Background
Escherichia coli inhabit the large intestine of healthy humans and other warm-blooded animals, but in some instances it can produce a wide range of extraintestinal infections. This bacterium can easily acquire virulence factors (VFs) and mobile genetic elements from related bacteria that leads to different pathogenicity [
1]. Extraintestinal pathogenic
E. coli (ExPEC) isolates are highly complex and have a variety of VFs and may belong to different phylogenetic lineages. These strains cause complicated urinary tract infections (UTIs), bacteremia, and sepsis [
2].
UTI is one of the most common infectious diseases accounting for approximately 40% of all nosocomial infections and 10–20% of hospital-acquired infections [
3]. UTI is associated with considerable morbidity and costly health problems. They cause a variety of clinical signs from asymptomatic bacteriuria to pyelonephritis, cystitis, and septic shock with multi-organ systems failure [
4]. The most common pathogen causing UTI is a heterogeneous group of ExPEC, named uropathogenic
E. coli (UPEC) [
5]. UPEC strains cause 75–95% of uncomplicated and 40–50% of complicated UTIs [
3]. Based on the available literature, UPEC strains evolve from non-pathogenic strains by acquiring new VFs through horizontal gene transfer (HGT) [
6]. The
E. coli genome consists of a main core genome and a mobile gene pool that determine pathotype or ecotype specific traits [
3]. Various VFs have been attributed to UPEC pathogenesis; however, there is no general agreement regarding the definitive discriminatory virulence factors within this pathotype. UPEC isolates need VFs for colonizing or invading host cells, escaping or disrupting hosts’ immune systems, damaging host tissues, and/or stimulating inflammatory responses. Among variety of VFs, some are generally accepted to be more associated with UPEC [
7].
fim operon,
pap operon, and
sfa genes encode type I fimbriae, P fimbriae and S fimbriae respectively [
8,
9]. These structural VFs are the main attachment factors associated with colonization of organism to host cells [
7]. Apart from adhesins, some virulence genes encode toxins such as hemolysin (
hly gene), cytotoxic-necrotizing-factor (
cnf1 gene), and sidrophores (
fyuA gene) that are mainly involved in intracellular survival, iron-acquisition, escape from immune system, inflammatory response, and host tissue damage [
8‐
10]. UPEC isolates may carry pathogenicity-associated islands (PAIs) which carry sets of different virulence associated genes [
8]. Some of these virulence genes are also found in the commensal isolates and are not specific to pathogenic isolates. However, most available studies have only investigated the prevalence of virulence genes in UPEC associated isolates.
Due to variability in the gene content and the possibility of HGT among different
E. coli isolates, it is vital to understand the genetic basis of differences between commensal and UPEC isolates, to be able to prevent ExPEC and UPEC infections more effectively. This information can be acquired through case-control epidemiological studies [
4,
11]. Therefore, in the present study, we investigated the frequency and relationship between different VFs and phylogenetic groups of UPEC and commensal isolates.
Discussion
To have a better knowledge on the pathogenesis of the UPEC, it is necessary to identify virulence markers of various strains that cause UTIs. VFs, as the potential clinical predictors, help clinicians to manage patients and anticipate the evolution of infection in the host body [
6,
13]. However, except for the genetic characteristics of the virulence strains, host factors play an important role in the incidence and outcomes of the infection [
14,
15].
Fimbriae and adhesins are frequently reported as VFs in the UPEC isolates [
11,
16,
17]. Fimbriae have an important role in establishing and progression of UTI. P-fimbrial adhesins with binding capacity to renal cell receptors cause the specific signaling pathways that trigger mucosal inflammation and tissue damage [
18,
19]. Although, in the current study all investigated P fimbriae genes were more prevalent in the UPEC isolates, than the commensal isolates, but the prevalence of
papAH and
papEF genes was statistically significant. Correlation association analysis between virulence genes revealed a positive association between
papAH and
papEF genes. The
pap genes are usually chromosomal [
2]; therefore, these two genes were probably transmitted together through the chromosome.
Among adhesin genes,
fimH, the gene that encodes type 1 fimbriae, was common among the UPEC isolates. This VF is attributed to cyctitis-associated UPEC strains and helps to adhere, invade, and form the intracellular bacterial communities (IBCs) [
14,
16,
20]. The prevalence of
fimH was found to be 62.3% that was lower than some studies in Iran [
16,
21], but it’s consistent with some previous reports from Mexico, Tunisia, and Iran [
6,
22,
23]. In the current study, the
fimH gene had the most prevalence among virulence genes, which may indicate its critical role in producing UTI. Therefore, FimH could be considered as a potential vaccine candidate. Besides, some studies are investigating this issue. For instance, it has been previously shown that antibodies against FimH can prevent the colonization of UPEC in urinary tract system [
24,
25]. The
fimH gene showed a positive correlation with PAI, indicating the genetic linkages between them.
Although, both siderophore genes of
fyuA and
iutA were prevalent in more than 50% of UPEC isolates; the frequency of yersiniabactin (
fyuA gene) was statistically significant in the UTI producing isolates more than commensal isolates. Moreno et al. also reported a strong association of
fyuA with urine versus fecal sources [
26]. Drawn heat map revealed a strong positive association between two
fyuA and
iutA siderophore genes, which indicates the importance of iron absorption systems in pathogenic isolates.
The
traT was another gene that was statistically significant in UPEC isolates. It expresses a transfer protein that inhibits the classical pathway of complement activation [
2]. This gene is a part of
tra operon of the F-like conjugative plasmids and leads to serum survival [
2]. In the other study from Iran, consistent with our results,
traT,
fyuA, and
fimH genes were the most frequently detected VFs in UPEC isolates [
27].
ExPEC strains, particularly UPEC isolates, usually contain multiple PAIs with a distinctive combination of VFs; therefore, some isolates may have multiple copies of a VF [
13]. In this way, PAIs can play an important role in increasing the pathogenicity of bacteria.
Although, the frequency of other genes was not statistically significant between two groups of UPEC and commensal isolates; but, in the current study, gene expression levels were not investigated. The rate of genes transcription, expression, or the copy number of each gene might be different in these two groups of isolates. Also, to detect virulence genes the PCR method was used, however, due to mutation, some virulence genes may not be accurately detected. Therefore, positive PCR results indicated the presence of genes; but, a negative result does not essentially equivalent to the absence of the corresponding genes, although this phenomenon is scarce [
6].
Phylogenetic analysis revealed that the B2 and D (to a lesser extent) were dominant phylogroups of UPEC isolates. The prevalence of VFs was higher among group B2 isolates taken from the urine of patients with acute cystitis than fecal isolates of healthy people [
28,
29], which is in line with previous studies conducted in Ethiopia [240], Denmark [
30], Pakistan [
31], South Korea [
18], Poland [
32], and Mexico [
22]. Therefore, in the producing-UTI isolates, most of the VFs were more frequent than commensal isolates. On the other hand, the UPEC isolates mostly belonged to group B2. Thus, the association between VFs and phylogenetic groups was investigated.
Distribution of VFs in the phylogenetic groups indicated the presence of some genes, including
papC,
fyuA,
iutA,
kpsMT K5, and
kpsMTII were positively associated with group B2,
fimH and PAI with both B2 and D groups, and
papG allele I
′a was associated with group D. Such associations are also reported in other studies [
14,
33‐
35]. Using such evidence scientists can investigate these phylogenetic groups (B2 and D) for VFs, which as-yet is undefined [
13].
VF scores of UPEC isolates were higher than commensal isolates. Group B2 in both commensal and UPEC isolates had the highest aggregative VF scores, followed by group D (intermediate), and groups A and B1 (the lowest). Based on the obtained results, it can be concluded that isolates related to the B2 phylogenetic group had a pathogenic potential, regardless of their origin. In this way, these isolates can be a possible candidate for developing a vaccine or drugs.
Co-selection or direct genetic linkage of VFs leads to the common simultaneous appearance of certain VFs [
11,
13,
36]. In the current study, the Goodman and Kruskal tau coefficient method and heat map were used to find the strength of the associations of virulence gens. If different studies investigate and identify the associations between genes; it would be feasible to have a better understanding of the role of these genes in how pathogenesis occurs.
UPEC isolates are diverse due to the presence of different virulence genes carried by plasmids, transposons, PAIs, and bacteriophages. These genetic elements may carry antibiotic resistance genes in addition to virulence genes. Plasmids belonging to the IncF incompatibility group were found to encode both VFs and antibiotic resistance genes [
37]. However, some researchers believe that multidrug resistant isolates are significantly less virulent than susceptible isolates since antibiotic resistance and virulence do not usually co-evolve simultaneously [
38]. Although some studies have found the positive association between virulence traits including iron scavenger receptors with antibiotic resistance [
39,
40], there are many contradictions in this regard. For example, in some studies the presence of
hly gene was associated with sensitivity to fluoroquinolones [
41‐
43] and in other study resistance to fluoroquinolones [
8]. Thus, future comprehensive studies are necessary to elucidate the relationship between VFs and antibiotic resistance and the evolutionary direction of bacteria.
The clustering results demonstrated that the UPEC or commensal
E. coli isolates are genotypically highly heterogeneous. These different patterns of clusters are probably due to chromosomal or plasmid location of virulence genes and vertical (within-lineage) or horizontal (among-lineage) gene transfer phenomena [
13]. Additionally, these data emphasize the findings relay on the fact that many UPEC isolates originate from commensal strains without considerable virulence genes content as previously reported in some women, and UTI may cause by a high prevalence of relatively low virulence
E. coli strains in the fecal reservoir [
4]. Also, commensal strains can be potentially pathogenic, when colonizing extra-intestinal tissues. Therefore, by reducing the intestinal colonization of UPEC strains and dealing with
E. coli virulence mechanisms, the UTIs may be prevented [
26]. The epidemiological aspects of the UPEC in different regions need further investigation to find the spread of different isolates and to understand the dissemination of these pathogens to hosts [
17].
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