|Year : 2020 | Volume
| Issue : 3 | Page : 421-429
Molecular, phylogenetic and antibiotic resistance analysis of enteroaggregative escherichia coli/uropathogenic Escherichia coli hybrid genotypes causing urinary tract infections
Vinay Modgil, Harpreet Kaur, Balvinder Mohan, Neelam Taneja
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
|Date of Submission||10-Aug-2020|
|Date of Decision||02-Sep-2020|
|Date of Acceptance||25-Sep-2020|
|Date of Web Publication||4-Nov-2020|
Dr. Neelam Taneja
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh
Source of Support: None, Conflict of Interest: None
Background: Horizontal gene transfer of virulence genes (VGs) from different Escherichia coli pathotypes results in the evolution of hybrid strains. Hybrid genotypes of enteroaggregative E. coli and uropathogenic E. coli (EAEC/UPEC) have been reported in sporadic infections and outbreaks of extraintestinal origin. Yet, their association with routine infections is still underrated. Materials and Methods: In this study, we analysed 163 isolates of E. coli from cases of urinary tract infection seeking hybrid (EAEC/UPEC) strains. Using multiplex polymerase chain reaction, we investigated VGs (adhesive and toxin genes) of UPEC along with EAEC marker genes (aap and agg R), ast A (toxin genes) and serine protease autotransporters of Enterobacteriaceae, pet (plasmid-encoded toxin) and pic (mucinase gene). Those UPEC strains which had characteristic defining genes of EAEC (agg R/aap or their combination) were considered UPEC/EAEC hybrids. Results: Molecular predictors of EAEC (aap and aggR) were detected in 20.2% (33/163) of the strains. The pap C was also detected in 36% of the EAEC/UPEC hybrid strains. Phylogenetic analysis revealed that hybrid strains belonged to Group D (60.6%). Nearly 73.8% of UPEC and 75.7% of UPEC/EAEC hybrid strains were multidrug-resistant. Among UPEC isolates, 72.3% and in hybrid UPEC/EAEC, 78.7% isolates were able to produce biofilm. Conclusions: Our results indicated a closer relationship among EAEC and UPEC, which suggested that some EAEC strains can be potential uropathogens. Ours is a first study documenting the existence of EAEC pathotypes VGs in UPEC strains of nosocomial origin; further studies are required to understand the diarrhoeagenic potential of these hybrids.
Keywords: Enteroaggregative Escherichia coli, hybrid strain, multiplex polymerase chain reaction, phylogenetic group, serine protease autotransporters of Enterobacteriaceae, uropathogenic Escherichia coli
|How to cite this article:|
Modgil V, Kaur H, Mohan B, Taneja N. Molecular, phylogenetic and antibiotic resistance analysis of enteroaggregative escherichia coli/uropathogenic Escherichia coli hybrid genotypes causing urinary tract infections. Indian J Med Microbiol 2020;38:421-9
|How to cite this URL:|
Modgil V, Kaur H, Mohan B, Taneja N. Molecular, phylogenetic and antibiotic resistance analysis of enteroaggregative escherichia coli/uropathogenic Escherichia coli hybrid genotypes causing urinary tract infections. Indian J Med Microbiol [serial online] 2020 [cited 2021 Jan 27];38:421-9. Available from: https://www.ijmm.org/text.asp?2020/38/3/421/299841
| ~ Introduction|| |
Three different health disorders can be caused by pathogenic Escherichia coli: diarrhoea, urinary tract infection (UTI) and sepsis/meningitis. The term Extraintestinal pathogenic E. coli (ExPEC) was coined to describe the E. coli strains isolated from UTIs, neonatal meningitis and bacteremia. UTI is the most common extra-intestinal E. coli infection and is mainly due to the transmission of uropathogenic E. coli (UPEC) strains from the gut to the urinary tract. Commensal and pathogenic E. coli strains are categorised into four major phylogroups, namely A, B1, B2 and D. Epidemiological considerations showed that ExPEC mainly belongs to either phylogroup B2 or D. In contrast, commensal strains are grouped into phylogroup A or B1. Each phylogroup can include heterogeneous collections of strains and distinctive clonal populations that cause complex epidemiological relations between phylogroups and human infections.
Diarrhoeagenic E. coli (DEC) strains are characterised into the following six well-characterised pathotypes: enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, diffusely adherent E. coli and enteroaggregative E. coli (EAEC)., Among DEC, EAEC is known to carry the virulence plasmid pAA, defined by gene aat A, aap and aggR (master regulator gene), on a molecular scale. The aap gene produces an anti-aggregation protein that creates a bacterial capsule capable of preventing bacterial accumulation and allows bacterial dispersion. The agg R gene also produces a transcription activator for several pathogenic genes. The aat A gene produces a membrane protein which is part of a transmitter system for aat PABCE and essential for translocation of pathogenic proteins such as aap. Cordeiro et al. have shown that detection of any of the three genes by multiplex polymerase chain reaction (M-PCR) defines a strain as EAEC.
UTIs are usually endogenous infections with UPEC virulence markers triggered by intestinal microbiota. These UPEC strains contain various virulence factors, such as adhesins, toxins and siderophores, which lead to the development of the disease. The widely accepted theory today is that UPEC evolved from non-pathogenic strains by gaining new virulence factors from the horizontal transfer of accessory DNA located on chromosome or plasmid. UPEC virulence factors implicated in UTIs can be divided into two groups: the bacterial surface virulence factors (e.g., Type 1 fimbria, P Fimbriae, S fimbriae and afimbrial adhesines) and the factors which are secreted and exported to the site of action.
The exceptional plasticity of E. coli strain genomes allows the appearance of hazardous strains with unique virulence gene (VG) arrangements. Among all DEC virulence markers, EAEC VGs are the most common markers present in ExPEC strains, which are derived from sporadic cases of extraintestinal infection and outbreaks. To classify pathogenic E. coli strains retaining phenotypic and genetic determinants from more than one pathotypes, the term 'hybrid strains' has now been adopted. Because EAEC is frequently found in healthy individuals, this can reach their urinary tract and cause UTI., Indeed, EAEC has recently emerged as a causative agent of UTI. In Copenhagen, Denmark, a clonal EAEC strain caused a cluster of predominantly community-acquired UTI. All strains shared the same serotype, O78:H10, and phylogenetic group.
It is important to search for the existence of EAEC virulence markers in UPEC collections isolated from UTI patients to understand the epidemiology of EAEC in UTI., Furthermore, both UPEC and EAEC share an important pathogenetic mechanism by biofilm formation. Biofilm formation in UPEC strains is a dynamic process that combines several adhesive factors, including (afa C, fim H, pap C and sfa). Because limited data is available from India, in this study, we examined a collection of UPEC strains causing nosocomial UTI (NUTI) for the presence of characteristic virulence properties of UPEC. We determined whether some of these UPEC strains carry virulence factors characteristic of EAEC (hybrids of UPEC and EAEC). Using M-PCR, we investigated VGs (adhesive and toxin genes) of UPEC along with EAEC marker genes (aap and agg R), ast A (toxin genes) and members of serine protease autotransporters of Enterobacteriaceae (SPATE) pet (plasmid-encoded toxin), pic (EAEC mucinase gene). We also compared the hybrids with UPEC in terms of their antibiotic resistance, biofilm formation capacity and phylogeny.
| ~ Materials and Methods|| |
All the culture media and antibiotic discs utilised in this study were procured from HiMedia. All primers were obtained from Sigma-Aldrich Co., St. Louis, MO, USA. PCR reagents (dNTP, Taq polymerase, buffer, Mgcl2) were taken from GeNeiTM Bangalore.
Samples and strains
A total of 163 consecutive UPEC isolates were isolated in significant numbers (>105 colony-forming units/ml) from 1636 admitted cases of NUTIs at our tertiary care hospital, Postgraduate Institute of Medical Education and Research Chandigarh (PGIMER) in 2017. NUTI was defined as UTI developing in patients 72 h after hospitalisation in a patient whose urine culture was sterile or when a patient was asymptomatic at the time of admission. Pure cultures were stored frozen at −80°C in Luria–Bertani (LB) broth with 10% glycerol. These strains were revived for the study. The study was approved by the PGIMER Institutional Ethics Committee (Ref. No. MK/2856/Ph. D/8678).
In 50-ml deionised water, a single colony of biochemically confirmed E. coli isolates was emulsified and boiled for 5 min. For 1 min, the mixture was centrifuged at 10,000 ×g, and the then DNA-containing supernatant was collected and stored at 4°C until further usage.
Polymerase chain reaction amplification for uropathogenic Escherichia coli specific genes
UPEC-specific primers were used to amplify the fim H, pap C, sfa, afa, hly C and iuc sequences. The list of sequences of each primer, sizes of the amplified products and specific annealing temperatures are shown in [Table 1]. M-PCR was performed for the detection of pap C, sfa and afa genes. DNA amplification was done in a total volume of 25 μl containing 1 μl of DNA, 200 μM of dNTP, 10 pmol of each of the primers and 1U Taq DNA polymerase in 1X PCR buffer containing 1.5 mM MgCl2.
|Table 1: Uropathogenic Escherichia coli and enteroaggregative Escherichia coli primers used in this study|
Click here to view
The PCR (Applied Biosystem, Veriti 96 Well Thermal Cycler) conditions were as follows: initial denaturation at 94°C for 10 min, followed by 35 cycles of denaturation at 94°C for 2 min, annealing at a specific temperature for the 30 s [Table 1], and extension at 72°C for 1 min. A 10-μl aliquot of the PCR product underwent gel electrophoresis on 2% agarose, followed by staining with ethidium bromide (EtBr) solution. Amplified products were studied using 1.5% agarose gel electrophoresis and visualised with EtBr staining.
Polymerase chain reaction condition for enteroaggregative Escherichia coli-specific genes
The following EAEC VGs were detected by M-PCR in UPEC isolates aggR (EAEC virulence regulator gene) and aapA (dispersin gene), astA (toxin gene), pet (plasmid-encoded toxin) and pic (EAEC mucinase gene). The combination of agg R and aap A genes was used as predictor genes to define EAEC, as shown in [Table 1]. In this study, the presence of aggR/aapA or their combination was taken as a defining criterion as EAEC.
EAEC-specific genes were amplified to a total volume of 25 μl comprising 2.6 mM of each dNTP, 0.5 mM each primer, 10X PCR buffer, 1.5 mM MgCl2, 1U Taq polymerase and 1 μl bacterial DNA. PCR parameters were as follows: (1) 2-min denaturation at 95°C, (2) 50 s denaturation at 94°C, (3) 1.5-min annealing, at 57°C and (4) 1.5-min extension at 72°C with 35 cycles returning to step 2. The final extension at 72°C was for 10 min. Amplified products were studied using 1.5% agarose gel electrophoresis and visualised with EtBr staining. The amplicon size was estimated by comparing them to the ladder of 100 kb of DNA included on the same gel.
Phylogenetic analysis via polymerase chain reaction
A triplex PCR was used to detect phylogenetic Groups A, B1, B2 and D by amplifying the following gene targets: chu A, yja A and a cryptic DNA fragment, TspE4C2. The classification was correlated with Clermont et al. dichotomous decision tree.
Detection of biofilm production by congo red agar method
The medium consists of brain–heart infusion broth (37 g/l), sucrose (5 g/l), agar (10 g/l) and Congo red dye (0.8 g/l). Congo red stain was prepared as a condensed aqueous solution, autoclaving for 15 min at 121°C. Then, it was added to 55°C autoclaved BHI agar with sucrose. The plates were inoculated for 24–48 h and were aerobically incubated at 37°C. Black colonies with dry consistency shown the production of biofilms. Isolates producing very dark-coloured colonies with dry crystalline appearance were interpreted as strong biofilm (+++) producers. Bacteria-forming black colonies were considered as moderate biofilm (++) producers, and medium black colonies were noted as weak biofilm (+) producers. Colonies with red colour were considered non-biofilm (−) producers.
Antibiotic sensitivity testing
Antibiotic sensitivity testing was performed with the disc diffusion method for the following antibiotics: ampicillin (10 μg), ciprofloxacin (5 μg), amikacin (30 μg), imipenem (10 μg), levofloxacin (5 μg), gentamicin (10 μg), cefepime (15 μg), piperacillin-tazobactam (10 μg), nalidixic acid (30 μg), ertapenem (10 μg), cotrimoxazole (25 μg), cefoxitin (30 μg) and ceftriaxone (30 μg) according to Clinical and Laboratory Standards Institute guidelines.
Defining criteria for uropathogenic Escherichia coli/ enteroaggregative Escherichia coli hybrids strains
Those UPEC strains which had characteristic defining genes of EAEC (agg R/aapA or their combination) were considered to be UPEC/EAEC hybrids.
A two-tailed Chi-square test was used to compare groups. If low predicted values constrained the study, Fisher's exact test was performed using the GraphPad PRISM version 5.0 software (GraphPad Software 2365 Northside Dr. Suite 560 San Diego, CA 92108).
| ~ Results|| |
Distribution of virulence genes among uropathogenic Escherichia coli and uropathogenic Escherichia coli/ enteroaggregative Escherichia coli hybrid isolates
Among 163 UPEC isolates, the aggR gene was found in 10 isolates, aap gene was present in 24 isolates and one strain carries both agg R with aap gene. Hence, a total of 33 UPEC/EAEC hybrid strains were obtained [Table 2]. Distribution of virulence markers from the UPEC isolates was as follows: iuc (65.3%) and fim H (60%) genes were the most frequently detected, followed by pap C (43.8%), ast A (29.2%) and afa C (14.6%) [Table 3]. In hybrid isolates, iuc (69.6%) was the most prevalent gene [Table 3]. Based on the distribution of the VGs, the pic gene (P = 0.0001) was statistically significant when genes among UPEC and hybrid isolates were compared [Table 3].
|Table 2: Characteristics of hybrid strains (uropathogenic Escherichia coli/enteroaggregative Escherichia coli) harbouring virulence genes of enteroaggregative Escherichia coli and uropathogenic Escherichia coli, biofilm formation, and their phylogroups.|
Click here to view
|Table 3: Distribution of virulence genes among uropathogenic Escherichia coli and uropathogenic Escherichia coli/ Enteroaggregative Escherichia coli hybrid isolates|
Click here to view
Phylogenetic analysis among uropathogenic Escherichia coli and enteroaggregative Escherichia coli/ uropathogenic Escherichia coli hybrid isolates
The phylogenetic groups were characterised using M-PCR assay. Regarding the phylogenetic analysis of UPEC strains, the predominant groups were A (53%) and D (33.8%), followed by group B1 (10%) and group B2 (3.07%). Whereas phylogroup D (60.6%) was predominant in UPEC/EAEC hybrid strains followed by A (27%), as shown in [Table 4]. There was a significant association of UPEC with phylogroup A (P = 0.01) and hybrids with phylogroup D (P = 0.008) [Table 4].
|Table 4: Distribution of phylogenetic group among uropathogenic Escherichia coli and enteroaggregative Escherichia coli/uropathogenic Escherichia coli hybrid isolates|
Click here to view
Biofilm potential of uropathogenic Escherichia coli and uropathogenic Escherichia coli/ enteroaggregative Escherichia coli strains
Of 130 UPEC isolates, 94 (72.3%) isolates were biofilm forming, out of which 6 (4.6%) were strong, 54 (41.5%) were moderate, 34 (26.1%) were weak biofilm producers and 36 (27.6%) were non-biofilm producers. Similarly, in 33 UPEC/EAEC hybrid isolates, 26 (78.7%) were biofilm forming, out of which 1 (3.3%) were strong, 16 (48.4%) were moderate, 9 (27.2%) were weak biofilm producers and 7 (21.2%) were non-biofilm producers [Figure 1] and [Supplementary Table S1].
|Figure 1: Biofilm-forming potential of uropathogenic Escherichia coli and uropathogenic Escherichia coli/enteroaggregative Escherichia coli strains|
Click here to view
Relationship between biofilm formation and expression of virulence factors in uropathogenic Escherichia coli isolates
We studied the association of biofilm formation with the presence of virulence factors. The presence of VGs and biofilm formation for the pic gene (P = 0.007) were significantly associated when UPEC and UPEC/EAEC strains were compared [Table 5].
|Table 5: Occurrence of biofilm formation and the presence of various virulence genes in uropathogenic Escherichia coli isolates|
Click here to view
Antibiotic resistance among uropathogenic Escherichia coli and uropathogenic Escherichia coli/ enteroaggregative Escherichia coli isolates
The antibiotic susceptibility test showed that the 121 (93%), 98 (75.3%), 95 (73%), 92 (70%) and 92 (70%) of UPEC isolates show resistance to nalidixic acid, co-trimoxazole, cefotaxime, cefoperazone and ciprofloxacin, respectively [Table 6]. In the case of UPEC/EAEC isolates, similar resistance similar level of resistance was found [Table 6]. Nearly 73.8% of UPEC and 75.7% of UPEC/EAEC hybrid strains were multidrug resistant (MDR).
|Table 6: Antibiotic resistance among uropathogenic Escherichia coli and uropathogenic Escherichia coli/enteroaggregative Escherichia coli isolates|
Click here to view
| ~ Discussion|| |
EAECs are recently described as sporadic etiological agents in extraintestinal infections, especially in UTI., Hybrid EAEC/UPEC strains were also associated with community-acquired UTI outbreaks in 1991 in Denmark. The aap and agg R genes are characteristic of EAEC and were used as a detection marker. We evaluated a set of 163 E. coli, which cause NUTI and detect 33 (20.2%) UPEC/EAEC hybrid strains based on agg R and aap. In a Brazilian study, using EAEC, markers (aat A and agg R) were detected in 3.4% (9/225) of UPEC/EAEC strains. In another study by Abe et al. from Brazil, on UTI, 3.5% (8/225) of the strains were classified as UPEC/EAEC hybrids on the presence of aat A virulence marker. The presence of these marker genes in UPEC defines the hybrid nature of these isolates. Along with other classical UPEC markers, Abe et al. found that 62.5% (5/8) of UPEC/EAEC strains also carried UPEC gene pyelonephritis-associated pilus (pap C), which highlighted the hybrid aspect of these strains. PapC is an essential external membrane protein in UPEC forPpili biogenesis correlated with pyelonephritis due to PapG adherence to host cell receptors. In our collection, 36% (12/33) of the hybrid strains were positive for pap C. We also demonstrated that the haemolysin encoding gene (hly C) and iuc encoding aerobactin were present in 9.0% and 69.6% of UPEC/EAEC isolates. Haemolysin is one of the best-known pore-forming toxins in UPEC, and the iuc gene encodes for aerobactin siderophore and is necessary to obtain iron from iron chelator molecules., In addition, we found that the distribution of adhesive factors, including (afa C, fim H, pap C and sfa), was similar in both UPEC and UPEC/EAEC hybrid strains. The results demonstrated a closer relationship between EAEC and UPEC that can be described by the phenomenal plasticity of the genome or horizontal gene transfer in E. coli strains that result in virulent strains conveying VGs from different pathotypes in one isolate.
Our study had a few similarities and differences with other studies on the distribution of EAEC VGs in UPEC., Toxin ast A gene, a characteristic gene of EAEC codes EAST1 (EAEC heat-stable enterotoxins), causes increased chloride secretion and is correlated with secretory diarrhoea. Notably, in our study, the ast A (42%) gene was the most prevalent toxin among UPEC/EAEC hybrid isolates. Whereas in UPEC, the prevalence of the ast A was 29.2%, which was higher than a study in Iran where they found that 9.8% of the 138 UPEC strains was carrying this gene. Besides, UPEC strains also possessed the SPATEs pic and pet, both of which are commonly found in EAEC., Pet is a toxin from the family of SPATE which was initially identified in EAEC and exhibits enterotoxic and cytotoxic properties. This toxin damages the cytoskeleton assembly of intestinal epithelial cells which result in severe inflammatory response. While pic exhibits mucinolytic properties, which causes host immune responses to modulate, in our findings, the prevalence of pet and pic in UPEC isolates was 9.2% and 0.7%, respectively. Although in previous reports, Abe et al. reported the prevalence of pic and pet toxins in 13.8% and 0.4% of UPEC strains, respectively. Whereas we demonstrated that in hybrid strains, UPEC/EAEC, the prevalence of SPATE toxins pet and the pic was 15.1% and 21%, respectively. EAEC VGs were frequently detected in UPEC isolates. Our findings revealed that the UPEC strains had specific profiles of EAEC VGs relative to other studies, suggesting that the profile of virulence depends on geographical and environmental or some other factors.,
In our findings, one of the EAEC/UPEC hybrid strains, characterised by a full collection of EAEC (aggR and aap) and UPEC (iuc, afa A, fim H and pap C)-specific genes, was classified as phylogroup D. Hybrid EAEC/UPEC strains with similar genetic constructs were also linked with UTI outbreaks in Denmark in 1991. Further genetic analysis revealed that 18 out of 19 strains were positive for EAEC-specific genes agg R and aap along with other UPEC and EAEC-specific genes. Olesen et al. found that this hybrid EAEC/UPEC outbreak strains belonged to intestinal phylogroup D, and these findings were similar to our results. We found that twenty (60.6%) EAEC/UPEC strains belonged to phylogroup D; these findings were in contrast with the hypothesis established in the literature that diarrhoeagenic virulence markers are rarely detectable in phylogenetic Group D. Whereas nine (27.2%) hybrid EAEC/UPEC strains belonged to the commensal group and classified as phylogroup A. On the other hand, E. coli lineages that frequently gather commensal strains from phylogroup A and B1 may also include highly virulent strains. This interesting finding demonstrated that the classic classification of strains into E. coli pathotypes is limited and inaccurate. We also found that UPEC strains in phylogroups A were more abundant (53%), indicating that Group A and other phylogroups could be pathogenic. The dominance of the phylogenetic Group A isolates in UPEC isolates, generally associated with commensal strains, indicates that the digestive tract is the primary source of colonising strains in the urinary tract., VGs and phylogroups are distributed differently between different countries; for example, phylogroup A was most prevalent in Russia, and also in China in UPEC, so our findings can be explained by geographical variation.
Besides, we also conducted a possible association between different VGs with biofilm formation among UPEC and UPEC/EAEC strains. Biofilm formation is considered a pivotal step in the development of infection in human mucosa. The ability to adhere to different surfaces and the formation of biofilms has been regarded as an important feature of E. coli pathogenicity in both UPEC and EAEC. Surface virulence factors of the bacterium, including different adherence factors, can contribute to bacterial adhesion and biofilm growth. Overall, among UPEC isolates, 72.3% and, in UPEC/EAEC, 78.7% of isolates were found to produce biofilm, similar to findings by Sharma et al. (2009), who found 67.5% of E. coli isolates to produce biofilms. In UPEC and hybrid strains, we observed a similar prevalence of adhesion and fimbrial genes that play a key role in biofilm formation. In our study, the prevalence of several adhesive and fimbrial factors of UPEC among biofilm-forming UPEC and hybrid strains revealed that the majority of isolates harbouredPand Type 1 fimbrial associated genes irrespective of their biofilm formation.
Furthermore, previous studies investigated the relationship between aap gene and its role in biofilm formation. We found that 80.7% of UPEC/EAEC hybrid strains were able to form biofilm formation in the presence of the aap gene. UPEC isolates possessing the VGs of EAEC (aap, ast A, pic and pet) and UPEC VGs in combination produced more biofilm. Because aggR is a master regulator of plasmid and chromosomally encoded genes in EAEC, UPEC isolates carrying aap, ast A, pic and pet enhance the biofilm formation potential of these isolates. Our findings also suggest that although type 1 and P fimbriae are essential adhesive factors for initial bacterial adhesion to biological surfaces, the occurrence of these genes is not the only determining factor for the formation of biofilms in UPEC and hybrid strains, but other genetic and environmental factors may be involved in the expression of these genes. Cases of UTI and epidemics involving hybrid EAEC/UPEC strains supported the suspicion that the EAEC VGs add uropathogenic properties to E. coli strains., A better knowledge of bacterial pathogenesis would allow the development of novel therapeutic strategies.
Antibiotic susceptibility testing revealed that antibiotic resistance in UPEC is higher, as reported in previous studies.,, In our study, very high levels of antibiotic resistance were found to nalidixic acid, co-trimoxazole, cefotaxime and ciprofloxacin in UPEC. Antibiotic resistance in hybrid UPEC/EAEC and UPEC isolates was similar. We also found that 73.8% of UPEC and 80% of UPEC/EAEC hybrid strains were MDR. Biofilm producers displayed higher antimicrobial resistance than non-biofilm strains. The increased antibiotic resistance among biofilm producers is induced by the slow growth and the presence of an exopolysaccharide protection cover, which affects the penetration of antibiotics into the biofilm and hinders the activity of antimicrobial agents.,
| ~ Conclusions|| |
This analysis shows that certain UPEC strains carry characteristic VGs of EAEC. This observation indicates that at least some faecal EAEC strains could represent possible uropathogens; therefore, their occurrence in the faeces of asymptomatic persons could explain controversies between epidemiological studies to examine the diarrhoeal potential of EAEC. Alternatively, UPEC strains could have developed EAEC markers as a possible cause of diarrhoea. Further research on EAEC/UPEC strains and other UPEC and EAEC genes are crucial to understand the function that plays a vital role in pathogenicity. Currently, there are few studies available suggesting that hybrid strains are more virulent than their parental pathotypes. Hence, it is highly important to address the nature of the pathogenicity of these pathogens. Although our study documents the existence of EAEC pathotypes VGs in UPEC strains, whether such genes are expressedin vivo and play some role in human UPEC infections remains to be addressed. Future research must explore and identify the function and expression levels of the biofilm-associated adhesive genes with the genetic context of UPEC and also the development of various types of UTIs. A close relationship between UPEC and EAEC, emerging enteropathogens capable of inducing intestinal and extraintestinal infections, particularly in developing countries, warrants attention in modern strain typing and epidemiological surveillance of E. coli infections. In addition, further studies regarding hybrid E. Coli isolates are needed to understand the pathogenesis of these pathogens better and to control their spread.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Terlizzi ME, Gribaudo G, Maffei ME. UroPathogenic Escherichia coli
(UPEC) infections: Virulence factors, bladder responses, antibiotic, and non-antibiotic antimicrobial strategies. Front Microbiol 2017;8:1566.
Doumith M, Day MJ, Hope R, Wain J, Woodford N. Improved multiplex PCR strategy for rapid assignment of the four major Escherichia coli
phylogenetic groups. J Clin Microbiol 2012;50:3108-10.
Dias RC, Marangoni DV, Smith SP, Alves EM, Pellegrino FL, Riley LW, et al
. Clonal composition of Escherichia coli
causing community-acquired urinary tract infections in the State of Rio de Janeiro, Brazil. Microb Drug Resist 2009;15:303-8.
Evans DJ Jr., Evans DG. Classification of pathogenic Escherichia coli
according to serotype and the production of virulence factors, with special reference to colonization-factor antigens. Rev Infect Dis 1983;5 Suppl 4:S692-701.
Nataro JP. Enteroaggregative Escherichia coli
pathogenesis. Curr Opin Gastroenterol 2005;21:4-8.
Monteiro BT, Campos LC, Sircili MP, Franzolin MR, Bevilacqua LF, Nataro JP, et al
. The dispersin-encoding gene (aap) is not restricted to enteroaggregative Escherichia coli
. Diagn Microbiol Infect Dis 2009;65:81-4.
Jenkins C, van Ijperen C, Dudley EG, Chart H, Willshaw GA, Cheasty T, et al
. Use of a microarray to assess the distribution of plasmid and chromosomal virulence genes in strains of enteroaggregative Escherichia coli
. FEMS Microbiol Lett 2005;253:119-24.
Cordeiro F, da Silva Gomes Pereira D, Rocha M, Asensi MD, Elias WP, Campos LC. Evaluation of a multiplex PCR for identification of enteroaggregative Escherichia coli
. J Clin Microbiol 2008;46:828-9.
Hebbelstrup Jensen B, Olsen KE, Struve C, Krogfelt KA, Petersen AM. Epidemiology and clinical manifestations of enteroaggregative Escherichia coli
. Clin Microbiol Rev 2014;27:614-30.
Bien J, Sokolova O, Bozko P. Role of uropathogenic Escherichia coli
virulence factors in development of urinary tract infection and kidney damage. Int J Nephrol 2012;2012:681473.
Johnson JR, Kuskowski MA, O'bryan TT, Colodner R, Raz R. Virulence genotype and phylogenetic origin in relation to antibiotic resistance profile among Escherichia coli
urine sample isolates from Israeli women with acute uncomplicated cystitis. Antimicrob Agents Chemother 2005;49:26-31.
Abe CM, Salvador FA, Falsetti IN, Vieira MA, Blanco J, Blanco JE, et al
. Uropathogenic Escherichia coli (UPEC) strains may carry virulence properties of diarrhoeagenic Escherichia coli
. FEMS Immunol Med Microbiol 2008;52:397-406.
Nyholm O, Halkilahti J, Wiklund G, Okeke U, Paulin L, Auvinen P, et al
. Comparative genomics and characterization of hybrid shigatoxigenic and enterotoxigenic Escherichia coli
(STEC/ETEC) strains. PLoS One 2015;10:e0135936.
Nunes KO, Santos ACP, Bando SY, Silva RM, Gomes TA, Elias WP. Enteroaggregative Escherichia coli
with uropathogenic characteristics are present in feces of diarrheic and healthy children. Pathog Dis 2017;75:(8).
Boll EJ, Struve C, Boisen N, Olesen B, Stahlhut SG, Krogfelt KA. Role of enteroaggregative Escherichia coli
virulence factors in uropathogenesis. Infect Immun 2013;81:1164-71.
Soto SM. Importance of biofilms in urinary tract infections: New therapeutic approaches. Adv Biol 2014;2014:1-13.
Zamani H, Salehzadeh A. Biofilm formation in uropathogenic Escherichia coli
: Association with adhesion factor genes. Turk J Med Sci 2018;48:162-7.
Khudaier BY, Tewari R, Shafiani S, Sharma M, Emmanuel R, Sharma M, et al
. Epidemiology and molecular characterization of vancomycin resistant Enterococci isolates in India. Scand J Infect Dis 2007;39:662-70.
Tarchouna M, Ferjani A, Ben-Selma W, Boukadida J. Distribution of uropathogenic virulence genes in Escherichia coli
isolated from patients with urinary tract infection. Int J Infect Dis 2013;17:e450-3.
Boisen N, Scheutz F, Rasko DA, Redman JC, Persson S, Simon J, et al
. Genomic characterization of enteroaggregative Escherichia coli
from children in mali. J Infect Dis 2012;205:431-44.
Clermont O, Christenson JK, Denamur E, Gordon DM. The Clermont Escherichia coli
phylo-typing method revisited: Improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep 2013;5:58-65.
Mathur T, Singhal S, Khan S, Upadhyay DJ, Fatma T, Rattan A. Detection of biofilm formation among the clinical isolates of Staphylococci: An evaluation of three different screening methods. Indian J Med Microbiol 2006;24:25-9.
] [Full text]
Ferreira AA, Tette PA, Mendonça RC, Soares A, de Carvalho MM. Detection of exopolysaccharide production and biofilm-related genes in Staphylococcus
spp. isolated from a poultry processing plant. Food Sci Technol 2014;34:710-6.
Murugan K, Usha M, Malathi P, Al-Sohaibani AS, Chandrasekaran M. Biofilm forming multi drug resistant Staphylococcus
spp. among patients with conjunctivitis. Pol J Microbiol 2010;59:233-9.
Clinical and Laboratory Standards Institute; 2019. p. 604.
Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli
. Nat Rev Microbiol 2004;2:123-40.
Olesen B, Scheutz F, Andersen RL, Menard M, Boisen N, Johnston B, et al
. Enteroaggregative Escherichia coli
O78:H10, the cause of an outbreak of urinary tract infection. J Clin Microbiol 2012;50:3703-11.
Lara FB, Nery DR, de Oliveira PM, Araujo ML, Carvalho FR, Messias-Silva LC, et al
. Virulence markers and phylogenetic analysis of Escherichia coli
strains with hybrid EAEC/UPEC genotypes recovered from sporadic cases of extraintestinal infections. Front Microbiol 2017;8:146.
Emody L, Kerényi M, Nagy G. Virulence factors of uropathogenic Escherichia coli
. Int J Antimicrob Agents 2003;22 Suppl 2:29-33.
Subashchandrabose S, Mobley HLT. Virulence and fitness determinants of uropathogenic Escherichia coli
. Microbiol Spectr 2015;3:4.
Ejrnæs K. Bacterial characteristics of importance for recurrent urinary tract infections caused by Escherichia coli
. Dan Med Bull 2011;58:B4187.
Ruan X, Crupper SS, Schultz BD, Robertson DC, Zhang W. Escherichia coli
expressing EAST1 toxin did not cause an increase of cAMP or cGMP levels in cells, and no diarrhea in 5-day old gnotobiotic pigs. PLoS One 2012;7:e43203.
Mirzarazi M, Rezatofighi SE, Pourmahdi M, Mohajeri MR. Occurrence of genes encoding enterotoxins in uropathogenic Escherichia coli
isolates. Braz J Microbiol 2015;46:155-9.
Boisen N, Ruiz-Perez F, Scheutz F, Krogfelt KA, Nataro JP. Short report: High prevalence of serine protease autotransporter cytotoxins among strains of enteroaggregative Escherichia coli
. Am J Trop Med Hyg 2009;80:294-301.
Heimer SR, Rasko DA, Lockatell CV, Johnson DE, Mobley HL. Autotransporter genes pic and tsh are associated with Escherichia coli
strains that cause acute pyelonephritis and are expressed during urinary tract infection. Infect Immun 2004;72:593-7.
Dautin N. Serine protease autotransporters of Enterobacteriaceae
(SPATEs): Biogenesis and function. Toxins (Basel) 2010;2:1179-206.
Dutta PR, Cappello R, Navarro-García F, Nataro JP. Functional Comparison of Serine Protease Autotransporters of Enterobacteriaceae
. Infect Immun 2002;70:7105-13.
Khaleque M, Akter S, Akhter H, Khan SI, Begum A. Analysis of diarrheagenic potential of uropathogenic Escherichia coli
isolates in Dhaka, Bangladesh. J Infect Dev Ctries 2017;11:459-69.
Moreno E, Andreu A, Pérez T, Sabaté M, Johnson JR, Prats G. Relationship between Escherichia coli
strains causing urinary tract infection in women and the dominant faecal flora of the same hosts. Epidemiol Infect 2006;134:1015-23.
López-Banda DA, Carrillo-Casas EM, Leyva-Leyva M, Orozco-Hoyuela G, Manjarrez-Hernández ÁH, Arroyo-Escalante S, et al
. Identification of virulence factors genes in Escherichia coli
isolates from women with urinary tract infection in Mexico. Biomed Res Int 2014;2014:1-10.
Grude N, Potaturkina-Nesterova NI, Jenkins A, Strand L, Nowrouzian FL, Nyhus J, et al
. A comparison of phylogenetic group, virulence factors and antibiotic resistance in Russian and Norwegian isolates of Escherichia coli
from urinary tract infection. Clin Microbiol Infect 2007;13:208-11.
Tong Y, Sun S, Chi Y. Virulence genotype and phylogenetic groups in relation to chinese herb resistance among Escherichia coli
from patients with acute pyelonephritis. Afr J Tradit Complement Altern Med 2014;11:234-8.
Reisner A, Krogfelt KA, Klein BM, Zechner EL, Molin S.In vitro
biofilm formation of commensal and pathogenic Escherichia coli
strains: Impact of environmental and genetic factors. J Bacteriol 2006;188:3572-81.
Ochoa SA, Cruz-Córdova A, Luna-Pineda VM, Reyes-Grajeda JP, Cázares-Domínguez V, Escalona G, et al
. Multidrug- and Extensively Drug-Resistant Uropathogenic Escherichia coli
clinical strains: Phylogenetic groups widely associated with integrons maintain high genetic diversity. Front Microbiol 2016;7:2042.
Paniagua-Contreras GL, Monroy-Pérez E, Rodríguez-Moctezuma JR, Domínguez-Trejo P, Vaca-Paniagua F, Vaca S. Virulence factors, antibiotic resistance phenotypes and O-serogroups of Escherichia coli
strains isolated from community-acquired urinary tract infection patients in Mexico. J Microbiol Immunol Infect 2017;50:478-85.
Ali I, Rafaque Z, Ahmed S, Malik S, Dasti JI. Prevalence of multi-drug resistant uropathogenic Escherichia coli
in potohar region of Pakistan. Asian Pac J Trop Biomed 2016;6:60-6.
Hung CS, Henderson JP. Emerging concepts of biofilms in infectious diseases. Mo Med 2009;106:292-6.
Hall MR, McGillicuddy E, Kaplan LJ. Biofilm: Basic principles, pathophysiology, and implications for clinicians. Surg Infect (Larchmt) 2014;15:1-7.
Suzart S, Aparecida T, Gomes T, Guth BE. Characterization of serotypes and outer membrane protein profiles in enteroaggregative Escherichia coli
strains. Microbiol Immunol 1999;43:201-5.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]