Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative coliti (UC), are chronic incurable gastrointestinal inflammatory disorders with unknown etiology (1). Previously, IBDs were regarded as a disease prevalent in industrialized countries. However, in the 21st century, as the epidemiological trend of this disease changed, IBDs have become a global problem, and new cases in developing countries in Asia, South America, and Africa are on the rise (2).

They are probably the result of improper and continuous initiation of the intestinal mucosal immune system due to the complicated interactions of genetics, microbial, and immunological agents (3).

As shown by previous experimental and clinical research, intestinal bacteria play a role in the development of IBD and the severity of the disease (4). According to the recent molecular studies on patients with IBD, beneficial bacteria, for instance, Firmicutes and Bacteroidetes, have reduced, and pathogenic bacteria, for example, Proteobacteriae, particularly Escherichia coli, have increased (5).

Recently, the links between IBD and adherent-invasive Escherichia coli (AIEC) strains have been discussed (6–9). It is possible to classify E. coli strains into commensal or pathogenic categories based on genetic and phylogenetic characteristics. The definition of the pathogenic potential of E. coli is changing to some extent. AIEC was recently recognized as a pathotype of E. coli (late 1990s), and the variable range of AIEC was different from the six primary diarrheagenic E. coli pathotypes (Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Shiga toxin-producing E. coli (STEC), diffusely adherent E. coli (DAEC), Enterotoxigenic E. coli (ETEC), and Enteroaggregative E. coli (EAEC) (10). AIEC is well-known for its role in clinical and experimental epidemiological studies in IBD pathogenesis (11–14). These strains have the ability of adherence and invasion to intestinal epithelial cells (IECs) and extensive survival in macrophages by secreting high levels of tumor necrosis factor alpha (TNF-a) (15). Adherence of this pathotype through type 1 pili expression in the bacterial surface and through cell adhesion molecule 6 (CEACAM6) is attributed to the presence of carcinoembryonic antigen in the ileal epithelial cells' apical surface (16, 17). CEACAM6 is an AIEC receptor that has been indicated to have an abnormal expression in the ileal epithelial cells of adult CD patients (18).

Genetically, AIECs are very close to extraintestinal pathogenic E. coli, which includes uropathogenic E. coli as well as neonatal meningitis-related strains (5, 19). Based on the strong evidence concerning AIEC role in the promotion of gut inflammation and exacerbation of IBD pathology, most of the AIEC isolates belong to the D and B₂ phylogenetic groups of E. coli, as shown by genomic studies. In addition, based on the distribution of AIEC strains from the phylogenetic perspective, the dominant force in the formation of this pathotype is convergent evolution (11, 12). Previous research has shown that AIEC encodes a large subunit of propandiol dehydratase (a fermentation product of 1,2-propandiol fucose) that is elevated in the microbiome of patients with CD and directs intestinal T cell inflammation induced by AIEC (20).

Moreover, Rath et al. reported that the main role of AIEC in Crohn's disease is to impair the mitochondrial function of epithelial cells. Additionally, different types of intestinal inflammation have shown mitochondrial dysfunction, and it has been indicated that mutations in mitochondria modulating genes are susceptible to IBD (21).

To enhance information about the epidemiology of AIEC in IBD, comprehensive study on the prevalence of AIEC in IBD patients worldwide is believed to be of great value. Therefore, the current systematic review and meta analysis aimed to investigate the prevalence of AIEC isolates and their phylogenetic in IBD patients compared with the controls and the association of colonization by AIEC with IBD, which was quantified by estimating pooled odds ratio (OR) along with 95% confidence interval for OR.

A total of 205 articles were retrieved by the initial search of databases, of which 182 were removed following selection based on titles, abstract, and index review, and 23 studies were selected for full-text analysis. After assessment of the 23 reviewed studies, three studies collected samples from stool specimen (9, 25, 26), one study had a methodology problem (27), one study collected samples from animal sources (28), two studies did not report the prevalence of AIEC isolates (12, 29), results of a study was unclear in terms of the number of patients and biopsy samples (30), one study was performed on standard isolates (6), and one study did not report the results of control group (31).

Finally, 13 case–control studies met the eligibility criteria for inclusion in the meta analysis. Figure 1 shows a flow diagram illustrating the searching procedure for the selection of eligible studies (16, 32–43). Also, the detailed features of the included articles are accessible in Tables 1, 2. All of the included studies used intestine biopsy samples, bacterial adhesion, invasion assay, bacterial survival, and replication in macrophages methods for the detection of AIEC. All of the articles were case–control studies published between 2004 and 2020.

Totally, there were 465 IBD cases (348 CD and 117 UC) and 307 controls. Given that the three articles did not mention the sex of patients, the participants were almost 242 men and 239 women.

The current study was a comprehensive systematic review, and meta analysis was conducted to investigate the association of colonization by AIEC with IBD, IBD UC, and DC types. Based on previous studies, the mucosa-associated E. coli may be important in the pathogenesis of IBD, UC, and CD.

Additionally, AIEC has the ability to invade Peyer's patches and the lamina propria through M cells (44). AIEC could be adopted into macrophages, replicate, and survive within them because of the host autophagy defect. It then triggers the secretion of TNF-α through activating infected macrophages and increasing proinflammatory cytokine expression (45). Based on the studies reviewed, the incidence of IBD between colonized individuals with AIEC was different. These differences may be explained by the distribution and composition of different intestinal microbiota depending on the involvement of host and/or environmental factors (43).

In recent years, several studies have shown the role of intestinal microbiota in the development of IBD. It has been shown that in patients with IBD, the balance of intestinal bacteria is disturbed and the number of beneficial bacteria such as Bifidobacteria, Lactobacilli, and Firmicutes is reduced and the number of possible pathogenic bacteria such as Bacteroides and E. coli is increased (46). Studies have shown that an increase in Bacteroides and E. coli and a change in intestinal microbiota composition due to a high-fat/high-sugar diet increases the sensitivity to AIEC and intestinal inflammation in CEABAC10 transgenic mice (34, 45).

Majority of included studies in our systematic review and meta analysis showed significant association between IBD and the presence of AIEC; however, there were few studies with insignificant results. The non-significant results in these studies may be attributed to the low sample size and high type 2 statistical error rate. Totally, the estimated pooled OR from all included studies in our meta analysis resulted in significant relationship between AIEC rate and IBD, in which the strength of association was 2.93 and 3.07 for IBD (irrespective of disease type) and IBD CD type, respectively.

Based on literature review, individuals with high levels of CEACAM6 and CHI3L1 receptors, which are overexpressed in inflammation, promote AIEC adhesion and invasion to IECs located in the ileum via the type-1 pili's FimH adhesion (44, 47) or colonic IECs via the chitinase ChiA (48), and subsequently, the colonization of AIEC strains promote the secretion of IFN-γ and TNF-α by macrophages, which are likely to stimulate granuloma formation and is a common histological feature of CD (45). Overall, these findings suggest that AIEC strains in CD patients can promote their own colonization and the ensuing inflammatory amplification cycle (47).

Meta regression analysis indicated the association of AIEC with IBD and CD does not significantly confound by age.

Our results suggested that the affect by IBD in patients colonized with the AIEC is not random, it is a specific disease-related pathovar. That is because this pathovar has the ability to invade epithelial cells and attach to receptors of CEACAM with specificity for the oncofetal carbohydrate antigens that are overexpressed by mucosal glycoconjugates in the inflammation condition. AIEC is pertinent in IBD due to the contribution of genetic mutations associated with defects in handling intracellular microbes in the disease pathogenesis and to the intestinal injury. Thus, the mucosal environment of an individual susceptible to IBD could be exploited by these bacteria; otherwise, their proliferation might be an outcome of a normal flora depletion (49).

However, in patients undergoing AIEC colonization, the use of antibiotics may be effective in certain conditions. Antibiotics such as ciprofloxacin and rifaximin are safer alternatives for patients with CD concomitant AIEC because they have fewer side effects than immunosuppressive drugs (50). On the other hand, heptilmenoside derivatives have been shown to have strong antiadhesion effects and in vivo/in vitro protective effect against colitis, which means that they can be useful compounds for the treatment of patients with AIEC colonized patients (51). Moreover, according to previous report, the effect of diet on intestinal homeostasis and AIEC severity suggests that combined dietary use can be used to alter the availability of luminal nutrients, along with drug therapies, to limit AIEC growth and implantation (17).

In the present work, E. coli strains included A, B₁, B₂, and D phylogroups based on the availability of chuA, yjaA, and TspE4.C2 genes (52). These were described on the basis of their multilocus enzyme electrophoresis patterns (MLEE). Subsequently, they were grouped by DNA-based multilocus sequence typing (MLST), and whole genome sequences confirmed it (53). B₂ and D groups were the main parts of extraintestinal pathogenic strains of E. coli. However, A and B₁ groups were the lowest pathogenic E. coli strains and are defined as non-human enteropathogenic strains (54).

Researchers have recently compared non-AIEC and AIEC strains of the same phylogroup and identified three genomic regions in all the B₂ phylogroup AIEC strains, which are absent in AIEC strains of other phylogroups and commensal strains with any phylogenetic origin (e.g., B₂) (12). Nevertheless, it is not known if these regions are only specifically present in B₂-AIEC strains or are also available in other pathogenic groups with the same phylogenetic origin, like B₂ ExPEC strains (12). Our results implied that most frequent AIEC phylogroup in the IBD, CD, and control groups was B₂ and the least frequent phylogroup in the IBD and CD was B₁, which was D in the control group. Therefore, it could be concluded that AIEC strains belonging to phylogenetic groups B₂ might have the ability to colonize and survive in epithelial cells and macrophages in patients, particularly those with chuA gene.

In summary, this meta analysis revealed that colonization by AIEC is more prevalent in IBD. Our meta-analysis results indicated that there is a significant association between colonization by AIEC with IBD totally and IBD CD type. In addition, the most prevalent AIEC phylogroup among the IBD patients was B₂ and the least prevalent one was B₁. Meanwhile, the most frequent AIEC phylogroup among the control group was B₂ and the least frequent one was D. Our results suggested that the affect by IBD in patients colonized with the AIEC pathovar is not random, it is in fact a specific disease-related pathovar. Based on our findings, AIEC is neither commensal nor a real pathogenic strain, but it is a pathobiont that expands rapidly in the host and can apply special pathogenic influences. Although, our study showed a significant association between colonization by AIEC and IBD, these findings are based on case–control studies therefor the cause-and-effect relationship as well as directional dependency cannot be inferred. Longitudinal prospective studies will provide more reliable evidence about the directional association. However, these findings provide evidence on the importance of this strain in the treatment of IBD patients.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

This study used publicly available data. The studies involving human participants were reviewed and approved at the time of original publications (please see references and data availability statement). Written informed consent was not necessary to obtain for this study.

HF and AF conceived the study. AF, RK, MH, and PA wrote the study protocol and data analysis plan. RK and MH did the systematic review, requested individual participant data, and did the study quality assessments. AF did the statistical analysis. AF, HF, MH, RK, and PA interpreted the data. MH, RK, HF, and AF wrote the first draft of the manuscript. All authors reviewed and approved the final manuscript and had full access to all the data in the study and final responsibility for the decision to submit for publication.

This study was supervised by AF and supported in part by a grant from Isfahan University of Medical Sciences [Grant no. 199279, Ethics Code: IR.MUI.RESEARCH.REC.1399.287].

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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