrated that an infection of the urinary tract with the Afa/Dr IH11128 strain led to significant histopathological changes, corresponding to renal tubule atrophy, interstitial nephritis and renal parenchymal fibrosis. Urinary tract infections caused by DAEC strains can be particularly dangerous for pregnant women. In an experimental mouse model of chronic glomerulonephritis caused by Dr-positive strains, Kaul et al. [39] demonstrated that almost 90% of pregnant female mice infected with a Dr-positive strain bore offspring prematurely compared to 10% of mice infected with E. coli strains which did not have the Dr adhesin.

The epidemiology of DAEC strains responsible for diarrhoeas remains poorly understood due to the lack of commonly available laboratory methods for the identification of these strains. Nevertheless, DAEC strains have been identified as the aetiological factor of diarrhoea in children in Chile [45], Mexico [25], Brazil [54], Peru [65], Iran [1], Colombia [29] and the United States [17]. Furthermore, DAEC strains have been detected in children suffering from diarrhoea in Thailand [21], Bangladesh [3], Japan [59, 84], China [52], New Caledonia [26], Australia [33] and in African [66, 67, 83] and European countries, e.g. in the United Kingdom [41] and France [37, 71]. DAEC strains are isolated from healthy children and adults as often as from children suffering from diarrhoea, which makes it difficult to unequivocally determine their role as an aetiological factor of digestive tract infections. So far, the reservoir of DAEC strains and the routes of their transmission also remain unknown. In a small number of studies, the presence of DAEC strains was confirmed in drinking water samples [36] and samples containing water from irrigation systems [2]. There is also a paucity of data on the incidence of DAEC strains demonstrating the presence of the AfaE-I adhesin – AfaE-III, Dr and F1845 in animals. Meanwhile, DEAC strains carrying genes the AfaE-VII and AfaE-VIII afimbrial adhesins have been detected in calves with diarrhoea or sepsis [47, 48]. Afa-8-positive strains and strains which had genes associated with the afa/dr operon have been identified among E. coli isolates from pigs, poultry and cattle [26, 35, 49, 50, 56], but there is no evidence of their transmission from animals to humans.

Although the relationship between DAEC strains and infections, particularly those of the urinary tract, has been known for a long time, DAEC are omitted in the routine diagnosis of infections of the digestive tract and the urinary tract. This is related to the huge genotypic diversity of these strains. Phenotypically, the vast majority of DAEC strains manifests a mannose-resistant, diffuse adherence to epithelial cells [10, 60, 61]. However, an in vitro adherence test is time-consuming and quite expensive, which is why it is not used in routine diagnostics. Besides, diffuse adherence may also be manifested by other E. coli pathotypes [11, 46], which disqualifies its use in routine diagnostics. Due to the exceptional diversity of adhesins present among DEAC strains, molecular test also seem to be of little use in routine diagnostics. Nevertheless, epidemiological studies and studies on the pathogenic potential of DAEC strains are being undertaken as part of scientific research around the world. Numerous probes and primers have been developed for the purposes of genetic research on DAEC that make it possible to detect adhesins from the Afa/Dr family which are common among these strains, e.g. daaC, daaE, afaB and afaC [7, 50]. Unfortunately, recent research has shown that some of the developed probes, e.g. the daaC probe, commonly used for detection of Afa/Dr strains, cross-reacts with EAEC strains that also cause diarrhoea, which limits its use [54, 79]. An additional difficulty in diagnosing infections caused by DAEC strains is their prevalence among healthy individuals.

DAEC strains are associated with diarrhoea in babies and with urinary tract infections. The pathogenesis and diffuse adherence of this group of E. coli is determined by specific fimbrial and afimbrial adhesins, reacting with a wide range of receptors on the surface of host epithelial cells. Numerous studies have confirmed the pro-inflammatory effect of DEAC adhesins on intestinal epithelium. The latest data indicate a possible contribution of DAEC strains in the process of the development of colorectal cancer and Crohn’s disease. On the other hand, DAEC strains are isolated from patients as often as they are isolated from healthy people, which suggests that the pathogenic potential of this group of E. coli depends to a large extent on the virulence factors produced by them and the age of infected individuals. The lack of commonly available diagnostic methods hinders the correct identification of infections caused by DAEC, and therefore, the possibility of treatment which, in the case of some infections, e.g. infections of the urinary tract, may lead to serious clinical consequences.