Presence, Pathogenicity, Antibiotic Resistance, and Virulence Factors of Escherichia coli: A Review

Abstract

Escherichia coli (E. coli) is a Gram-negative, commensal/pathogenic bacteria found in human intestines and the natural environment. Pathogenic E. coli is known as extra-intestinal pathogenic E. coli (ExPEC) or intestinal pathogenic E. coli (InPEC). InPEC E. coli strains are separated into six pathogenic groups, known as enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), enteroaggregative (EAEC), enterohaemorrhagic (EHEC), and diffusely adherent (DAEC), that have various virulence factors that cause infection. Virulence factors refer to a combination of distinctive accessory traits that affect a broad range of cellular processes in pathogens. There are two important virulence factors that directly interact with cells to cause diarrhoeal diseases within the intestines: adhesion and colonization factors and exotoxins. Virulence factors are crucial for bacteria to overcome the host’s immune system and result in antibiotic resistance. Antibiotics are used to combat the symptoms and duration of infection by pathogenic E. coli. However, the misuse and overuse of antibiotics have led to the global concern of antibiotic resistance. Currently, the antibiotic colistin is the last-resort drug to fight infection caused by this bacterium. Antibiotic resistance can be achieved in two main ways: horizontal gene transfer and mutation in different genes. The genetic basis for developing antibiotic resistance in E. coli occurs through four mechanisms: limiting drug uptake, modification of the drug target, inactivation of the drug, and active efflux of the drug. These mechanisms use different processes to remove the antibiotic from the bacterial cell or prevent the antibiotic from entering the bacterial cell or binding to targets. This prevents drugs from working effectively, and bacteria can acquire antibiotic resistance. E. coli is classified into different phylogenetic groups (A, B1, B2, D1, D2, E, and clade I). It is a very versatile bacterium that can easily adapt to different environmental factors. The present review gathered information about the pathogenicity, antimicrobial resistance, and phylogenetics of E. coli. These aspects are interconnected; thus, it will provide information on tracking the spread of pathogenic strains and antibiotic resistance genes of different strains using phylogenetics and how antibiotic resistance genes evolve. Understanding genetic variation in E. coli will help in monitoring and controlling outbreaks and in developing novel antibiotics and treatment. The increasing rate of antibiotic resistance, and the ability of E. coli to evolve rapidly, suggest that in-depth research is needed in these areas.

1. Introduction

Escherichia coli (E. coli) is a Gram-negative, non-sporing, aerobic and facultative anaerobic, rod-shaped bacterium that belongs to the Enterobacteriaceae family [1,2,3]. It is an intestinal bacterium that is found in warm-blooded animals, humans, water, the environment, and in some instances, in contaminated food [1,4,5,6]. This bacterium is highly adaptable and can survive, thrive, and multiply under various environmental conditions [7].

E. coli isolates have been subdivided into pathogenic and commensal strains [1,8]. Commensal E. coli can adapt to different hosts and abiotic factors and is able to evolve to become pathogenic [4,9]. Pathogenic E. coli causes several human diseases that affect the extra-intestinal regions and gastrointestinal tract, including diarrhoeal diseases and urinary tract infections [9,10]. Pathogenic E. coli is separated into different pathotypes based on characteristics such as virulence factors, antibiotic resistance, and environmental influences [11,12,13]. Pathotypes include enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusely adherent E. coli (DAEC) [14].

Virulence factors enable the bacterial cells to colonize the host and spread, resulting in infection [15]. Specific genes found on bacterial chromosomes or mobile genetic elements (MGEs) encode for virulence factors [16,17]. Virulence factors include adhesions, exotoxins, and colonization [14,18]. In addition to virulence factors, antibiotic resistance is becoming more prevalent. Bacteria need virulence factors to disable hosts’ defence systems, and antibiotic resistance prevents drugs from fighting illnesses or infections; therefore, bacteria are able to adapt [19]. Antibiotic resistance is a result of the overuse and misuse of antibiotics in healthcare, animal husbandry, and agriculture [20,21]. Mobile genetic elements carry genes that are resistant to antibiotics and can be passed on to other bacteria via horizontal gene transfer [22,23]. This means that bacteria of the same species can share resistant traits between each other and potentially have a pool of antibiotic-resistant genes [24]. This ability emphasizes the importance of understanding the evolution and spread of antibiotic resistance [25]. Additionally, multiple studies have demonstrated that various factors such as environment, human population dynamics, and ecological conditions result in variability in E. coli populations [26,27]. This suggests that E. coli is able to adapt to different conditions.

To find relevant documents describing the presence, pathogenicity, antibiotic resistance, and virulence factors of E. coli, various searches were conducted on these different areas of interest on Google Scholar, mostly from 2010 to 2025. This review outlines the impact of pathogenic E. coli on humans and animals, the use of antibiotics to combat illnesses caused by the bacteria and its role in antibiotic resistance, and the impact of high rates of evolution on genomic variation. Understanding these subjects is essential to implement or improve on current prevention strategies and control outbreaks.

1.1. Presence of Escherichia coli in Animal Products

Livestock are a major source of foodborne pathogens [28]. The human population is increasing and, as result, the demand for the consumption of more animal products has led to a surge in animal production and processing [29]. This demand could result in flawed processing practices and the risk of contamination by foodborne pathogens from production to consumption [30]. E. coli pathogens can cause human illnesses or infections when contaminated or undercooked animal products are consumed [31,32]. From a microbiological perspective, the quality of livestock products depends on (1) the quality of products used during processing operations, (2) efficacy of the cooking process, (3) hygiene during processing and packaging, i.e., ensuring refrigeration from the point of processing to the point to the purchase, and (4) hygiene during handling at the retail point [33].

In a study, 600 raw milk and dairy products were analysed to determine the prevalence of E. coli [34]. The results reported that 30.16% (181 out of 600 samples) harboured E. coli. The O157 E. coli serogroup (43.75%) and the O26 E. coli serogroup (37.50%) were identified most frequently [34]. In a separate study by Davis and colleagues (2018), chicken and turkey retail meats were tested to identify the presence of E. coli. It was reported that 91%, 88%, and 18% of meat samples from turkey, chicken, and prepacked retail meat products (beef, chicken, pork, turkey, venison, veal, and mixed minced pork and beef), respectively, contained E. coli isolates. This study also determined that 18% (17/97) of the samples were ESBL-E. coli isolates [35]. These studies indicate that at some point during production and processing, the samples were contaminated with E. coli. This also means that if the animal products are not cooked or handled correctly, there is a risk of illness or infection. Therefore, it is vital to practice good hygiene from production to consumption.

1.2. Presence of Escherichia coli in Raw Fresh Produce

Fresh produce contains nutrients and important functional properties that can alleviate chronic illnesses and diseases [36]. Hence, recently there has been an increase in the consumption of fresh produce [37]. This high demand is associated with foodborne outbreaks as a result of bacterial contamination [38,39]. Fresh produce is exposed to its environment and handling from seed to table [40]; the risk of contamination could be due to manure that is poorly composted, exposure to contaminated water from irrigation/flooding, or by faecal matter from animals [41]. Post-harvest decontamination techniques have been used to decrease the risk of contamination; however, research has shown that it is not effective and can lead to cross-contamination [42,43]. E. coli are able to colonize plants internally, making it difficult for the bacteria to be washed away or removed using disinfectants, resulting in dissemination of the organism to uncontaminated products and eventually the food production chain via consumable products that are infested [44]. As a result, if decontamination is not effective or possible, consumption is not safe [45].

Recently, a study by Pakbin and colleagues identified the prevalence of E. coli in 115 vegetable salad samples collected from various areas in Iran [46]. E. coli was identified in 3.4% of the samples [46]. In another study, 100 raw lettuce heads were sampled from the kitchen of a military hospital in Tunis [47]. Microbiological quality testing showed that 80% of the samples had satisfactory quality, 10% was good enough for consumption, and the remaining 10% was not satisfactory [47]. Between 1973 and 2012, 18% of leafy-vegetable outbreaks associated with Shiga toxin-producing Escherichia coli (STEC) have been reported in the United States. The research mentioned above indicates that fresh produce farms need to practice or improve their hygiene and management to avoid E. coli dissemination. Hence, there is a need for more effective decontamination methods.

1.3. Presence of Escherichia coli in the Environment

Intestinal systems are the primary habitats for E. coli to survive; however, the bacterium is also able to survive in various environments [48,49]. E. coli has been examined to a great extent in laboratory conditions; however, very little is known about its behaviour in the natural environment [48]. Some E. coli strains can generate filamentous structures to attach to cell surfaces, enabling colonisation of E. coli from sources such as soil, water, manure, and contaminated seeds [50,51]. However, E. coli strains show a great ability to grow and survive in diverse environments [52]. For that reason, it is vital to understand the various environmental conditions, such as fluctuating temperatures, limited moisture, levels of oxygen, and low or high pH, and their effect on the rate of survival of E. coli [7].

In a study at broiler and laying hen farms, a higher prevalence of E. coli (77% of samples) was identified in soil from laying hen farms compared with broiler farms. In contrast, the surface water adjacent to broiler farms was more prevalent (63% of samples) than that of laying hen farms [53]. E. coli has also been located in river and sea water in Malaysia at levels above the recommended limits. Water samples were collected from 15 different sites along the Kelantan River, adjacent coastal water, and estuaries [54]. E. coli was prevalent at the Kelantan River, adjacent coastal water, and estuaries. The average abundance of E. coli present at the sample sites was above the recommended limits. E. coli found at Kota Bahru had the highest prevalence in river water, and E. coli found in the Semarak River had the highest prevalence at estuaries and coastal sites [54]. In 2021, Nguyen and others investigated the effects of steady and fluctuating nutrients on bacterial growth rate using single-cell microscopy [55]. There was an approximate 50% reduction in growth rate in samples that had a fluctuation in nutrients compared with a steady influx of nutrients [55]. These studies reflect the ubiquitous nature of E. coli and its ability to adapt to various environments and conditions.

2. Pathogenesis of Escherichia coli

In E. coli species, there are pathogenic and commensal strains: commensal E. coli strains are located in the human gut and are essential for human health [56,57], and pathogenic E. coli strains are able to colonize other animal species [1,58]. Depending on the clinical manifestations and site of infection in humans, the pathogenic strains are further sub-grouped.

Pathogenic E. coli is categorised as intestinal pathogenic E. coli (InPEC) or extra-intestinal pathogenic E. coli (ExPEC) and differentiated based on virulence factors [9,59]. ExPEC colonises the intestinal tract and does not cause gastroenteritis, but results in infections of regions external to the intestinal tract and is associated with septicaemia, urinary tract infections, and neonatal meningitis [32]. Conversely, InPEC strains can cause various forms of gastroenteritis. InPEC E. coli strains are sub-grouped into six groups: enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), enteroaggregative (EAEC), enterohaemorrhagic (EHEC), and diffusely adherent (DAEC) [58,60]. Each pathogenic sub-group is associated with different infection mechanisms and symptoms [9,21]. Different E. coli groups express specific virulence factors and are therefore associated with various diseases [61]. Understanding their localization, cell interaction, pathogenesis, and adherence patterns has the potential to assist in advancing medication and treatment processes, thereby protecting humans from disease [62]. In this review, attention is drawn to the InPEC E. coli pathogenic groups.

2.1. Enteropathogenic Escherichia coli

EPEC pathogens are associated with fatal diarrhoea in children, vomiting, fever, and nausea [63,64,65]. EPEC pathogenesis is a three-stage model, which includes (1) adhesion of bacterial cells, (2) signal transduction, and (3) intimate attachment [62,66]. EPEC pathogens do not directly attack enterocytes, but form attaching and effacing (A/E) lesions on the surfaces of the host’s intestinal epithelial cells (IECs) [32,59,67,68]. The A/E lesion in EPEC pathogenesis occurs by the bacteria attaching to and effacing the microvilli, resulting in the formation of actin pedestals at the site of attachment and damage to the microvilli [59,64,69]. A chromosomal pathogenicity island (PAI), which is approximately 35 kb of virulence genes known as the locus of enterocyte effacement (LEE) island, is necessary for virulence [32,70]. The formation of A/E lesions relies on the LEE island, which encodes the type III secretory system (T3SS), which translocates the bacterial effector protein into the host [63,70]. Intimate attachment occurs by interactions between intimin (bacterial outer membrane protein) and the intimin receptor (Tir) (bacterial receptor protein) via T3SS to produce actin pedestals [32,69]. This secretion process is necessary for EPEC pathogenesis [59]. The initial attachment of EPEC to enterocytes involves the bundle-forming pilus, which is encoded on the E. coli adherence factor (EAF) plasmid [63,65]. The EPEC bundle-forming pili enable EPEC to form microcolonies for adherence and bind to receptors on host cell surfaces [63,71].

2.2. Enterotoxigenic Escherichia coli

Enterotoxigenic E. coli (ETEC) causes travellers’ diarrhoea and diarrhoea in children from developing countries, especially in locations with poor sanitation [59,63,65,67]. ETEC infections cause watery diarrhoea, nausea, vomiting, abdominal pain, and fever [72,73]. ETEC can produce two forms of enterotoxins, known as heat-stable (ST) and heat-labile (LT), and colonization factors (CFs) [58,59,69,74]. ETEC strains inhabit the small intestine and adhere to the microvilli, mediated by colonization factors such as fimbrial, non-fimbrial, helical, and fibrillar types [58,59,63]. Following adherence, either one or two of the enterotoxins are produced and bind to the cell surface, causing diarrhoea [63,72]. Subunit A activates adenylyl cyclase, which increases cyclic adenosine monophosphate (cAMP) in cells and results in the secretion of electrolytes and water in the intestinal lumen, and subunit B binds to the GM₁ ganglioside, which allows the cell to absorb the toxin [59,63,71,72]. Guanylyl cyclase receptors bind to microvilli, which results in the ST enterotoxin accumulating cyclic guanosine monophosphate (cGMP) [14,32]. The increase in cGMP in cells results in reduced absorption of sodium chloride, causing an increased secretion of water into the intestinal lumen and passing of diarrhoeal stool [32,62,69]. It has been proposed that virulence factors and plasmid-borne toxins are the major factors that make ETEC E. coli pathogenic [72,75].

2.3. Enteroinvasive Escherichia coli

Enteroinvasive E. coli (EIEC) causes severe diarrhoea that is watery and has blood and mucus present, and in some instances, abdominal cramps [32,63,71]. EIEC pathogens are more similar to Shigella species than other E. coli pathogens in terms of taxonomy and physiology [63,65]. EIEC and Shigella species are known as facultative intracellular pathogens and have the same pathogenic mechanisms [65,76]. These are extremely invasive pathogens that reproduce in the large intestine [59,68]. EIEC pathogens attack the human colonic mucosa, microfold cells (M cells), macrophages, and epithelial cells [32,71,76]. When contaminated food or water is consumed, infection occurs once the pathogens reach the colon via M cells to reach the colonic submucosa, and the pathogen is then taken up through macrophages [62,63,76]. The pathogens enter and reproduce in the colonocytes after escaping from the macrophages [63,65]. Endotoxins and virulence factors are secreted by T3SS into the host cell, causing infection [32,63].

2.4. Enteroaggregative Escherichia coli

Enteroaggregative E. coli (EAEC, also known as EAggEC) is also a pathogen that causes travellers’ diarrhoea [59,68,69,77]. EAEC causes persistent diarrhoea in human immunodeficiency virus (HIV) patients and children, and often the stool is watery and may discharge with blood and mucus. Other symptoms are vomiting, low fever, and abdominal pain [58,67]. EAEC is phenotypically characterised by an aggregative adherence to Hep-2 cells, forming a dense cluster that has a brick-like pattern [63]. There is a three-part model that has been developed for the pathogenesis of EAEC: (1) adherence to the microvilli in the intestinal mucosa via fimbriae, (2) increased mucus production that forms a biofilm, and (3) increased production of toxins and mucosal inflammation [59,63,71,78]. EAEC pathogenesis is established by adherence to the microvilli of the intestinal mucosa by association with the afimbrial adhesins and fimbria (aggregative adhesion fimbria (AAF)) [79]. There are many strains of EAEC that contain various diverse afimbrial adhesins, also known as outer membrane proteins that have an association with aggregative adhesion [79,80]. The major subunit of the AAF has five different variants, aggA (AAF/I), aafA (AAF/II), agg3A (AAF/III), agg4A (AAF/IV), and agg5A (AAF/V), that are associated with different EAEC strains [81]. The second step is the production of cytotoxins and enterotoxins, which results in microvillus vesiculation, increased epithelial cell extrusion, and enlarged crypt openings [82,83]. EAEC strains produce three main toxins: Shigella-like enterotoxin (ShET1), plasmid-encoded toxin (Pet), and EAEC heat-stable enterotoxin (EAST1) [63]. EAST1 accumulates cGMP by the activation of guanine cyclase, which results in secretion of water into the intestinal lumen. Pet causes a disruption in the enterocyte cytoskeleton, which results in cell detachment, and ShET1 also plays a role in the secretion of water [78,84,85]. Inflammation depends on the strain of EAEC and the host’s immune system; thus, when the gastrointestinal tract is infected, the adhesion of EAEC to the intestinal epithelial cells results in inflammatory diarrhoea [86].

2.5. Enterohemorrhagic Escherichia coli

Enterohemorrhagic E. coli (EHEC) is a Shiga toxin-producing E. coli (STEC) that causes haemolytic uraemic syndrome and haemorrhagic colitis [9,63,65,68,69]. Most outbreaks are food-associated and cause abdominal pains and bloody diarrhoea [59,65]. Common foods that are undercooked, such as meat, dairy products, and raw vegetables, are known to be the reservoirs for EHEC, and infection can also occur via contact with animals that are infected [32,59]. LEE encodes the eae gene, which produces intimin on the bacterial outer membrane, and Tir, which is moved via the T3SS into the host cell [66]. An intimate attachment is formed when Tir attaches to the bacterial intimin protein and is characterised by A/E lesions when the pathogen colonizes the large bowel [65,68]. After attachment, the T3SS passes the bacterial effecter proteins into the host cell [63]. This allows STEC to modify the host cell environment, leading to infection. The major virulence factor for EHEC is the phage-encoded Shiga toxin (Stx) and is sub-grouped into Stx1 and Stx2 [59]. Stx is an AB₅ toxin that consists of five B subunits and is bound non-covalently to an A subunit that is enzymatically active [63,65]. The B subunits of Stx receptors bind to kidney epithelial cells and the globotriaosylceramides (Gb3s) in the intestinal mucosa [14,87,88,89]. An interaction between the Stx subunit B and the Gb3 causes membrane invaginations to enable the toxin to internalize; it is then transported to the Golgi apparatus, where the subunit A (RNA-glycosidase, which inhibits protein synthesis) removes an adenine nucleotide, which results in cell death [63,68,89]. The Stx does not inhibit protein synthesis or initiate necrosis, but reduces chemokine expression, thus suppressing inflammatory responses [90].

2.6. Diffusely Adherent Escherichia coli

Diffusely adherent E. coli (DAEC) pathogens colonize in the small bowel and are possibly the cause of watery diarrhoea in infants and children that are younger than five years [63]. DAEC pathogens attach to host cell surfaces (HeLa and Hep-2 cells) in a diffuse adherence pattern that is mediated via fimbrial (Dr and F1845) and afimbrial adhesins (Afa) causing diarrhoea [59,74]. On epithelial cells (intestinal and urinary), there is an interaction of Afa–Dr adhesins with brush border-associated complement decay-accelerating factor (DAF), causing rearrangement or destruction of the cytoskeleton structures of the microvilli [91]. A few DAECs also bind with the Carcino-Embryogenic Antigen-Related Cell Adhesion Molecule (CAECAM-1 and CAECAM-6), and when this binding occurs, the pathogens are internalized into the epithelial cells [14]. Microtubules and lipid draft of DAF-expressing cells allow internalization, resulting in pathogen colonization [14]. After attachment, secreted autotransporter toxin (SAT) creates lesions on epithelial cells at tight junctions, which results in an increase in cellular permeability, causing the induction of watery diarrhoea; however, uncertainty still surrounds this induction mechanism [63].

3. Intestinal Escherichia coli Pathogens’ Virulence Factors

Most E. coli strains are commensal; however, there are some pathogenic strains that cause infection or diseases in humans [9,92,93]. As mentioned previously, the well-known intestinal pathogenic E. coli are EPEC, EHEC, ETEC, EAEC, EIEC, and DAEC. Infection or disease caused by intestinal E. coli pathogens are a result of various virulence factors [9,92,93]. The most important virulence factors that are associated with intestinal E. coli pathogens are adhesion and colonization factors and exotoxins, which have direct interactions with epithelial cells [9,93]. Surface structures such as fimbrial adhesins and afimbrial adhesins mediate the adhesion to epithelial cells [93]. Exotoxins are not secreted; however, they are proteins that are able to target the cell metabolism, cell cytoplasmic membrane, or the cell skeleton [93].

3.1. EPEC Virulence Factors

Initially, bundle-forming pili were suspected to be involved in adherence; however, recently they are considered to assist bacteria with attaching together to form microcolonies, also known as localized adherence patterns [92]. They are also involved in intestinal colonization and may also have a part in protecting bacteria from antimicrobial agents [94]. The T3SS is crucial for Gram-negative pathogenic bacteria to disrupt host signal pathways by directly injecting virulence proteins [95]. The LEE is a pathogenicity island that plays an important role in forming the A/E lesions [92]. The LEE pathogenicity island contains essential functional genetic clusters that are involved in the pathogenic process by using the T3SS. The espA, espB, and espD genes are integral to the physical structure of the injectisome, enabling the bacteria to directly inject virulence factors into the host cell. The translocated intimin receptor (tir) gene produces the effector Tir, which binds to intimin, creating an intimate attachment of the bacteria to the host cell, and the experimental autoimmune encephalomyelitis (eae) gene codes for the intimin adhesin [92,93]. The injectisome has a dual role in both adherence and effector protein translocation [95].

3.2. EHEC Virulence Factors

Key virulence factors of EHEC are Shiga toxin (Stx), also known as verotoxin (VTx) families, known as Stx1 and Stx2 [74,96]. Stx1 and STx2 are approximately 60% similar and comprise five B subunits that are soluble [97,98,99]. Once the toxin is released, it binds to the glycolipid globotriaosylceramide (Gb3) on the target cell surface [74]. This results in catalytic inactivation of the ribosomal subunit, thereby inhibiting protein synthesis in endothelial or epithelial cells [9,96]. Additionally, verotoxins are also able to induce cell death in many cellular types [96]. Verotoxins are associated with haemolytic–uremic syndrome in people and oedema disease in pigs [92,95]. The genes encoded for VTx are found on phages and enable bacteria to colonize the intestines [92,93,100].

3.3. ETEC Virulence Factors

Toxins are released by activating the guanylate cyclase pathway, which increases the production of cyclic guanosine monophosphate (cGMP), disrupting ion transport [92,101]. AB toxins comprise two different subunits, one A subunit and one or more B subunits, and other AB toxins are made of two subunits that correspond to the A and B subunits [92]. The A subunit is the toxic element, and subunit B is responsible for binding toxins to receptors [92]. The toxic activity of the AB toxin is activated by enzymatic cleavage, causing interference with the secretion pathways, DNA metabolism, cytoskeleton integrity, or various protein syntheses [93]. Heat-labile enterotoxin (LT) involves the type 2 secretion system, which transports proteins to remove signal sequences [92]. LT consists of an A subunit and B subunits. The B subunit is responsible for binding to specific receptors (the main receptor is ganglioside GM1) on the apical surface of enterocytes, initiating receptor-mediated endocytosis [92]. The A subunit undergoes a conformational change and translocates to the cytoplasm, activating adenylate cyclase and leading to an increase in cAMP levels within the host cell [92]. Heat-stable enterotoxin (STa and STb) and LT increase the secretion of intracellular messengers such as chloride, carbonate ions, and water and inhibit the absorption of sodium, resulting in watery, cholera-like diarrhoea [9,89,92,102].

3.4. EIEC Virulence Factors

Infection by EIEC occurs by proteins such as IpaB and IpaC entering the host epithelial cells via penetration using the T3SS [62]. IpaC plays a role in induing actin polymerization and activating the GTPases Cdc42 and Rac, which forms cell extensions [74]. IpAs change cell extensions via binding to vinculin, which results in actin depolymerization, allowing bacterial cells to enter [74]. IpgD is involved in the internalization of bacteria into host cells during infection [62,103,104]. After this, the bacteria reproduce inside the cells and enter and damage surrounding epithelial cells, resulting in infection [104,105,106].

3.5. EAEC Virulence Factors

In EAEC, virulence factors are coded on pathogenicity islands and plasmids known as the plasmid of aggregative adherence (pAA) in the chromosome [107]. The most important virulence factor is AggR, a member of the transcriptional regulator family (AraC), which is found on the pAA to control the expression of virulence genes [108]. EAEC involves the attachment and adherence to the intestinal mucosal cells and the secretion of EAST1 [79]. AAF, which are encoded by AAF genes, are involved in aggregative adherence [79]. The toxin secretion results in enlarged crypt opening, microvillus vesiculation, and epithelial cell extrusion, causing watery diarrhoea [68].

3.6. DAEC Virulence Factors

The diffused adherence pattern is mediated by the fimbrial adhesion family, which is made up of six variants, which are Dr, Afa, Nfa, M, AAF, and F1845. The genes for fimbrial adhesins are coded on chromosomes or plasmids [83,93]. The Afa, Dr, and M variants are true fimbrial adhesins, while the F1845 and AAF code for small fibrillae and the Nfa variant generates capsule-like structures that surround the bacterial cell. Fimbrial adhesins bind to the glycoprotein known as decay-accelerating factor (DAF), which is distributed on urinary, intestinal, human, and haemopoietic endothelial cells [93]. These fimbriae break down the actin network, causing long cellular extensions, elongation, and malfunction of the microvilli in intestinal cells [89,91,109]. There are some Afa/Dr adhesins that bind to the epithelial cell surface, resulting in CDC42 activation, CEACAM aggregation during adhesion, microvilli effacement, and internalization of the bacterial pathogen, causing infection of the intestinal epithelial cells [14,91].

4. Antibiotics: Uses and Resistance Concerns

Antibiotics are compounds that have antimicrobial activity and occur naturally or are synthetic or semi-synthetic [110]. These compounds are administered orally, parenterally, or topically [111]. Antibiotics are used for purposes such as growth promotion in food animals, in human medicine and veterinary medicine for major medical procedures, and to prevent disease [112,113]. Additionally, antibiotics are used in feed and water of animals that are bred for food consumption to increase the growth and size of the animal and in human and veterinary medicine to control the spread of disease, prevent infections, and treat sick individuals [114,115]. The focus here is on antibiotics used in human medicine, specifically, to treat diarrhoeal diseases.

Antibiotics are vital in the control and treatment of E. coli infections, especially in cases were diarrhoea is persistent, with the aim of managing and reducing the symptoms and duration of infection [116,117]. The antibiotic used for treatment depends on the susceptibility of E. coli. Antibiotics such as fluoroquinolone or trimethoprim/sulfamethoxazole (TMP/SMZ) are commonly used to treat diarrhoeal diseases to shorten the time period of infection [118]. Infections caused by Shiga toxin-producing varieties are significantly reduced by using the azithromycin antibiotic [119]. Other antibiotics used are carbapenems (imipenem and meropenem, ertapenem, and doripenem), Cephalosporins, aminoglycosides, tigecycline, colistin, Fosfomycin, and a combination of antibiotics [120]. When diarrhoeal diseases last for more than one day and the individual experiences fever or dehydration for more than one week with the use of antibiotics, faecal specimens should be evaluated [121]. This may be a result of antibiotic resistance. Although using antibiotics to treat diarrhoeal diseases is successful, antibiotic resistance is rising at an alarming rate and becoming a major concern [115,122].

Humans have become more vulnerable to diseases due to antibiotic resistance, which is a result of infections being treated poorly the and overuse and negligent use of antibiotics; treatment is difficult because pathogens are immune to the available antibiotic drugs [123,124,125,126]. Over time, E. coli has become resistant to the antibiotics that are used to treat diarrhoeal diseases, and there is a lot of interest around the antibiotic resistance in E. coli because of intestinal infections that cause diarrhoea [9,127]. It is important to determine how resistance is developed and how resistant genes are transferred between various bacteria as well as between bacteria and hosts [128]. Several countries have established programs to monitor antibiotic resistance; however, misuse and mismanagement is still an issue [129]. Understanding the manner in which antibiotic resistance is spread is important to developing effective management strategies [129]. Figure 1 shows how resistant bacteria are developed and spread to humans, animals, food, and the environment. It is also of particular concern the role that animals play in being a reservoir for resistant genes or pathogens that may result in human diseases [130].

The frequency of antimicrobial resistance is increasing, and the lack of development of new drugs poses a serious threat to the treatment of infection. Gram-negative bacteria pose the greatest threat to human health, since resistance is developed at a greater rate and there are few antibiotics available for infections associated with this type of bacteria [131]. Multidrug-resistant Gram-negative bacteria such as carbapenemase-producing Enterobacteriaceae have sparked an interest in the colistin antibiotic, which is used for treating Gram-negative infections [132]. Colistin is an antibiotic that is widely used in veterinary medicine and rarely used in human medicine [133]. This antibiotic interacts with the outer cell membrane of Gram-negative bacteria [134]. Currently, in human medicine colistin is the last therapeutic option for the treatment of infections caused by carbapenemase-producing bacteria. The first report of the colistin resistance gene mcr-1 was identified in China in 2015 and was detected in livestock animals, raw meat, and humans [135]. Since this discovery, there have been various studies conducted in Asia, North Africa, Europe, and North and South America to determine the global spread of the mcr-1 gene. The mcr-1 gene has been detected in Salmonella, Shigella, Klebsiella, Vibrio, and Enterobacteria; however, it is most often detected in E. coli. A study by Grami and colleagues in 2016 [136] showed that the mcr-1 gene mediated colistin resistance between countries through the trading of food-producing animals and retail meat. This indicates that the worldwide spread of the plasmid-encoded colistin resistance gene mcr-1 reflects a major topic at the interface between human and animal health [136]. Thus, it is critical to determine the prevalence of the mcr-1 gene.

4.1. Genetic Basis of Antimicrobial Resistance in E. coli

The genetic makeup of bacterial pathogens allows them to thrive in extreme environmental conditions, even in the presence of antibiotic molecules [137]. Resistance is naturally present or can be obtained in bacteria via different mechanisms associated with several genetic loci [138,139,140]. Antibiotic resistance can be attained in two main ways: (1) horizontal gene transfer (acquiring foreign resistant DNA) and (2) mutation in different genes [137,141].

4.2. Horizontal Gene Transfer

Antimicrobial resistance by horizontal gene transfer is an important factor for bacterial evolution [137]. DNA can be transferred in three ways: (1) transformation, (2) transduction, and (3) conjugation by drivers such as bacteriophages, plasmids, and extracellular DNA [25,69,137,142]. Horizontal gene transfer by transformation occurs when extracellular DNA is recombined into a host cell [25]. When bacterial genes are passed through viral particles, then transduction has taken place [25]. The transfer of genetic material through conjugation involves cell-to-cell contact and takes place in the gastrointestinal tract [137]. Mobile genetic elements such as plasmids (transferable DNA elements), integrons, and transposons play a vital role in developing and distributing antimicrobial resistance [69,137]. Antibiotic resistance and multiple-drug resistance can be explained by the ability of bacteria to exchange genetic material [140]. In other words, when environmental selective pressures are strong, antibiotic resistance genes are acquired by bacteria, which decreases their susceptibility to antibiotics [25].

4.3. Mutations

Bacterial pathogens that are susceptible to antibiotics can develop mutations in genes as a result of random events such as replication errors or when damaged DNA is incorrectly repaired in cells that are actively dividing [143]. These mutations affect the activity of the antibiotics, which increases the survival rate of the bacteria in the presence of that antibiotic [137]. In bacterial cells, antibiotics have various targets, access, and protection pathways; thus, several genes may be involved in antibiotic resistance [144]. Essentially, mutations that result in antibiotic resistance will eradicate bacteria that are susceptible and allow the resistant bacteria to thrive [145]. Mutations that result in antimicrobial resistance modify the antibiotic action through one of these mechanisms: (1) modifying the antimicrobial target, (2) reducing the drug uptake, (3) activating efflux mechanisms to remove the harmful molecule, or (4) altering essential metabolic pathways through the modulation of regulatory networks [137,140]. Therefore, resistance acquired through mutational changes is diverse and varies in its complexity [137].

4.4. Mechanism of Antibiotic Resistance in E. coli

In general, Gram-negative bacteria can acquire or develop antibiotic resistance through four mechanisms, which include (1) limiting drug uptake, (2) modification of the drug target, (3) inactivation of the drug, and (4) active efflux of the drug [146,147,148]. All four mechanisms occur on the chromosomal genes of bacteria within a species or from other bacteria by horizontal gene transfer [128,146]. Figure 2 shows the general antimicrobial resistance mechanism, and

Table 1. Summary of antibiotic resistance mechanisms found in Escherichia coli species [59].

Antimicrobial Agents	Mechanisms of Resistance	Genetic Basis
β-lactams	β-lactamases inactivate drugs	ampC
Penicillins	Active efflux	bla genes-plasmid
Cephalosporins		(TEM, SHV, CTX-M, NDM)
Monobactams		acrAB(tolC), acrAD(tolC)
Carbapenems
Aminoglycosides	Aminoglycoside-modifying enzymes	aac, ant, aph-plasmid
Amikacin	Modify target-16S rRNA	amrA, rmtB
Gentamicin	Active efflux	mdtEF(tolC)
Tobramycin
Tetracyclines	Limiting uptake	ompF
Tetracyclines	Active efflux	acrAB(tolC)
		tetA, tetB-plasmid
Chloramphenicol	Limiting uptake	ompF
	Active efflux	acrAB(tolC)
Fluoroquinolones	Limiting uptake	ompF
Ciprofloxacin	Modified target-gyrase	gyrA
Norfloxacin	Modified target-topoisomerase IV	parC
	Active efflux	acrAB(tolC), acrEF(tolC)
		mdtABC(tolC)
Metabolic pathway inhibitors	Target enzyme modification	TMP-dhfr
Trimethoprim/Sulfamethoxazole		SXT-dhps

shows the summary of antibiotic resistance mechanisms found in E. coli species.

• (1) Limiting drug uptake

Bacteria can limit the uptake of antimicrobial molecules by decreasing the absorption, increasing the discharge, or using both mechanisms concurrently [146]. The outer membranes of bacteria form a permeability barrier which antibiotics must pass to reach their target [144,146]. Hence, when the antimicrobial’s ability to enter the bacterial cell is reduced, it inhibits the antimicrobial from meeting the target [141]. Gram-negative bacteria comprise a lipid bilayer and porins that serve as the route of entry for hydrophobic (aminoglycosides, quinolones, and macrolides) and hydrophilic (β-lactams) antibiotics to pass through the bacterial outer membrane [59,137,144]. Mutations at porin sites cause inactivation or downregulation of porin proteins like OprD, leading to reduced permeability to antimicrobials [149]. The quantity and variety of these porins significantly affect the ability of hydrophilic antimicrobials to penetrate the bacterial cell, ultimately affecting the antibiotic susceptibility of the bacterium [150,151]. The access of drugs is limited by changes in the porin channels in two ways: (1) reduction in the number of porins and (2) reduction in the binding properties or function of the porin channel due to a change in charge [59,128,148]. In E. coli species, the production of porin may decrease or stop, or they may produce a different porin as a result of mutations [128,145]. Antimicrobial agents that are susceptible to limitation of drug uptake in E. coli are tetracyclines, erythromycins, fluoroquinolones, and chloramphenicol due to the decrease in permeability and increase in efflux [59,144]. Antimicrobials must be able to reach the target site for binding to prevent bacterial growth; thus, any mechanism that hinders this process will lead to ineffective treatment [145].

• (2) Modification of the drug target

Gram-negative bacteria modify drug targets to allow resistance to antimicrobial groups such as β-lactams, fluoroquinolones, aminoglycosides, and the combination drug trimethoprim/sulfamethoxazole (TMP/SXT) [59,128,137,152]. Spontaneous mutations in the genes encoding the protein targets cause drug targets to be modified [145]. Hence, antibiotics are prevented from binding, and this results in treatment failure. Drugs generally target the bacterial ribosome, which results in the inhibition of protein synthesis. There are many mechanisms that protect the ribosomal units of bacteria from being attacked by the drug, such as ribosomal mutation, ribosomal methylation, or ribosomal protection, which inhibit the ability of the drug to bind to the ribosome [146,148,153]. Aminoglycoside drugs bind to ribosomes and inhibit protein synthesis. The targets on ribosomes are modified in bacteria through methylation, which results in the bonding ability of the drug decreasing [128,146]. Many methyltransferases have been characterized and identified. The armA and rmtB genes encode for aminoglycoside resistance methylase and ribosomal methyltransferase, respectively [59,128]. Fluoroquinolone drugs hinder nucleic acid synthesis by competing for the DNA binding site of DNA gyrase or topoisomerase IV [59,137,148]. Resistance to fluoroquinolone drugs is a result of mutations in the chromosomally encoded ParC subunit of topoisomerase IV (parC gene) or the GyrA subunit of gyrase (gyrA gene), which reduces the binding ability [147]. Research has also demonstrated that low-level resistance may develop through plasmids carrying quinolone resistance (qnr) genes [137,154,155]. Penicillin-binding proteins (PBPs) are peptidases that play a role in formation of the peptidoglycan cell wall [59,137,146]. Gram-negative bacteria produce PBPs that are β-lactam resistant. Resistance to β-lactam drugs occurs through alterations in the structure or number of PBPs or both [147]. This results in the inability of drugs to bind. There are numerous PBPs that are naturally produced by E. coli, and certain E. coli species decrease their binding affinity to certain β-lactam drugs. This mechanism enables bacteria to acquire antibiotic resistance, which presents a major challenge in the treatment of infections [145].

• (3) Inactivation of the drug

One of the most effective methods bacteria use for protection is to either degrade or change the active component of the antimicrobial drug [145]. There are two main ways that drug inactivation is achieved in bacteria: (1) degradation of the antibiotic or (2) transfer of a chemical group to the antibiotic [59,144,148]. Drug inactivation against aminoglycoside and β-lactams is used by Gram-negative bacteria. β-lactamase enzymes inactivate β-lactam drugs, which results in degradation of the drugs, and E. coli produces many β-lactamase enzymes [59,146]. The β-lactam drugs’ main structure comprises a four-sided ring known as a β-lactam ring [148]. β-lactamases inactivate the drugs by hydrolyzation at a particular site in the β-lactam ring [137,156]. This results in the opening of the ring and inactivation of the drug because the drug cannot bind to the target proteins. There are many β-lactamases that have been identified to inactivate β-lactam drugs, and Gram-negative bacteria use this mechanism against β-lactam drugs [137,146,157]. Transferring a chemical group to the drug also results in drug inactivation. Modifying enzymes catalyse biochemical reactions such as adenylation, acetylation, and phosphorylation [128,148]. These are transfer mechanisms that are used against the aminoglycosides, chloramphenicol, streptogramins, and fluoroquinolone drugs [148]. Aminoglycosides-modifying enzymes (AMEs) are located in mobile genetic elements and provide the best example of drug inactivation [128,137,158]. AMEs are classified based on their biochemical activity. These enzymes include the adenyltransferases (ANTs, ant genes), phosphotransferases (APHs, aph genes), and acetyltransferases (AACs, aac genes) [128,137,147,158]. The AMEs covalently alter the amino or hydroxyl group, making it inactive [145].

• (4) Active efflux of the drug

For a drug to have an effect, it needs to be in the bacteria’s system for a long time in high concentrations; however, some bacteria use efflux pumps to remove drugs as soon as they enter [159]. Efflux pumps are common mechanisms used to remove toxic substances outside the bacterial cell [137,147,156]. Bacteria use efflux pumps to export antibiotics, and this is an essential contributor to Gram-negative bacteria’s intrinsic resistance [145]. Efflux pumps have encoded genes that are either constitutively expressed or the expression is promoted by the environment [59,148]. Several substances can be transported through the efflux pump; thus, it is known as a multidrug resistance (MDR) mechanism [146,160]. There are five efflux pump groups known, which are the small multidrug resistance (SMR), multidrug and toxic compound extrusion (MATE) family, resistance-nodulation cell division (RND), ATP-binding cassette (ABC), and major facilitator superfamily (MFS) [59,137,148].

The SMR efflux pumps are hydrophobic and are powered by the proton-motive force (H⁺) that exports lipophilic cations [148,161]. The genes encoding for SMR efflux pumps are located in chromosomal DNA and mobile genetic elements like plasmids and transposons [148]. This efflux pump has a narrow substrate range and contributes resistance to β-lactams and some aminoglycosides [148].

The MATE efflux pumps use a sodium ion gradient as the energy source in the bacterial membrane [148,162]. MATE pumps have been shown to efflux fluoroquinolones and aminoglycosides, which are important treatments for infections [148,163]. The NorE pump is a MATE efflux pump found in E. coli that transports fluoroquinolones. However, whether the clinical significance of the NorE pump has an influence on antimicrobial resistance is still questionable [164]. MATE efflux pumps are found mostly in Gram-negative bacteria, which often face more complex challenges with antibiotic resistance due to the presence of the outer membrane [148].

The ABC (ATP-binding cassette) transporter family is one of the most significant because it has an uptake and efflux system [148,159]. To transport amino acids, drugs, ions, polysaccharides, proteins, and sugars, the pumps use ATP hydrolysis [148,165]. This transport system consists of six transmembrane segments comprising α-helices, functions as either homodimers or heterodimer pairs in the membrane, and works in combination with cytoplasmic ATPases [148].

In Gram-negative bacteria, RND efflux pumps have clinical significance and determine resistance to many antimicrobials [59,137,148]. The RND efflux pumps use a substrate/H⁺ antiport mechanism to remove substrates [148,161]. This pump plays a role in removing antibiotics, detergents, heavy metals, solvents, and dyes [148,166]. Some RND pumps are substrate specific, for example, Tet pumps for tetracycline and Mef pumps for macrolides [148]. The RND efflux pumps are tripartite: (1) an inner membrane transporter, (2) an outer membrane porin, and (3) a periplasmic accessory protein [59,137,144]. This pump functions by connecting to an OMP, which is stabilized by MFPs [167]. The genes for the RND pumps are arranged as an operon, and the gene for the regulator is next to the MFP gene, which is next to the main pump gene followed by the OMP gene [148]. There are five known RND pumps in E. coli: AcrAD-TolC and AcrAB-TolC to efflux β-lactams and aminoglycosides, MdtABC-TolC to efflux quinolones, MdtEF-TolC to efflux erythromycin, and AcrEF-TolC to efflux quinolones and tigecycline [59,137,161].

The MFS efflux pumps use either the proton-motive force (H⁺ gradient) or sodium gradient (Na⁺) for the active transport of anions, drugs, metabolites, and sugars [148,168]. This indicates that MFS pumps have a great substrate diversity; however, individual pumps are substrate specific [148]. E. coli have individual MFS pumps for macrolides (MefB), fluoroquinolones (QepA), and trimethoprim (Fsr) [148]. In E. coli, there are five MFS efflux pumps that are known that can transport fluoroquinolones (QepA2, EmrAB-TolC, and MdfA pumps), macrolides (MefB and MdfA pumps), chloramphenicol (MdfA pump), trimethoprim (Fsr pumps), and tetracycline (EmrAB-TolC and MdfA pumps) [169].

5. Phylogenetics

E. coli strains are classified into the phylogenetic groups A, B1, B2, D1, D2, E, and clade I [170,171,172]. Non-pathogenic E. coli that inhabit the gastrointestinal tract belong to group A; commensal and certain pathogenic strains belong to group B1; InPEC belong to groups D1, D2, and E; most ExPEC belong to group B2; and strains that are phenotypically indistinguishable and genetically diverse belong to clade I [12,173,174]. Categorizing the E. coli strains into different phylogenetic groups provides important information on E. coli populations and helps to explain relationships between strains and diseases [175]. E. coli is one of the most adaptable bacteria because it can colonize and persist in the host [74]. It has a high genome plasticity, which enables the bacteria to evolve by gaining and losing genes via genetic changes, leading to the development of pathogenic strains from the commensal strains. In the evolutionary process, E. coli strains require virulence factors to become pathogenic [74]. Most virulence genes are found on transmissible genetic elements; thus, genes can be exchanged via horizontal gene transfer and can rearrange in other bacteria. Studies on phylogenetics of the core genome of various E. coli pathotypes have shown interrelated genetic structures [21]. Furthermore, researchers have shown that InPEC lineages have evolved numerous times, under different genetic makeups, and joined into a specific pathotype [176,177,178,179]. Environmental factors have a strong influence on E. coli evolution; this means that genes and mobile elements that are acquired from environmental bacteria play a role in the diversification of the E. coli genome [21]. During this process of evolution, commensal and pathogenic strains can function as genetic sources that gain or lose DNA to become pathogenic or revert to commensalism, respectively [180]. Environmental factors also have a great influence on gaining or losing genes or mobile elements, causing bacterial genome diversification [21]. The in-depth analysis of whole genomes is possible because sequencing technology is always advancing; thus, E. coli genome evolution can be studied extensively [181]. Gene content is the most important source of variation between genomes and is due to specific selective pressures experienced during evolution [182]. For example, 5.5 Mb genome of two E. coli strains (O157:H7 EDL933 and K-12 MG1655) shared a core genome of 4.1 Mb, indicating that the majority of the sequences were highly similar [181]. Also, analysis of more E. coli strains showed that a significant percentage of genes have been laterally acquired, and this means that there is a shared core genome [181]. Thus, phylogenetic analysis enables us to understand how the bacterium evolves over time and identify genetic variations or similarities. This will be beneficial for studying disease outbreaks and developing new drugs to combat diseases. Furthermore, phylogenetic analysis can provide information about genetic changes that contribute to antibiotic resistance and its dissemination, which ultimately will help to improve management and prevent emerging infections.

6. Conclusions

Every person requires food to survive, and with the continuous increase in the population, there is a higher demand on farming practices to meet the need. Studies have shown that E. coli is present in animal products and fresh produce, and it can survive in multiple environments. E. coli isolates acquire specific virulence attributes by adapting to the environment, thus allowing a wide range of infections [92]. Additionally, to meet the increasing meat demands, livestock production commonly uses antibiotics as growth promoters and to control bacterial pathogens that may cause diseases [183]. However, the overuse of antibiotics results in the rapid increase in resistant bacteria, which decreases the efficacy of antibiotics [184]. This has led to infection by E. coli becoming more prevalent in communities and hospitals in recent years [120]. In pharmaceutical industries, a major challenge is the lack of development of novel drugs for the treatment of bacterial infections [185]. As a result of the rise in resistance to antibiotics, the ineffectiveness of the available treatments for bacterial infections could possibly become fatal [185]. The State of the World’s Antibiotics report in 2015 showed that the rise in drug-resistant Gram-negative bacteria such as E. coli has led to reliance on the colistin antibiotic as a last line of defence. Spreading of antibiotic resistance can be limited by practising good hygiene from production to consumption, using antibiotics rationally, screening, treating, and raising awareness/education to prevent spread from person to person [186]. The above-mentioned prevention measures are good to contain the spread of antibiotic resistance; however, in-depth research to develop new antibiotics and increase the efficiency of existing drugs is necessary and will be beneficial [187]. Importantly, it should be noted that to date, no new antibiotics have been discovered to fight the bacteria that are resistant to antibiotics [188,189]. The disease-causing aspects of pathogenic E. coli is commonly studied; however, they can also differ in evolution [182]. It is evident that E. coli isolates have evolved numerous times through HGT, recombination, and/or mutation, which contributes to the genetic diversity of bacteria [182,190]. As a result, the emergence of new E. coli isolates will always be a challenge. Knowing the phylogenetic history of the core genome of E. coli isolates compared with the variable genome will provide meaningful and updated information of pathotypes [179]. E. coli is a widely studied bacterium that is highly adaptable, and thus, its characteristics are always evolving [191]. This rate of evolution means that researchers need to constantly be analysing E. coli strains to determine zoonotic transmission potential and its implications for human health. Thus, future research should focus on identifying the genetic composition of E. coli strains via phylogenetics to aid in controlling outbreaks and monitoring the spread of antibiotic resistance, ensuring development of new drug therapies and treatment strategies, and identify possible future antibiotic-resistance patterns [190].