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. 2023 Dec 1;55(1):1–9. doi: 10.1007/s42770-023-01195-9

Detection and characterization of potentially hybrid enteroaggregative Escherichia coli (EAEC) strains isolated from urinary tract infection

Shima Moazeni 1, Mahdi Askari Badouei 1,✉, Gholamreza Hashemitabar 1, Seyedeh Elham Rezatofighi 2, Fahimeh Mahmoodi 2
PMCID: PMC10920591  PMID: 38036848

Abstract

Uropathogenic Escherichia coli (UPEC) have the potential to receive the virulence markers of intestinal pathotypes and transform into various important hybrid pathotypes. This study aimed to investigate the frequency and characteristics of hybrid enteroaggregative E. coli (EAEC)/UPEC strains. Out of 202 UPEC strains, nine (4.5%) were detected as hybrid EAEC/UPEC. These strains carried one to four iron uptake systems. Among nine investigated pathogenicity islands (PAIs), PAI IV536, PAI II536, and PAI ICFT073 were found in 9 (100%), 3 (33.3%), and 1 (11.1%) strains, respectively. The chuA and sitA genes were detected in 5 (55.5%) and 3 (33.3%) hybrid strains, respectively. Six hybrid strains were found to be typical extraintestinal pathogenic E. coli (ExPEC) according to their virulence traits. Most of the hybrid strains belonged to the phylogenetic group E (6/9). Among the hybrid strains, seven (7/9) were able to form biofilm and adhere to cells; however, only two strains penetrated into the HeLa cells. Our findings reveal some of the virulence characteristics of hybrid strains that lead to fitness and infection in the urinary tract. These strains, with virulence factors of intestinal and non-intestinal pathotypes, may become emerging pathogens in clinical settings; therefore, further studies are needed to reveal their pathogenicity mechanisms and so that preventive measures can be taken.

Keywords: Uropathogenic Escherichia coli, Enteroaggregative Escherichia coli, Pathogenicity islands, Iron acquisition systems, Extraintestinal pathogenic E. coli

Introduction

Escherichia coli is a facultative anaerobic microorganism of the intestinal flora of humans and many warm-blooded animals. However, in some cases, it can acquire some virulence factors (VFs) and cause intestinal or extraintestinal infections [1]. Intestinal pathogenic E. coli (IPEC) strains are categorized into six major pathogroups including enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), and adherent-invasive E. coli (AIEC). The members of extraintestinal pathogenic E. coli (ExPEC) strains include uropathogenic E. coli (UPEC), septicemic E. coli (SEPEC), avian pathogenic E. coli (APEC), and neonatal meningitis-associated E. coli (NMEC) [2].

EAEC is known as a cause of acute and chronic diarrhea in children, travelers, and outbreaks in the developed countries [1, 3, 4]. This pathotype is defined by an aggregative adherence (AA) pattern on HEp-2 cells named “stacked brick” [1]. The pathotype EAEC has a heterogenous pathogenicity, so that in addition to diarrheal samples, it has been found in the feces of healthy people without causing disease [1, 5, 6]. Also, clones of this pathotype are reported to cause extraintestinal infections. A number of community-acquired UTIs were reported in Denmark during the year 1991 which was due to a particular EAEC clonal lineage [7, 8]. Some studies investigated the presence of hybrid EAEC among E. coli strains isolated from patients with UTIs and analyzed phenotypically and molecularly their virulence characteristics [9–12]. However, some characteristics of these hybrid strains, including the type and variety of pathogenicity islands (PAIs) they carry, are unknown.

PAIs are a group of large (>10 kb) insertion segments which encode virulence gene(s) that are present in the genome of pathogenic strains but not in the non-pathogenic bacteria of the same or related species [13–16]. PAI specific to the EAEC pathotype has not been reported; however, in some cases, it carries PAI related to other pathotypes or some members of Enterobacterales [17]. High pathogenicity island (HPI) is a PAI originally detected in Yersinia enterocolitica [18, 19], which is also prevalent in EAEC [20]. This PAI encodes genes related to the synthesis of the siderophore yersiniabactin and its receptor proteins [21]. PAI pic/set, also known as PAI IICFT073, is the other island identified in EAEC as well as in UPEC, EIEC, EHEC, and EPEC; however, the genome composition is different in these types [13]. This PAI carries pic and set genes encoding mucinolytic-hemagglutinin protein and Shigella enterotoxin 1, respectively [17, 22]. The other reported PAIs in EAEC pathotype are LAA PAI and SHI-O PAI [13]. UPEC strains also carry PAIs [13]. Most virulence factors including S and P pili, Iss (serum resistance protein], siderophores such as salmochelin, yersiniabactin, and aerobactin, K capsule, toxins such as a-hemolysin, and colibactin are varied and encoded by PAIs [13].

Iron, an essential cofactor, is involved in many metabolic processes [23]. E. coli isolates of intestinal flora have access to more sources of iron, while ExPEC isolates have to cope with iron deficient environment of the host. In the human body, iron is mainly found in ferric (Fe3+) form which has low solubility. The ferric iron is usually combined with transferrin, lactoferrin, ferritins, or bond to the heme of hemoglobin [13]. This complexity leads to the limitation of free iron for the bacteria; instead, pathogenic bacteria have developed various strategies to acquire iron from the host body. Several iron uptake systems have been found among ExPEC strains including siderophores such as aerobactin, entrobactin, salmochelin, and yersiniabactin, transport systems such as ChuA, Hbp, Hma, IreA, and Iha [24]. Many iron uptake systems are encoded by PAIs and these islands play a very important role in fitness and increasing pathogenicity of bacteria [13].

Several studies have been conducted on the molecular and phenotypic characteristics of EAEC and UPEC isolates; however, a very limited number of research are available on EAEC/UPEC hybrids. In the present study, we aimed to investigate the frequency of hybrid EAEC/UPEC strains, and molecular properties including the presence of PAIs, iron uptake systems, virulence factors, and phylogenetic groups, also some phenotypic characteristics associated with virulence such as biofilm production, adhesion and invasion ability.

Material and methods

Sample collection

A total of 165 E. coli positive urine culture samples were collected from Ghaem and Imam-Reza hospitals of Mashhad city, Iran, from March to June 2019. In addition, 37 UPEC archived strains were investigated. These strains were isolated from patients in 2016 and 2017 and archived in microbial culture collection at the Microbiology department of Ferdowsi University of Mashhad. The study was approved by the Ethics Committee of Ferdowsi University of Mashhad. Before collecting information, participants or parents (for children cases) were asked to read, accept and sign an informed consent form. All strains were screened and confirmed by conventional biochemical tests including culture on MacConkey agar, Eosin Methylene Blue, sulfur indole motility, Simmon’s Citrate, triple sugar iron agar, methyl red/Voges-Proskauer.

Detection of hybrid EAEC strains

DNA was extracted from the UPEC strains using boiling method. The molecular markers of aggR and pCVD432 were used for detection of hybrid EAEC/UPEC strains. PCR primers and conditions were applied as described by Toma et al. [25].

Identification of EAEC virulence genes

The strains which were positive for one of the molecular predictors aggR, pCVD432, or both were considered for the next stages of the study. The hybrid EAEC/UPEC strains were analyzed for the presence EAEC VGs of app, aafA, astA, pet, and aggA using PCR [26–28].

Molecular classification of ExPEC and UPEC strains and iron uptake systems

The hybrid strains were evaluated for the presence of ExPEC VGs including iss [29], hlyA [30], and tsh [31] using PCR. Spurbeck et al. proposed a multiplex PCR for molecular detection of UPEC potential strains according to identification of vat, yfcV, fyuA, and chuA genes. The strains harbored three or more of these genes were considered to have uropathogenicity potential [32]. The hybrid strains were designated molecularly as potential ExPEC if they possessed at least 2 of 5 virulence markers, including papAH and/or papC, afa/draBC, iutA, sfa/focDE, and kpsMT II [33]. Three multiplex PCR and one simplex PCR were also applied for detection of iron uptake genes including fecA, iroN, chuA, iha, iucD, fyuA, tonB, irp2, iutA, and sitA [34, 35].

Phylogenetic grouping and serotyping

Phylogrouping of hybrid EAEC/UPEC strains were performed by a quadruplex PCR assay designed by Clermont et al. According to presence or absence of yjaA, chuA, arpA, and tspE4.C2 the strains assign to one phylogenetic group of A, B1, B2, C, D, E, F, or Clade I. The strains which were indistinguishable phylogroup in the first step, a duplex PCR was carried out [36].

A multiplex-PCR assay was applied for detection of O antigens prevalent including O1, O2, O18, and O78 as described previously [37].

Detection of PAIs

Detection of PAIs was performed according to a method of Sabate et al. with some modification [38]. In order to increase the sensitivity of the reactions, three multiplex PCR was performed instead of two multiplex. In the first multiplex reaction, PAI IICFT073, PAI III536, and PAI IV536 were amplified. In the second reaction, PAI ICFT073, PAI II536 and PAI IJ96 were detected. Third multiplex PCR reaction was used for detection of PAI I536 and PAI IIJ96. The PCR conditions were similar to those recommended by Sabate et al. with the difference that in third reaction, the time of extension step was increased from 1 min to 3 min.

Enterobacterial repetitive intergenic consensus (ERIC)-PCR

ERIC-PCR was applied to find the clonal variability and phylogenetic closeness of hybrid EAEC/UPEC strains. The ERIC primers and PCR condition were performed as described by Weigel et al. [39]. After running the PCR products on gel electrophoresis, the presence or absence of PCR bands were recorded as binary value of 1 or 0, respectively. To construct dendrogram, SIMQUAL program in NTSYS-pc, version 2.2 was used. The genetic relationships among hybrid strains were calculated by Jaccard’s similarity coefficients and UPGMA.

Biofilm assay

Fresh Luria-Bertani (LB) culture of hybrid strains was prepared and adjusted to 0.5 MacFarland. Then, 5 μl of each strain was added to 200 μl LB containing 0.45% glucose in wells of 96 microtiter plate and kept for 24 h at 37 °C. Subsequently, after washing the wells with PBS, they were stained with 0.5% crystal violet for 5 min. The excess dye was removed and the wells were carefully washed by PBS. To remove and solubilize the dye of bacteria, 200 μl of 96% ethanol was added to each well for 10 min. Then, the supernatants were collected to measure the Optical density (OD) at 570 nm by an ELISA reader (BioRad; USA). The hybrid strains were grouped to strong, moderate, weak, and no biofilm producers as previously interpreted [3].

Adhesion and invasion assays

Adhesion and invasion assays were performed on HeLa cell line. To adhesion, the HeLa cells were seeded in 24-well plates in the presence of Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Scotland), pen-strep (100 U/mL) (Gibco, Scotland) fed with 10% fetal bovine serum (FBS) (Gibco, Scotland) and incubated at 37 °C under 5% CO2 to obtain ~80% confluence. Then, the culture medium was replaced with fresh DMEM without antibiotic, supplemented with 2% inactivated FBS and 1% D-mannose. Subsequently, 35 μL of each fresh bacterial culture adjusted to OD600=2 was inoculated to cells and kept for 3 h under previous condition. For quantitative adhesion assay, after washing the wells with PBS, 100 μL of 1% Triton X-100 was added for 10 min to lyse the cells and release the adherent bacteria. Afterward, the suspensions were gently homogenized with 900 μL LB and subjected to colony count. The adherence index was calculated by the ratio of number of adherent bacteria (colony forming unite (CFU)) to total bacteria at the third hour (CFU). The adherence index was expressed as a percentage [3].

To find the adherence pattern, after washing the wells, inoculated cells were fixed by methanol and stained with 10% Giemsa. An inverted microscope (Olympus; Japan) was used to record the adherence pattern [3].

The invasion assay was similar to adhesion assay with an extra step. After washing the infected cells, fresh medium containing 250 μg/mL amikacin was added for 1 h to kill the adherent bacteria. The next steps were the same for the adhesion assay. The invasion index was defined by the ratio of invasive bacteria (CFU) to adherent bacteria (CFU). The invasion index was expressed as a percentage [3].

Results

Detection of hybrid EAEC/UPEC strains and VGs of EAEC

Out of 202 UPEC strains, 9 (4.5%) carried aggR, pCVD432, or both and considered hybrid EAEC/UPEC. The pCVD432 marker was detected together with aggR in 8 strains; however, one strain carried only pCVD432. Analysis of EAEC virulence genes showed that all hybrid strains harbored app gene, while were negative for aafA, astA, pet, and aggA genes (Fig. 1).

Fig. 1.

Fig. 1

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Enterobacterial repetitive intergenic consensus (ERIC) dendrogram, genotypic and phenotypic characters of hybrid enteroaggregative Escherichia coli/Uropathogenic E. coli strains. Numbers at the terminal branches are isolate name. The colors mean as follows: Inline graphic : negative; Inline graphic : positive; Inline graphic : strong; Inline graphic : moderate; Inline graphic : weak; Inline graphic : no biofilm; EAEC: Enteroaggregative Escherichia coli; ExPEC: Extrapathogenic E. coli; UPEC: uropathogenic E. coli; VG: Virulence gene: PAI: Pathogenic Island

Molecular classification of ExPEC and UPEC strains

To find potential ExPEC and UPEC, the hybrid strains were evaluated for the presence of virulence marker genes. The VGs of papC, sfa/focDE, afa/draBC, iutA and kpsMT II were found in 7 (77.8%), 0 (0%), 0 (0%), 4 (44.5%), and 6 (66.7%), respectively. In this way, 6 (66.7%) hybrid strains had at least two or more of these genes and were considered potential ExPEC (Fig. 1).

Gene markers of potential UPEC including vat, yfcV, fyuA, and chuA were found in 0 (0%), 0(0%), 9 (100%), and 5 (55.6%) hybrid strains, respectively. The other investigated VGs including iss, hlyA, and tsh were positive in 0 (0%), 0 (0%) and 1 (11.1%) hybrid strains.

Detection of genes associated with iron uptake systems

The frequency of fyuA (yersiniabactin receptor), iutA (ferric aerobactin receptor), and chuA (heme receptor) genes among hybrid strains was reported above. However, the other investigated genes including fecA (ferric citrate), iroN (salmochelin), iha (enterobactin), iucD (aerobactin biosynthesis), tonB (energy transducer), irp2 (yersiniabactin biosynthesis), and sitA (iron transport) were found in 0 (0%), 2 (22.2%), 1 (11.1%), 5 (55.5%), 9 (100%), 9 (100%), and 3 (33.3%) hybrid EAEC/UPEC strains, respectively (Fig. 1).

Phylogenetic grouping and serotyping

Out of 9 hybrid EAEC/UPEC strains, 6 (66.6%) were related to group E. The other three strains were placed in C, A, and B2 phylogroups. PCR results revealed that none of hybrid strains carried molecular markers of O1, O2, O18, and O78 antigens (Fig. 1).

Detection of PAIs and plasmids

All hybrid strains carried PAI IV536. PAI II536 and PAI ICFT073 were found in 3 (33.3%) and 1 (11.1%) hybrid strains, respectively. The other investigated PIAs (IICFT073, III536, IIJ96, I536, and IJ96) were not detected (Fig. 1).

ERIC-PCR

The ERIC-PCR revealed different banding patterns from 8 to 12 bands with size from 100 to 2500 bp. UPGMA classified the hybrid strains into 4 clusters at a coefficient of 0.81 (Fig. 1). However, although strains 161u and 269u showed completely similar ERIC-PCR band pattern, some differences in terms of virulence profile were found. It is probably due to the presence of different genetic elements such as plasmid and PAI. ERIC-PCR results showed that some of these hybrid strains could be the similar clones, which have gradually changed by horizontal gene transfer mechanisms (HGT).

Biofilm, adhesion, and invasion assays

The ability to produce biofilm can help bacteria to increase pathogenicity and escape from antimicrobial compounds. The semi-quantitative microtitre plate assay showed 2 (22.1%), 3 (33.3%), and 3 (33.3%) hybrid EAEC/UPEC strains were strong, moderate, and weak biofilm producer, respectively; however, one strain was not able to produce biofilm. Adhesion to the cell is an important factor for bacteria to initiate the disease. Microscopic examination of the hybrid EAEC/UPEC strains showed that 7 (77.8%) strains have aggregative adherence (AA) pattern. The ability of these strains to adhere to the cells was variable, so that 4 (44.4%) strains had a strong adherence, 3 (33.3%) were weak, and 2 (22.2%) were unable to adhere to the cells. Bacteria that have the ability to penetrate into the cell can escape from the attack of host immune system and most drugs. These bacteria, in some cases, cause recurrent infections. Out of 9 hybrid EAEC/UPEC strains, only 2 (22.2%) were able to penetrate into the cells and show strong invasion potential (Index > 0.05%). The others could not be invasive into the HeLa cells (Fig. 1 and Fig. 2).

Fig. 2.

Fig. 2

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Adherence and invasion indexes obtained by hybrid UPEC/EAEC strains on HeLa cell line

Discussion

Enteroaggregative E. coli (EAEC) can be considered an intriguing diarrheagenic E. coli pathotype as some studies have reported UPEC strains with the predictor molecular markers of EAEC. This becomes more important when these strains lead to outbreaks. An EAEC strain was reported as responsible for an outbreak of UTI in Denmark in 1991 [8]. Various studies have reported different frequencies for hybrid EAEC/UPEC strains isolated from UTIs including 0.4% in Germany [40], 20.2% in India [12], 3.5% [11], 1.5% [9], and 7.1% [10] in Brazil, and 13.2% [41] and 10.5% [42] in Iran. Among the 202 UPEC strains, we found 9 (4.5%) hybrid EAEC/UPEC. The mentioned reports reveal that a part of UTIs can be caused by EAEC strains.

In the present study, the identified hybrid strains, except for one, had aap-pCVD432-aggR profile. These genes are located on the pAA plasmid and can be received by UPEC to form hybrid EAEC/UPEC [11]. The aggR is a transcriptional activator gene which regulates several chromosomal virulence genes in addition to pAA encoded genes [22]. The aap gene codes for dispersin, a protein which acts as an anti-aggregation and prevents bacterial accumulation [12, 43]. The relationship between aap gene and biofilm formation has been reported in some studies. In the present study, 88.9% (8/9) of hybrid strains which carried aap was a biofilm producer from weak to strong. The pCVD432 segment also named aatA gene encodes a membrane protein which is essential for exporting of dispersin [44]. Cordeiro et al. suggested that any of these three genes can be considered a molecular marker for EAEC detection [45]. We could not find the other EAEC virulence genes including aafA, astA, pet, and aggA. The astA can be carried by other plasmids than pAA and is present in 41% of EAEC, as well as in 38% of commensal E. coli isolates [17, 46]. AAF fimbriae which mediate biofilm formation, AA pattern, and haemagglutination, has five variants including AggA (AAF/I), AafA (AAF/II), Agg3A (AAF/III), Agg4A (AAF/IV), and Agg5A (AAF/V). AAF biogenesis is regulated by transcription factor of AggR [47]. In the present study, AAFI and AAFII were not detected. The aggA is needed for expression of AAF fimbriae [26, 48]. It should be noted that many EAEC strains do not express these fimbriae, but has been considered pathogenic [26, 48]. The pet gene encoded an enterotoxin, located on pAA plasmid and is found in 18–44% of EAEC isolates [49, 50]. Modgil et al. reported that 15.1% of hybrid EAEC/UPEC strains harbored pet gene [12].

Dudley et al. (2006) using proteomic and microarray analysis identified that AggR activates the expression of pheU PAI [51]. The pheU island is widely distributed in EAEC isolates, especially in those that are positive for aggR and other plasmid-encoded members of the AggR regulon [44]. We searched common PAIs associated with ExPEC among hybrid EAEC/UPEC strains. All hybrids carried PAI IV536, also termed High-Pathogenicity Island (HPI). PAI IV536 encoded genes needed for the synthesis of a major bacterial iron uptake system [20, 52], siderophore yersiniabactin and its receptors. All hybrid strains carrying PAI IV536 were positive for fyuA and irp2 genes which probably transferred by this island. In addition to UPEC, PAI IV536 is prevalent in EAEC [17, 18]. Galardini et al. showed that the presence of HPI is highly associated with death in mice, beyond other genetic factors related to iron acquisition, such as the aerobactin and the sitABCD operons [52]. Dobrindt et al. reported that 67 to 92% of the ExPEC isolates and 17 to 28% of the intestinal pathogenic isolates were positive for PAI IV536 [15]. The acquisition of PAI is not a random process, but a sequential, programmed mechanism. Sabate´ et al. believed that PAI IV536 in isolates from phylogroup B2 is always, with one exception, associated with PAI ICFT073 [38]. It is according with our findings. Only one hybrid EAEC/UPEC strain belonged to group B2 which also carried PAI IV536 in combination with PAI ICFT073. In the rest of the hybrid strains, PAI IV536 was found either alone or in association with PAI II536.

PAI II536 was identified in 3/9 (33.3%) hybrid strains. This island encoded virulence factors of α-haemolysin and P-related fimbriae. All three hybrid strains were negative for hlyA, but two of them carried papC. All three strains belonged to group E, while in the study of Sabate´ et al. [38], all isolates with PAI II536 were placed in phylogroup B2. Dobrindt and coworkers reported that PAI III536 was more common than PAI II536 among UPEC isolates. However, in our study, PAI III536 were not identified in hybrid strains, while 3 had PAI II536. According to a research of Dobrindt et al., PAIs I536, II536, and III536 are usually not detectable in DEC isolates and are a part of the ExPEC specific gene pool [15].

The presence of PAI markers and absence of their virulence factors may be due to intrinsic genetic instability of PAI. The deletion frequency of virulence genes on specific PAIs is higher than the normal rate of mutation. In this type of mutation, the large portions of a PAI including several virulence genes or even the entire of PAI are lost. It may occur during cultivation of pathogens in vitro, or obtain from infected individuals, for example during persistent infections [53].

PAI ICFT073 was found in one hybrid strain. This island carry genes encoding α-Hemolysin, P pilus, Iha, Sat, and aerobactin virulence factors [24]. The genes related to siderophore aerobaction (iutA and iucD) were identified in this strain; however, the other iutA or iucD encoding hybrid strains were negative for PAI ICFT073. The genes can be encoded by plasmids instead of chromosome [24]. Although one strain harbored iha gene, but was negative for PAI ICFT073.

The hybrid EAEC/UPEC strains were investigated for the presence of several iron uptake systems. As mentioned earlier, siderophore yersiniabactin which may be carried by PAI IV536 plays a very important role in the establishment of UTI by iron uptake required for bacteria. It also helps to biofilm production and reduce the formation of reactive oxygen species by the cells of the immune system using trapping iron and preventing Haber–Weiss reactions [54, 55]. We found the fyuA and irp2 genes among all hybrid strains. These genes are encoded more by ExPEC than DEC. Nascimento and coworkers found fyuA among seven (7/9) hybrid EAEC/UPEC strains [9].

Out of 9 hybrid EAEC/UPEC strains, 7 (77.8%) harbored iucD and/or iutA aerobactin biosynthetic genes. These genes were more frequently detected in ExPEC than commensal strains and their incidence has been associated with highly pathogenic strains [56]. Modgil et al. reported that iuc encoding aerobactin was present in 69.6% of UPEC/EAEC isolates [12]. Aerobactin is able to acquire iron from transferrin more effectively than enterobactin [23].

The other siderophore, salmochelin is contributed to virulence of ExPEC strains [23]. The genes encoding salmochelin are generally on ColV or ColBM virulence plasmids, although sometimes encoded by chromosomal PAI III536 [16]. The iroN was detected in 2/9 (22.2%) hybrid strains and these were not located on PAI III536. It is reported that enterobactin is found in both pathogenic and nonpathogenic E. coli isolates and plays a minor role in the pathogenicity of ExPEC [23]. The iha gene was found in one hybrid strain.

Another way for acquiring iron is to use the heme receptor system. The chuA gene encodes a specific outer membrane receptor, hemin receptor ChuA, which has important role in the pathogenicity of ExPEC specially UPEC, but not DEC [56]. In the present study, chuA was found in 55.5% (5/9) of hybrid strains. In the study of Nascimento et al., one (1/9) hybrid EAEC/UPEC strain harbored chuA gene [9].

ExPEC use the Sit systems to transport manganese and ferrous iron into cytoplasm. It has been shown that the expression of sit genes is increased during human UTI and contributes to urofitness [13]. The sitA gene was detected in 3/9 (33%) strains.

Iron transport using siderophores or heme uptake systems, in addition to receptors, needs an energy transducer. The required energy is provided by the TonB protein, which is placed in the cytoplasmic membrane. The role of TonB and TonB-dependent iron transport systems in UPEC virulence has been revealed [57]. The most iron uptake systems including siderophore transporters, heme receptors, and several transferrin/lactoferrin receptors are dependent on TonB and its accessory proteins. We found the presence of tonB gene in all hybrid strains, indicating its importance for iron uptake systems.

The phylogenetic assessment of hybrid strains showed that most of them (6/9:66.7%) belonged to the phylogenetic group E. This finding is important because phylogroup E strains are uncommon in human (less than 1 to 3.6%) [58–60]. ExPEC typically belong to the phylogroup B2 and strains that belong to group E are rare (< 0.5%) [61, 62]. However, EHEC, EPEC, ETEC and EIEC are frequently found to be in group E [63]. Clermont et al. found that phylogroup E strains have the largest genomes of the species due to the presence of mobile genetic elements [58]. Phylogroup E emerged between group D and groups A/B1 [60]. Clermont et al. believe that group E strains are mainly intestinal and susceptible to antibiotics, but few of them can be very virulent and resistant [58]. Although they did not investigate the EAEC pathotype in their study and did not comment on this, it seems that the identified hybrid EAEC/UPEC strains were of intestinal origin and were also virulent.

Many strains of EAEC and UPEC are able to form biofilm. Biofilm formation is involved in the persistence infections, as well as, antimicrobial resistance [64]. The ability of bacteria to produce biofilm, adhere to and invade host cells are important mechanisms of pathogenesis [65]. Recurrent UPEC infections have been attributed to these characteristics. Among the hybrid strains, seven (7/9) were able to form biofilm and adhere to cells; however, only two strains penetrated into the HeLa cells. The phenotypic properties and other virulence genes detected in hybrid EAEC/UPEC strains reveal the pathogenic potential of these strains.

Conclusion

The ability of iron uptake helps to increase the virulence of bacteria. In the present study, at least one to four iron acquisition systems were identified in the hybrid EAEC/UPEC strains. The siderophore yersiniabactin which has a high ability to acquire iron, was identified in all hybrid strains. Our finding also showed the prevalence of PAIs, specially, PAI IV536 among these strains. It should be considered that hybrid strains have additional virulence mechanisms that differ from non-hybrid UPEC strains, which have acquired from other pathotypes. These characteristics may turn them into important pathogens in the world scenario; therefore, further studies are needed to reveal their virulence mechanisms and so that preventive measures can be taken.

Author contributions

MAB designed and supervised the study. SM conducted the main laboratory experiments and some complementary tests were carried out by SER and FM. SM, MAB, GH, SER and FM contributed in data analysis. SM, MAB and SER contributed in writing the initial draft and all authors edited and approved the manuscript.

Funding

The present study was partially funded by Ferdowsi University of Mashhad (Grant No. FUM. 48976).

Declarations

Ethics approval

The study was approved by the Ethics Committee of Ferdowsi University of Mashhad. The study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The research was performed on bacterial isolates collected from clinical samples that were already cultured as part of the routine work in the Microbiology laboratories of hospitals. Before collecting information, participants or parents (for children cases) were asked to read, accept and sign an informed consent form.

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher’s Note

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